PI: Faubion, William A Title: Inflammatory cascades disrupt Treg function through epigenetic mechanisms

Received: 07/02/2015 FOA: PA13-302 Council: 01/2016

Competition ID: FORMS-C FOA Title: RESEARCH PROJECT GRANT (PARENT R01)

2 R01 AI089714-06A1 Dual: DK Accession Number: 3841834

IPF: 4976101 Organization: MAYO CLINIC ROCHESTER

Former Number: Department: Internal Medicine

IRG/SRG: GMPB AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 6:

Year 7:

Year 8:

Year 9:

Year 10:

Animals: Y 

Humans: N 

Clinical Trial: N

Current HS Code: 10 

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Stephen Ekker PhD Mayo Clinic Other (Specify)-Other Significant 

Contributor

Thomas Smyrk MD Mayo Clinic Other (Specify)-Other Significant 

Contributor

Raul Urrutia MD Mayo Clinic Other (Specify)-Other Significant 

Contributor

William Faubion MD Mayo Clinic PD/PI 

Chandrashekhar Pasare PhD University of Texas Southwestern 

Medical Center at Dallas

Consultant

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Funding Opportunity Number: PA-13-302 . Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

OMB Number: 4040-0001

Expiration Date: 06/30/2016

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

3. DATE RECEIVED BY STATE State Application Identifier

1. TYPE OF SUBMISSION* 4.a. Federal Identifier

❍ Pre-application ● Application ❍Changed/Corrected

Application

b. Agency Routing Number

2. DATE SUBMITTED Application Identifier c. Previous Grants.gov Tracking Number

5. APPLICANT INFORMATION Organizational DUNS*:

Legal Name*: Mayo Clinic 

Department: Internal Medicine

Division: Gastroenterology 

Street1*: 200 First Street SW

Street2:

City*: Rochester 

County:

State*: MN: Minnesota 

Province:

Country*: USA: UNITED STATES

ZIP / Postal Code*: 559050001 

Person to be contacted on matters involving this application

Prefix: First Name*: David Middle Name: M LastName*: Moertel Suffix: 

Position/Title: Institutional Official 

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6. EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN)* 

7. TYPE OF APPLICANT* M: Nonprofitwith501C3IRSStatus(Other than Institution ofHigher 

Education)

Other (Specify):

Small Business Organization Type ❍Women Owned ❍ Socially and Economically Disadvantaged

8. TYPE OF APPLICATION*

❍ New ●Resubmission

❍ Renewal ❍ Continuation ❍Revision

If Revision, mark appropriate box(es).

❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration

❍D. Decrease Duration ❍E. Other (specify) :

Is this application being submitted to other agencies?* ❍Yes ●No What other Agencies?

9. NAME OF FEDERAL AGENCY*

National Institute of Allergy and Infectious Diseases

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

Inflammatory cascades disrupt Treg function through epigenetic mechanisms

12. PROPOSED PROJECT

Start Date* Ending Date*

07/01/2016 06/30/2021

13. CONGRESSIONAL DISTRICTS OF APPLICANT

MN-001
Funding Opportunity Number: PA-13-302 . Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

Contact PD/PI: Faubion, William A

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: William Middle Name: A LastName*: Faubion Suffix: MD

Position/Title: Associate Professor 

Organization Name*: Mayo Clinic 

Department: InternalMedicine

Division: Gastroenterology 

Street1*: 

Street2: 

City*: 

County: 

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*:

15. ESTIMATED PROJECT FUNDING 16.IS APPLICATION SUBJECT TO REVIEW BY STATE 

EXECUTIVE ORDER 12372 PROCESS?*

a. Total Federal Funds Requested* $ 

b. Total Non-Federal Funds* $0.00

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE 

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

c. Total Federal & Non-Federal Funds* $ DATE:

d. Estimated Program Income* $0.00

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR 

REVIEW 

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein 

are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with 

any resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to 

criminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001)

●I agree* * The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name:

19. AUTHORIZED REPRESENTATIVE

Prefix: First Name*: David Middle Name: M LastName*: Moertel Suffix: 

Position/Title*: Institutional Official 

Organization Name*: Mayo Clinic 

Department: RS-Research Services

Division: RS-Research Services

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: STATES

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*:

Signature of Authorized Representative* Date Signed*

David.Moertel 07/02/2015

20. PRE-APPLICATION File Name:

21. COVER LETTER ATTACHMENT File Name:
Contact PD/PI: Faubion, William A

424 R&R and PHS-398 Specific

TableOf Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1 

Table of Contents------------------------------------------------------------------------- 3 

Performance Sites--------------------------------------------------------------------------------------------- 4 

Research & Related OtherProject Information------------------------------------------------------------------ 5

Project Summary/Abstract(Description)----------------------------------------------------- 6 

Project Narrative------------------------------------------------------------------------- 7

Facilities & Other Resources-------------------------------------------------------------- 8 

Equipment--------------------------------------------------------------------------------- 9

Research & RelatedSenior/Key Person-------------------------------------------------------------------------- 10

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 34

PHS 398 Modular Budget---------------------------------------------------------------------------------------- 36

Personnel Justification------------------------------------------------------------------- 42

PHS 398 Research Plan----------------------------------------------------------------------------------------- 43

Introduction------------------------------------------------------------------------------ 44

Specific Aims----------------------------------------------------------------------------- 45

Research Strategy------------------------------------------------------------------------- 46

Progress Report Publications List--------------------------------------------------------- 58

Vertebrate Animals------------------------------------------------------------------------ 60

Bibliography & References Cited----------------------------------------------------------- 63

Letters Of Support------------------------------------------------------------------------ 68

Resource Sharing Plans-------------------------------------------------------------------- 73

Table of Contents Page 3
Contact PD/PI: Faubion, William A

Project/Performance Site Location(s)

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Project/Performance Site Primary Location ❍I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of 

organization.

Organization Name: Mayo Clinic

Duns Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*: MN-001

File Name

Additional Location(s)

Page 4

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date:

2015-07-02T14:04:51.000-04:00
Contact PD/PI: Faubion, William A

RESEARCH & RELATED Other Project Information

OMB Number: 4040-0001

Expiration Date: 06/30/2016

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6 If 

NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ● Yes ❍ No 

IACUC Approval Date:

Animal Welfare Assurance Number A3291-01 

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an ❍ Yes

environmental assessment (EA) or environmental impact statement (EIS) been performed?

4.d. If yes, please explain:

● No

❍ No

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes

5.a. If yes, please explain:

● No

6. Does this project involve activities outside the United States or partnership with international ❍ Yes

collaborators?*

6.a. If yes, identify countries:

6.b. Optional Explanation:

● No

Filename

7. Project Summary/Abstract* Project Summary_Abstract.pdf 

8. Project Narrative* Project_Narrative.pdf

9. Bibliography & References Cited Final_bibliography.pdf

10.Facilities & Other Resources Facilities_and_Other_Resources.pdf

11.Equipment Equipment.pdf

Page 5

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date:

2015-07-02T14:04:51.000-04:00
Contact PD/PI: Faubion, William A

Project Summary/Abstract Page 6

PROJECT SUMMARY/ABSTRACT:

The transcription factor FOXP3 is critical to the regulation of numerous debilitating human immune-mediated 

diseases. Very recently, the essential role for the histone methyltransferase (HMT) EZH2 in the epigenetic 

regulation and function of FOXP3 has been described. Inflammatory pathways modify EZH2 activity, and 

inflammatory signaling impairs Treg function in vivo and in vitro. The biological impact of the FOXP3-EZH2 

pathway to IBD is unknown. Our long-term goal is to dissect epigenetic mechanisms regulating Treg cellular 

differentiation and function, particularly within the setting of GI inflammatory diseases. These discoveries will 

facilitate design of human cell therapy trials for IBD. The objective of this grant is to characterize the role for 

EZH2 in Treg suppressive function. The central hypothesis is that EZH2 plays a critical role in the homeostasis 

of Treg cells, and the disruption of EZH2 function by inflammatory signaling pathways contributes to IBD. Our 

rationale is that identification of the mechanism(s) to restore Treg suppressive function in the setting of 

intestinal inflammation will offer new therapeutic opportunities. Our specific aims will test the following 

hypotheses: (Aim1) Repression of immunoregulatory gene networks by FOXP3 requires the formation of a 

complex between this transcription factor and EZH2; (Aim 2) Inflammatory stimuli, such as IL6 lead to EZH2 

phosphorylation and thereby disrupt the enzymatic activity of this epigenomic regulator; (Aim 3) Inhibition of the 

IL6 to EZH2 signaling pathway permits sustained Treg suppressive function in the setting of intestinal 

inflammation. Upon conclusion, we will understand the role for EZH2 in Treg loss of function in the setting of 

active inflammation. This contribution is significant since it will establish that several pathways targeted by 

available therapies (ie IL1β, IL6, TNFα) have the potential to regulate EZH2 HMT activity through posttranslational modifications. Furthermore, current Treg cell therapy trials, while promising have not addressed 

the key issue of in vivo inflammation-induced disruption of Treg function. The proposed research is innovative

because we investigate the effect of inflammatory signaling pathways on epigenetic complexes in Treg cells, a 

heretofore-unexamined process. Insight into epigenetic mechanisms is impactful as T cell progenitor cells 

inherit the parent transcriptional profile and unlike genetic change, they are modifiable by currently available 

therapy.
Contact PD/PI: Faubion, William A

Project Narrative Page 7

PROJECT NARRATIVE:

The proposed research is relevant to the public health because IBD, increasing in prevalence, represents a 

major national cost measured by both patient suffering and economic burden; and despite significant advances 

in care, clinical trial data demonstrate remission rates at best of 40%. Upon conclusion, we will understand the 

role for EZH2 in Treg loss of function in the setting of active inflammation, and this discovery will stimulate the 

opening of a new avenue in therapeutics directed at stimulation of autologous Treg cells to function within the 

inflammatory milieu.
Contact PD/PI: Faubion, William A

Facilities & Other Resources Page8

FACILITIES AND OTHER RESOURCES:

Mayo Clinic’s Research Resource/Core Service Facilities

Resource Director

Medical Genome Facility

Includes the following Resources/Cores: 

•Biospecimens Accessioning Processing 

(BAP)

•Pathology Research Core

•Gene Expression Core

•Cytogenetics Shared Resource (CSR)

•Genotyping Shared Resource Core Facility

•Sequencing Core

Eric D. Wieben, Ph.D.

• W. Edward Highsmith, Jr., Ph.D. 

& Mine Cicek, Ph.D.

• Thomas Flotte MD.

• Jin Jen MD, PhD.

• Patricia Greipp, DO.

• Julie Cunningham, Ph.D.

• Eric D. Wieben, Ph.D.

Analytical Nuclear Magnetic Resonance (NMR) Slobodan I. Macura, Ph.D.

Mayo Antibody Core Facility Rochester (MACFR) Mr. Thomas G. Beito

Biomathematics Resource Zeljko Bajzer, Ph.D. & Armando Manduca, Ph.D.

Biomedical Imaging Resource Richard A. Robb, Ph.D.

Electron Microscopy Resource Jeffrey L. Salisbury, Ph.D.

Flow Cytometry/Optical Morphology Resource Richard G. Vile, Ph.D.

Gene Targeted Mouse Core Facility (GTM-CF) Jan van Deursen, Ph.D.

Immunochemical Laboratory Core Facility Ravinder J. Singh, Ph.D.; Joseph P. McConnell, Ph.D.; & 

George G. Klee, M.D., Ph.D.

Materials and Structural Testing Resource Kai-Nan An, Ph.D. & Kenton R. Kaufman, Ph.D.

Mayo Clinic Cancer Center:

Includes the following Resources/Cores: 

•Biostatistics

•Cancer Informatics

•Clinical Research Office

•Gene and Virus Therapy

•Immunotherapy

•Pharmacology

•Pharmacy

•Survey Research

Robert B. Diasio, M.D.

• Daniel J. Sargent, Ph.D. & Vernon S. Pankratz, Ph.D.

• James R. Cerhan, M.D. & Christopher G. Chute, M.D.,

• Steven R. Alberts, M.D. & Janet Olson, Ph.D.

• Mark J. Federspiel, Ph.D.

• Dennis A. Gastineau, M.D.; Eugene Kwon, M.D.; & 

Stanimir Vuk-Pavlovic, Ph.D.

• Matthew M. Ames, Ph.D.

• Mr. Darryl C. Grendahl

• Timothy J. Beebe, Ph.D.

Mayo Proteomics Research Center (MPRC) Daniel J. McCormick, Ph.D.
Contact PD/PI: Faubion, William A

Equipment Page 9

EQUIPMENT:

The PI’s laboratory is equipped with most items for modern biochemistry, cell and molecular biology, and 

cellular immunology including 5 conventional PCR machines, 2 real time fluorescent PCR machines, a 

Phospholmager, 2 HPLC, FPLC, a spectrophotometer, electroporator, nucleofection unit, an ultracentrifuge, a 

mid-speed centrifuge and rotors, sonicator, scintillation counter, -20°C and -80°C freezers, a speed vac 

concentrator and a slab gel dryer. A Zeiss fluorescence confocal microscope is also available, fully equipped 

with an Eppendorf microinjection system for semi-automatic injection into single cells, along with an Icyte® 

Imaging Cytometer. There is also a cell biology and tissue processing laboratory, which has a biohood, cell 

incubators, centrifuge, refrigeration equipment, and purified water supplies. A computational biology laboratory 

with 5 Silicon Graphic Stations and 50 parallel CPU is used to model mutations. Biophysical equipment 

include for circular dicroism and isothermal titration calorimetry. Two fully equipped tissue culture facilities with 

2 hoods each and a total of 12 incubators. The GI unit also has a fully equipped Becton Dickinson LSR II 

Fluorescent Activated Cell Sorter (FACS) on the floor. Kodak X-omat X-ray film processor and dark room are 

located on the floor.
Contact PD/PI: Faubion, William A

Page 10

Funding Opportunity Number: PA-13-302 . Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

OMB Number: 4040-0001

Expiration Date: 06/30/2016

PROFILE - Project Director/Principal Investigator

Prefix: First Name*: William Middle NameA Last Name*: Faubion Suffix:MD

Position/Title*: Associate Professor 

Organization Name*: Mayo Clinic 

Department: InternalMedicine

Division: Gastroenterology

Street1*: 

Street2:

City*: 

County: 

State*: 

Province:

Country*:

Zip / Postal Code*:

Phone Fax Number: E-Mail*: 

Number*:

Credential, e.g., agency login:

Project Role*: PD/PI Other Project Role Category:

Degree Type: MD Degree Year:

File Name

Attach Biographical Sketch*: Biosketch_Faubion_6 26 2015.appdpflication/pdf

Attach Current & Pending Support:
Contact PD/PI: Faubion, William A

Page 11

Funding Opportunity Number: PA-13-302 . Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

PROFILE - Senior/Key Person

Prefix: First Middle Name Last Name*: Pasare Suffix: PhD

Name*: Chandrashekhar

Position/Title*: Associate Professor

Organization Name*: University of Texas Southwestern Medical Center at Dallas

Department: 

Division: 

Street1*: 

Street2:

City*: 

County: 

State*: 

Province:

Country*:

Zip / Postal Code*:

Phone Fax Number: E-Mail*: 

Number*:

Credential, e.g., agency login:

Project Role*: Consultant Other Project Role Category:

Degree Type: PhD Degree Year:

File Name

Attach Biographical Sketch*: Biosketch_PasareJune_2015.pdf

Attach Current & Pending Support:

PROFILE - Senior/Key Person

Prefix: First Name*: Stephen MiddleNameC Last Name*: Ekker Suffix: PhD

Position/Title*: Professor

Organization Name*: Mayo Clinic

Department: Biochem & Molecular Biology

Division: Biochem & Molecular Biology

Street1*: 

Street2:

City*: 

County: 

State*: 

Province:

Country*:

Zip / Postal Code*:

Phone Fax Number: E-Mail*: 

Number*:

Credential, e.g., agency login:

Project Role*: Other (Specify) Other Project Role Category: Other Significant Contributor

Degree Type: PhD Degree Year:

File Name

Attach Biographical Sketch*: Ekker_biosketchJune 2015.pdf

Attach Current & Pending Support:
Contact PD/PI: Faubion, William A

Page 12

Funding Opportunity Number: PA-13-302 . Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

PROFILE - Senior/Key Person

Prefix: First Name*: Thomas MiddleNameC Last Name*: Smyrk Suffix:MD

Position/Title*: Professor 

Organization Name*: Mayo Clinic 

Department: DLMP

Division: Anatomic Pathology

Street1*: 

Street2:

City*: 

County: 

State*: 

Province:

Country*:

Zip / Postal Code*:

Phone Fax Number: E-Mail*: 

Number*:

Credential, e.g., agency login:

Project Role*: Other (Specify) Other Project Role Category: Other Significant Contributor

Degree Type: MD Degree Year:

File Name

Attach Biographical Sketch*: Biosketch_SmyrkJune_2015.pdf

Attach Current & Pending Support:

PROFILE - Senior/Key Person

Prefix: FirstName*:Raul Middle Name Alfredo Last Name*: Urrutia Suffix: MD

Position/Title*: Professor 

Organization Name*: Mayo Clinic 

Department: Internal Medicine

Division: Gastroenterology

Street1*: 

Street2:

City*: 

County: 

State*: 

Province:

Country*:

Zip / Postal Code*:

Phone Fax Number: E-Mail*: 

Number*:

Credential, e.g., agency login:

Project Role*: Other (Specify) Other Project Role Category: Other Significant Contributor

Degree Type: MD Degree Year:

File Name

Attach Biographical Sketch*: Biosketch_UrrutiaJune_2015.pdf

Attach Current & Pending Support:
Contact PD/PI: Faubion, William A

Biosketches Page 13

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Faubion, William

POSITION TITLE: Associate Professor of Medicine

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date 

MM/YYYY

FIELD OF STUDY

Dartmouth College B.A. 1988 Geology

University of Texas at Houston Medical School M.D. 1992 Medicine

University of Texas at Houston Medical School 1992-1996 Int Med/Pediatrics

Mayo Clinic, Rochester, Minnesota 1996-2001 Gastroenterology

Beth Israel, Harvard, MA 2001-2003 Mucosal Immunology

A. Personal Statement

I am an expert in both pediatric and adult inflammatory bowel disease with a laboratory-based 

research program on the mucosal immunology of chronic intestinal inflammation. My current NIH 

funding supports research relevant to the developmental pathways towards T-regulatory cell 

development. Specifically, we study coupling of KLF family transcription factor binding to chromatin 

modifying complexes leading to epigenetic change in gene networks regulating T cell phenotypes. 

We have published key epigenetic mechanisms regulating stability of FOXP3 gene transduction and 

T regulatory cell development. We correlate biophysical and biochemical mechanisms to function 

using animal models of colitis. I have multiple models in the lab of chronic intestinal inflammation 

which serve to dissect molecular mechanisms of T-cell phenotypes related to chronic inflammation. I 

am a joint member in the Department of Immunology, and hold the rank of Associate Professor of 

Immunology (in addition to Medicine and Pediatrics), a rank at Mayo Clinic given upon merit of 

accomplishments specifically within the field of Immunology. In addition to this ongoing NIH-funded, 

laboratory-based research program, I am a well-recognized expert in clinical inflammatory bowel 

disease. I am the director of the Pediatric Inflammatory Bowel Disease Clinic and co-investigator in 

the adult IBD Clinic, where we collectively see over 4000 patients per year.

B. Positions and Honors

Positions and Employment

1992-1996 University of Texas at Houston, Internal Medicine/Pediatrics Residency

1997-1998 Mayo Clinic, Rochester, MN, Center for Basic Research in Digestive Diseases,

Gastrointestinal Fellow, Laboratory of Gregory J. Gores, M.D.

1996-2000 Mayo Clinic, Pediatric and Adult Gastroenterology, Fellowship

2000-2003 Instructor of Medicine, Mayo Medical School

2001-2006 Mayo Foundation Scholar

eRA COMMONS USER NAME:
Contact PD/PI: Faubion, William A

Biosketches Page 14

2001-2003 Instructor of Medicine, Division of Immunology, BIDMC, Harvard Medical School 

2003-2012 Assistant Professor of Medicine, Mayo Clinic College of Medicine

2012-present Associate Professor of Medicine, Mayo Clinic College of Medicine 

2012-present Associate Professor of Pediatrics, Mayo Clinic College of Medicine 

2013-present Associate Professor of Immunology, Mayo Clinic College of Medicine 

2013-present Director, T32 Training Grant

2014-present Research Chair, Division of Gastroenterology and Hepatology, Mayo Clinic

Other Experience and Professional Memberships

2007 -2013 Member, American Board of Pediatrics

2007-present Associate Editor, Inflammatory Bowel Disease Journal 

2007-present Associate Editor, Pediatric Inflammatory Bowel Disease

Member, American Board of Internal Medicine 

2011-present Member, American Gastroenterological Association 

2011-present Member, American College of Gastroenterology

Member, North American Society for Pediatric Gastroenterology and Nutrition 

2011-present NIH Study Section Reviewer: Digestive Diseases and Nutrition

2012-present CCFA Career Development & Research Fellowship Committee Member 

2015-2018 CCFA Research Fellowship Awards Committee Member

Honors

2000 J. Arnold Bargen Award – for outstanding achievement by a fellow in gastroenterology, 

Mayo Clinic

2005 REGAL (Research Excellence in GI and Liver)–for third year fellows or junior faculty 

members who are less than five years out of their fellowship or residency in GI who 

demonstrate the ability to conduct important research in the areas of upper GI, lower GI, 

outcomes, or hepatobiliary research

2005 Berry Family Foundation Scholar Award–to support Mayo GI staff who pursue 

educational opportunity at other institutions and then return to Mayo to set up their own 

research

C. Contribution to Science

1. The SLAM family regulates colitis through both adaptive and innate immune mechanisms.

Upon joining the Terhorst laboratory in 2001, the SLAM family of immune receptors was known to be 

relevant to lymphoproliferative syndromes however precise mechanisms of immunoregulation were 

unknown. As part of this collaborative team, we defined the role for SLAM, CD48, and Ly108 in T cell and 

innate immune cellular function in colitis. As SLAM is a measles virus receptor, the major impact of our work 

on SLAM as a regulator of phagosome function is on the field of virology and retargeting of measles virus. 

a. Wang, N. Satoskar, A., Faubion, W.A., Howie, D., Okamoto, S., Feske, S., Gullo, C., Clark, K., Sosa, 

M.R., Sharpe, A.H., Terhorst, C. (2004). The cell surface receptor SLAM controls T cell and 

macrophage functions. The Journal of Experimental Medicine, 199(9):1255-64. PMCID:2211908.

b. Howie, D., Laroux, F.S., Morra, M., Satoskar, A.R., Rosas, L.E., Faubion, W.A., Julien, A., Rietdijk, 

S., Coyle, A.J., Fraser, C., Terhorst, C. (2005). Cutting edge: the SLAM family receptor Ly108 

controls T cell and neutrophil functions. Journal of Immunology, 174(10):5931-5.PMID:15879084.

c. Abadia-Molina, A.C., Ji, H., Faubion, W.A., Julien, A., Latchman, Y., Yagita, H., Sharpe, A., Bhan, 

A.K., Terhorst, C. (2006). CD48 controls T-cell and antigen-presenting cell functions in experimental 

colitis. Gastroenterology, 130(2):424-34. PMID:16472597.

d. Berger, S.B., Romero, X., Ma, C., Wang, G., Faubion, W.A., Liao, G., Compeer, E., Keszei, M., 

Rameh, L., Wang, N., Boes, M., Regueiro, J.R., Reinecker, H.C., Terhorst, C. (2010). SLAM is a 

microbial sensor that regulates bacterial phagosome functions in macrophages. NatureImmunology, 

11(10):920-7. PMCID: PMC3338319.

2. Traditional innate immune receptors regulate function of human FOXP3+ Treg cells.
Contact PD/PI: Faubion, William A

Biosketches Page 15

The FOXP3+ Treg cell was defined during the time frame of my post-doctoral training with the Terhorst 

laboratory, and we were the first to characterize the effect of colitis on Treg thymic developmental steps. 

Upon my return to Mayo clinic, we continued on with Treg related research with a particular focus on human 

physiology. With unique experience in innate immune receptors and Treg biology, we were the first to 

demonstrate a role for both cell membrane (TLR10) and intracellular (NOD2) pathogen recognition 

receptors on human Treg cellular function. In particular, the work on TLR10 and FOXP3 led to our 

subsequent deeper line of investigation on FOXP3 gene transcriptional events.

a. Faubion, W.A., De Jong, Y.P., Molina, A.A., Ji, H., Clarke, K., Wang, B., Mizoguchi, E., Siimpson, 

S.J., Bhan, A.K., Terhorst, C. (2004). Colitis is associated with thymic destruction attenuating 

CD4+25+ regulatory T cells in the periphery. Gastroenterology, 126(7):1759-70.

b. Abadia-Molina, A.C., Mizoguchi, A., Faubion, W.A., De Jong, Y.P., Rietdjik, S.T., Comiskey, M., 

Clarke, K., Bhan, A.K., Terhorst, C. (2005). In vivo generation of oligoclonal colitic CD4+ T-cell lines 

expressing a distinct T-cell receptor Vbeta. Gastroenterology, 128(5):1268-77.

c. Bell, M.P., Svingen, P.A., Rahman, M.K., Xiong, Y., Faubion, W. A. Jr. (2007) FOXP3regulates 

TLR10 expression in human T regulatory cells. Journal of Immunology, 173(3):1893-900. 

PMID:17641056.

d. Rahman, M.K., Midtling, E.H., Svingen, P.A., Xiong, Y., Bell, M.P., Tung, J., Smyrk, T., Egan, L.J., 

Faubion Jr, W.A. (2010). The pathogen recognition receptor NOD2 regulates human FOXP3+ T cell 

survival. Journal of Immunology, 184(12):7247-56. PMCID: 38886856.

3. KLF family members regulate FOXP3 and Treg function through coupling to epigenetic complexes. 

Careful characterization of the FOXP3 core promoter led to the earliest recognition of epigenetic 

mechanisms leading to FOXP3 gene activation and Treg development. Our work on the coupling of KLF 

transcription factors to chromatin modifying complexes has advanced the understanding of Treg biology,

CD8+ T cell function, and intestinal stem cell function. The greatest impact has been the first recognition of 

the role for the histone methyltransferase EZH2 in Treg biology.

a. Xiong, Y., Khanna, S., Grzenda, A.L., Sarmento, O.F., Svingen, P.A., Lomberk, G.A., Urrutia, R.A., 

Faubion Jr, W.A. (2012). Polycomb antagonizes p300/CREB-binding protein-associated factor to 

silence FOXP3 in a Kruppel-like factor-dependent manner. Journal of Biological Chemistry, 

287(41):34372-85. PMCID: PMC3464543.

b. Xiong, Y., Svingen, P.A., Sarmento, O.F., Smyrk, T.C., Dave, M., Khanna, S., Lomberk, G.A., 

Urrutia, R.A., Faubion Jr, W.A. (2014). Differential coupling of KLF10 to Sin3-HDAC and PCAF 

regulates the inducibility of the FOXP3 gene. American Journal of Physiology: Regulatory, 

Integrative and Comparative Physiology, 307(6)R608-20. PMCID: PMC4166759.

c. Sarmento, O.F., Svingen, P.A., Xiong, Y., Xavier, R.J., McGovern, D., Smyrk, T.C., Papadakis, 

K.A., Urrutia, R.A., Faubion, W.A. (2015). A novel role for KLF14 in T regulatory cell differentiation. 

Cellular and Molecular Gastroenterology and Hepatology, 1(2):188-202. PMCID: PMC4349492.

d. Papadakis, K.A., Krempski, J., Reiter, J., Svingen, P., Xiong, Y., Sarmento, O.F., Huseby, A., 

Johnson, A.J., Lomberk, G.A., Urrutia, R.A., Faubion, W.A. (2015). Kruppel-like factor KLF10 

regulates transforming growth factor receptor II expression and TGF-beta signaling in CD8+ T 

lymphocytes. The American Journal of Physiology- Cell Physiology, 308(5):C362-71. PMCID: 

PMC4346734.

e. Dave, M., Hayashi, Y., Gajdos, G.B., Smyrk, T.C., Svingen, P.A., Kvasha, S.M., Lorincz, A., Dong, 

H., Faubion, W.A., Jr., Ordog, T. (2015). Stem cells for murine interstitial cells of cajal suppress 

cellular immunity and colitis via prostaglandin e2 secretion, 148(5):978-90.PMCID:PMC4409492.

A full list of research-related published work can be found at: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/william.faubion.1/bibliography/40757558/public/?sort=date&direction=

ascending
Contact PD/PI: Faubion, William A

Biosketches Page 16

D. Research Support

Ongoing Research Support

R01AI089714-02 Faubion (PI) 2/15/11 – 1/31/16

The goal of this study is to investigate transcriptional regulation of regulatory T cell (Treg) developed 

by Kruppel-like factor 10 (KLF10) and its role in immune homeostasis.
Contact PD/PI: Faubion, William A

Biosketches Page 17

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Chandrashekhar Pasare

POSITION TITLE: Associate Professor of Immunology

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date 

MM/YYYY

FIELD OF STUDY

Bidar Veterinary College, Bidar, India

Indian Veterinary Research Institute, Bareilly, India 

National Institute of Immunology, New Delhi, India 

Yale University School of Medicine, New Haven, CT

B.V.Sc

M.V.Sc

Ph.D.

Postdoctoral

06/1992

05/1994

09/2000

05/2006

Veterinary Medicine

Immunology and 

Microbiology 

Immunology and 

Molecular Biology 

Innate control of 

Adaptive Immunity

A. Personal Statement 

I have been working in the field of innate immunity and innate control of adaptive immunity for about 14 

years. In addition to a Ph.D. in Immunology, I have had post-doctoral training at Yale University, where I 

worked on understanding the importance of the TLR signaling pathway in activation of adaptive immune 

responses. This work led to understanding of TLR mediated regulation of T and B cell responses leading to 

publications in Science (1928 citations), Immunity (325 citations) and Nature (537 citations). My laboratory 

continues to work in the area of innate control of adaptive immunity and our more recent work has also 

focused on understanding molecular details of TLR signaling and molecular mechanisms by which the 

innate immune system regulates the adaptive immune responses. My lab has extensive experience in 

studying TLR mediated activation of adaptive immunity and our recent work has led to new understanding 

of regulation of immune responses in the gut (ref # 2a), discovery of a new TLR signaling adapter that links 

TLRs to PI3K activation (ref # 3a) and discovery of a rapid NLRP3 inflammasome activation pathway 

controlled by IRAK-1 (ref # 4a). We have also recently demonstrated that plasma membrane and surface 

TLRs have different functions in regulation of CD8 T cell responses against pathogens (ref # 1c).

B. Positions and Honors

Positions and Employment

2000-2005 Post-doctoral Associate, Howard Hughes Medical Institute, Yale University School 

of Medicine, New Haven, CT

2005-2006 Associate Research Scientist, Section of Immunobiology, Yale University School of 

Medicine, New Haven, CT

2006-2007 Scientist, Immunology, Genentech, Inc. South San Francisco, CA

2007-2014 Assistant Professor, Department of Immunology, The University of Texas Southwestern 

Medical Center, School of Medicine, Dallas, TX

2014-present Associate Professor, Department of Immunology, The University of Texas Southwestern 

Medical Center, School of Medicine, Dallas, TX

eRA COMMONS USER NAME (credential, e.g., agency login):
Contact PD/PI: Faubion, William A

Biosketches Page 18

Professional Memberships

1999 Life member, Indian Immunology Society

2010 - Member, American Association of Immunologists

Honors/Awards

1992-1994 Indian Council of Agricultural Research’s Junior Research Fellowship

1994-1999 Senior Research Fellowship from Department of Biotechnology, India

2000-2005 Howard Hughes Medical Institute, Post-Doctoral Fellowship

2007 UT Southwestern Medical Center Endowed Scholar in Biomedical Research

2013 AAI early career faculty travel award

C. Contribution to Science

1. When I started my post-doctoral work in the laboratory of Ruslan Medzhitov about 14 years ago, TLRs 

were only just discovered and not much was known about the various mechanisms by which TLRs 

influence adaptive immunity. In addition there was renewed interest in a population of cells called 

regulatory T cells or suppressor T cells. These cells, characterized by surface expression of CD4 and 

CD25 markers, were shown to be potent suppressors of naïve T cell activation. The general consensus 

was that these cells block activation of auto-reactive cells. A key question for us was: How do pathogen 

specific T cells over come the block induced by regulatory T cells to mount protective immune responses? 

We hypothesized that, since TLRs are major sensors of infections, TLR activation on DCs should be able 

to overcome suppression mediated by regulatory T cells (Treg cells). I used TLR-deficient and TLRsufficient dendritic cells for in vitro suppression assays to show that TLR activation on DCs overcomes the 

block induced by regulatory T cells. In addition I showed that surface maturation of DCs is not sufficient to 

overcome Treg cell mediated suppression. Finally I demonstrated that the cytokine IL-6 secreted by DCs 

in response to TLR ligands is responsible for providing critical signals necessary for naïve T cells to 

overcome Treg cell mediates suppression. In summary, I discovered that the Toll-pathway of DC activation 

controls adaptive immune responses by at least two major mechanisms; 1. By induction of surface 

maturation of DCs and 2: By inducing secretion of IL-6, which overcomes Treg cell mediated suppression. 

This study was described as ground breaking in a commentary that accompanied the published article in 

the journal “Science”. After gaining insights into the critical requirements for TLR activation on DCs in 

inducing activation of naïve T cells in vitro, I set out to understand the in vivo significance of these findings 

and to understand the dynamic interactions between innate and adaptive immune systems in a living 

animal. Immunologists over the years have been using protein antigens to study induction of T and B cell 

responses in vivo. I showed using pure protein preparations that, naïve T cells could not be activated in 

vivo if immunized in the absence of a TLR ligand. Most commercial protein preparations are contaminated 

with TLR ligands and hence induce T and B cell activation when immunized in depot forming adjuvant such 

as alum. I used MyD88-deficient mice to shown the importance of different aspects of DC activation for T 

cell priming in vivo. I showed that DCs mature and migrate to the draining lymph node in response to LPS 

in MyD88-deficient mice but fail to induce naïve T cell activation. This was very contrary to the current 

understanding that expression of MHC and co-stimulatory molecules is sufficient to prime antigen specific 

T cells. I showed that this is not the case and that Treg cells block activation of naïve T cells if DCs do not 

secrete cytokines during priming. I was thus able to define the critical requirements for naïve T cell 

activation. Further, I discovered that TLR and MyD88 dependent signals are necessary for induction of 

memory CD4 T cells. This is an important finding and there is ongoing research in my current lab to 

understand the cellular and molecular mechanisms of how TLR activation induces development of CD4 

memory T cells. In a related study dissecting the role of TLRs in regulating B cell responses I discovered 

that the poor antibody responses in MyD88 deficient mice were because of lack of TLR signaling in B cells 

rather than lack of T cell priming and differentiation. This study for the first time established the role of 

innate immune sensing in mounting T-dependent B cell responses and implicated B cell intrinsic TLR 

signaling in B cell activation and differentiation. I was a post-doctoral fellow for all these studies.
Contact PD/PI: Faubion, William A

Biosketches Page 19

a. Pasare, C., and Medzhitov, R. (2003). Toll pathway-dependent blockade of CD4+CD25+ T cellmediated suppression by dendritic cells. Science 299, 1033-1036. PMID: 12532024.

b. Pasare, C., and Medzhitov, R. (2004). Toll-dependent control mechanisms of CD4 T cell 

activation. Immunity 21, 733-741. PMID: 15539158.

c. Pasare, C., and Medzhitov, R. (2005). Control of B-cell responses by Toll-like receptors. Nature

438, 364-368. PMID: 16292312.

2. As an independent investigator I continued to focus on understanding the role of innate immune system in 

regulating adaptive immunity. In the first study published from the laboratory we found that the cytokine 

requirements for induction of the inflammatory Th17 lineage cells depend on the site of priming. We found 

that the mucosal and systemic immune systems have different rules for inducing differentiation of naïve T 

cells into Th17 cells. We discovered that IL-1R mediated, MyD88 dependent signaling in CD4 T cells is 

critical for generation of Th17 lineage cells in all lymphoid tissues. However, while there is a requirement 

for IL-6 for Th17 priming in the mucosal tissues such as the lamina propria of the intestines as well as the 

lungs, IL-6 is not required for generation of Th17 lineage cells in the peripheral lymphoid organs such as 

the spleen and lymph nodes, The differential requirement for Th17 priming in the peripheral lymphoid 

organs versus the lamina propria of the intestines is true both during steady state as well as during 

exposure to pathogens via oral or systemic route. We also demonstrate that lack of IL-6 leads to higher 

proportion of Foxp3 positive CD4 T cells selectively only in the lamina propria of the intestines. Finally we 

demonstrate that gut specific need for IL-6 is dictated by DCs resident in the lamina propria of the 

intestines as LP DCs from IL-6 deficient mice fail to prime Th17 lineage cells. DCs resident in the spleens 

of IL-6 deficient mice induce normal priming of Th17 lineage cells. This is the first demonstration of 

selective need of different cytokines based on priming micro-environments for any kind of helper T cell 

lineage. Our study challenges the concept that IL-6 is a master regulator of RORgt and Th17 differentiation

and provides novel insights into tissue specific requirements for T cell differentiation. Differential 

requirement for Th17 priming in spleen and gut lamina propria is a novel concept and has important 

implications for targeting systemic and mucosal tissue specific auto-immunity as well as for determining 

routes of vaccination. In a related study that deals with regulation of CD8 T cell responses by the innate 

immune system, we found that plasma membrane TLRs and endosomal TLRs have a differential role in 

inducing CD8 T cell priming and this is primarily dictated by population of DCs recruited to the site of 

priming

a. Hu, W., Troutman, T.D., Edukulla, R., and Pasare, C. (2011). Priming microenvironments 

dictate cytokine requirements for T helper 17 cell lineage commitment. Immunity 35, 1010- 

1022. PMID: 22137454. PMCID: PMC3246047.

b. Hu, W and Pasare, C (2013). Location, location, location: tissue specific regulation of immune 

responses. Journal of Leukocyte Biology. 94, 409-421. PMID:23825388. PMCID: 

PMC3747123.

c. Mandraju R, Murray S, Forman J and Pasare C. (2014). Differential ability of surface and 

endosomal TLRs to induce CD8 T cell responses in vivo. J Immunol. 192, 4303-15. PMID: 

24688022. PMCID: PMC4002505.

3. TIR domains are required for the initiation of TLR signaling and serve to link TLRs to their adaptors through 

homotypic interactions. The known adapters of TLR signaling MyD88 (and TIRAP) and TRIF (and TRAM) 

signal to activate NF-kB, MAP kinases and IRF proteins but it has never been clear how TLR signaling 

leads to PI3 kinase activation. Although NF-kB activation is important for inducing pro-inflammatory 

response and cytokine secretion by macrophages, PI3K activation in cells plays a critical role in regulating 

cell survival, cell cycling and proliferation. We have found that BCAP, via its TIR domain, links TLRs to 

PI3K activation, and is also responsible for negatively regulating TLR signaling. Our studies demonstrate 

that BCAP modulates pro-inflammatory responses and BCAP deficient mice have exaggerated 

inflammation in response to infections and are highly susceptible to colitis. The study of TLR mediated 

activation of NF-kB has yielded many important and seminal discoveries into the regulation of this 

inflammatory pathway. However, signaling via TLRs to PI3K is poorly characterized. Data using chemical 

inhibitors suggests contradictory roles for PI3K. Due to the central and important role of PI3K through many
Contact PD/PI: Faubion, William A

Biosketches Page 20

converging signaling pathways, genetic tools utilizing mice deficient for PI3K components carry the caveat 

of disrupted signaling through other signaling pathways. Our delineation of BCAP’s critical role in activation 

of the PI3K axis is important not only through our description of how BCAP regulates inflammation, but also 

because of the characterization of a signaling adapter critically required for the proximal bridging of TLRs to 

PI3K.

a. Troutman, T.D., Hu, W., Fulenchek, S., Yamazaki, T., Kurosaki, T., Bazan, J.F., and Pasare, C. 

(2012). Role for B-cell adapter for PI3K (BCAP) as a signaling adapter linking Toll-like receptors 

(TLRs) to serine/threonine kinases PI3K/Akt. Proc Natl Acad Sci USA 109, 273-278. PMID: 

22187460. PMCID: PMC3252926.

b. Troutman, T.D., Bazan, J.F., and Pasare, C. (2012). Toll-like receptors, signaling adapters and 

regulation of the pro-inflammatory response by PI3K. Cell Cycle 11. PMID: 22895011. PMCID: 

PMC3478307.

4. In the past few years, my laboratory also got interested in understanding the biogenesis of IL-1beta. The 

cytokines IL-1beta and IL-18 are made as pro-forms and are induced by TLR signaling. When a cell senses a 

toxin or virulence factor or is undergoing stress, this leads to activation of inflammasome causing caspase-1 

activation, which then cleaves IL-1beta and IL-18 for their release. The scientific understanding was that there 

is a priming signal (TLR activation) and an activation signal (NLR activation) separated by time, necessary for 

inflammasome activation. We hypothesized that when there is an infection by a virulent pathogen, the cells are 

likely to see both of these signals at the same time and that macrophages should be equipped to induce 

inflammasome activation independent of priming. Consistent with this hypothesis, we discovered a rapid 

NLRP3 inflammasome activation pathway that leads to caspase-1 activation in as little as 20 minutes following 

simultaneous sensing of TLR and NLRP3 ligands. More importantly we found that this activation is directly 

regulated by TLR signaling component IRAK-1 and that not only does IRAK-1 form part of the inflammasome 

complex, but its kinase activity is important for rapid NLRP3 inflammasome assembly. We also discovered that 

this rapid IRAK-1 dependent inflammasome activation pathway is critical to sense Listeria monocytogenes and 

induce rapid IL-18 dependent IFN-gamma production by NK cells and memory CD8 T cells. We believe that 

this rapid inflammasome activation pathway is critical for rapid innate defense and are continuing to work on 

understanding the molecular mechanism of this activation and its role in protection against a variety of virulent 

pathogens.

a. Lin KM, Hu W, Troutman, TD, Jennings M, Brewer, T, Li, X, Nanda, S, Cohen P, Thomas J and 

Pasare C. (2014) IRAK-1 by passes priming and directly links TLRs to rapid NLRP3 

inflammasome activation. Proc. Natl. Acad. Sci. USA. 111, 775-780. PMID: 24379360. PMCID: 

PMC3896167.

Complete List of Published Work in MyBibliography:

http://www.ncbi.nlm.nih.gov/sites/myncbi/1pmWT2zpU9bQM/bibliograpahy/46106384/public/?sort=date

&direction=ascending

D. Research Support

Ongoing Research Support

2015/04/15-2017/03/31

R21 AI115420, National Institute of Allergy and Infectious Diseases (NIAID, NIH) 

PASARE, CHANDRASHEKHAR (PI)

Role of Microenvironmental cues in CD8 T cell activation and memory generation

The major goals of this project are to investigate the mechanisms by which priming microenvironments 

influence activation and development of CD8 memory T cells.
Contact PD/PI: Faubion, William A

Biosketches Page 21

2010/08/15-2015/07/31

R01 AI082265, National Institute of Allergy and Infectious Diseases (NIAID, NIH) 

PASARE, CHANDRASHEKHAR (PI)

Toll-like receptor mediated regulation of effector and memory CD4 T cell responses

The major goals of this project are to investigate the mechanisms by which TLRs influence activation and 

differentiation of CD4 T cells into effector and memory cells.

Competed Research Support (ended in the last 3 years)

2010/02/15-2013/01/31

R43 AI089138, National Institute of Allergy and Infectious Diseases (NIAID, NIH) 

PASARE, CHANDRASHEKHAR (PI)

Synergistic vaccine adjuvants that stimulate both innate and adaptive immunity

The major goals of this project were to develop a glycoside adjuvant that can activate innate immunity and 

investigate its ability to induce protective adaptive immune responses to various pathogens.

Role: Principal Investigator
Contact PD/PI: Faubion, William A

Biosketches Page 22

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

NAME: Stephen C. Ekker

POSITION TITLE: Professor of Biochemistry and Molecular Biology 

EDUCATION/TRAINING

A. Personal Statement

My background in molecular genetics and genomics started with undergraduate research with a project to 

restriction map the genome of the Archaebacterium Sulfolobus solfataricus. I have been helping further the 

zebrafish (Danio rerio) as a model vertebrate to address major issues in human health and biology since 

training as a post-doctoral fellow. This system has the potential for diverse genetic and behavioral studies 

typically restricted to the world of the fly or worm but conducted within the biological framework of a vertebrate.

The major focus of my laboratory is on understanding our genome, using the zebrafish as a rapid 

molecular test system. We established the rapid use of morpholino sequence-specific knockdown technology 

for vertebrate functional genomics applications using the zebrafish as the pioneering model system fifteen 

years ago. In parallel, we developed vertebrate transposon tools including our protein trap gene-breaking 

vectors to generate a 700+ line collection of molecularly characterized and revertible mutant zebrafish lines, 

the first engineered conditional alleles in any organism outside the mouse. We have deployed transposons in 

diverse application areas including human T cells, zebrafish, and mice. Custom restriction endonucleases offer 

a third leg, targeted modification using genome editing tools. We continue to develop science behind these 

new engineering toolkits, working with an array of laboratories as diverse as rat, pig, mouse, nematode, and fly 

biologists in addition to regular colleagues that work in human cells and zebrafish.

As Director of the Genetics and Model Systems Core for the Center for Cell Signaling in Gastroenterology, 

I am delighted to support your project.

B. Positions and Honors

1986-1988 Undergraduate Research: Restriction Mapping the Genome of the Archaebacterium Sulfolobus 

solfataricus. University of Illinois; C.R. Woese, advisor

1988-1993 Graduate Dissertation Differential DNA Binding and the Specificity of Homeotic Gene Action, 

Philip A. Beachy, Advisor, Johns Hopkins School of Medicine (SOM) and HHMI, Baltimore, MD

1990-1992 Teaching Assistant, Dept. of Mol. Biol. and Genetics, Johns Hopkins University, Baltimore,MD 

1990-1993 March of Dimes Birth Defects Predoctoral Fellow

1993-1995 Postdoc, Biochemical Properties and Biological Activities of the Hedgehog Gene Family, P.A. 

Beachy, Advisor, R.T. Moon, Co-Advisor, Johns Hopkins SOM and HHMI, Baltimore, MD

1995-2007 Assistant, Associate, and full Professor, University of Minnesota, Minneapolis, MN 

1997-1999 March of Dimes Basil O’Connor Scholar

2000 Co-founder, Discovery Genomics, Inc.

1999-2007 Director, Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota 

2002-2007 Associate Head, Genetics, Cell Biology and Development, University of Minnesota

2007-present Adjunct Professor, Genetics, Cell Biology and Development, University of Minnesota 

2007-present Professor Dept Biochemistry and Molecular Biology and Consultant, Mayo Clinic,

Rochester,MN

eRA COMMONS USER NAME (credential, e.g., agency login):

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date 

MM/YYYY

FIELD OF STUDY

University of Illinois Urbana-Champaign, Urbana B.S. 05/1988 Electrical Engineering

University of Illinois Urbana-Champaign, Urbana B.S. 05/1988 Genetics & Dev. Biology

Johns Hopkins University, Baltimore, MD Ph.D. 05/1993 Molec. Bio. & Genetics

Johns Hopkins University, Baltimore, MD PostDoctoral 10/1995 Develop. Genetics
Contact PD/PI: Faubion, William A

Biosketches Page 23

2007-present Director, Mayo Clinic Zebrafish Facility

2007-present Mayo Predoctoral Education Programs Committees (CTS: 2007-present; BMB 2007-12) 

2008-present Editor-in-Chief, The Zebrafish journal

2009-present Director, Genetics and Model Systems Core, Mayo Center for Cell Signaling in 

Gastroenterology

2010-present Director, Mayo Addiction Research Center 

2012-present Founder, InSciEd Out Foundation

2014-present Chairman of the Board, InSciEd Out Foundation 

2013-present Member, Faculty of 1000

2013 Co-Organizer and Founder, 1st International Zebrafish for Personalized/PrecisionMedicine 

Conference, Toronto, Oct. 16-18, 2013

2013-present Founding member, Zebrafish Disease Models Society

2014-present Director, Model Systems Core, Mayo Translational PKD Center

Professional Memberships

AAAS (1992), Genetics Society of America (1992), SDB (1996), ASHG (2005), SFN (2010), IBANGS (2013)

Honors (selected) Member, National Honor Society (1983); James Scholar and Dean's List, University of 

Illinois (1983-88); Graduation with Honors, University of Illinois (1988); Member, Phi Kappa Phi and Eta Kappa 

Nu Honor Societies (1988); Predoc fellow, March of Dimes (1990-93); Basil O'Connor Scholar, March of Dimes 

(1997-99) Major Study Panels: NSF Dev Mech (2002-2004); NCRR 10/99; NHLBI SEP 06/00; NIGMS PPG

01/02, CDF4 06/02; Craniofacial Special Emphasis Panel 11/02; NIDDK SEP 11/02; Genome 02/03, 12/03; 

Innov. Tox Models SEP 07/03; NDPR 10/03, 06/04; Tools for Genetic Studies in Zebrafish 04/04, 03/09; 

Centers for Biomedical Computing SEP, 05/05; NCI Cancer Nanotech Platform SEP (section chair) 07/05; NIH 

MDCN SEP 07/05; NIH CAD&O 10/05, 06/06, 10/06; NHLBI 03/06; SBIR 04/06; Neurogenesis and Cell Fate 

Full member, 06/05 – 06/09; RC4 05/10; NHLBI 11/10; Member of the NIH College of CSR Reviewers 

01/2010-present; NIDA-K Full member 06/10-12 (end of panel); NIDA ZDA1 EXL-T Full member 06/10- 

present; NIH/NCATS Comparative Medicine (CM) Special Emphasis Panel (SEP) Feb 13-14, 2013; NIDA 

CEBRA Oct 2013, Mar 2014; NIH PPG review April 2014

Pertinent Science Education: served as Editor-in-Chief for two Special Issues of the Zebrafish on the topic 

‘Zebrafish in Education.’ I solicited funding to cover all publication charges (including NIH support), making all 

of these issues freely available to classroom teachers and students around the world. The first issue came out 

in 2009, the second in December of 2012.

Patents

Ekker,S.C. et al. Inhibition of Gene Expression using Polynucleotide Analogues. US Patent 6,867,349. 

Ekker,S.C. et al., Syndecans and Angiogenesis – US Patent 7,713,925.

C. Contributions to Science [125 publications; google h index 48]

1) Biochemical and biological activities of hedgehog signaling in Drosophila and vertebrates. During my 

post-doctoral training, I contributed to uncovering mechanistic understanding how this key pathway 

works in a variety of critical processes and in the cloning of vertebrate family members including the 

first work using zebrafish implicating shh signaling as defective in holoprosencephaly. 

a) Lee, J. J., Ekker, S. C., von Kessler, D. P., Porter, J. A., Sun, B. I. and Beachy, P. A. (1994). 

Autoproteolysis in hedgehog protein biogenesis. Science 266, 1528-1537.

b) Ekker, S .C., Ungar,A., Greenstein,P., von Kessler,D.P., Porter,J.A., Moon,R.T., and Beachy,P.A. 

(1995). Patterning Activities of Vertebrate hedgehog Proteins in the Developing Eye and Brain. 

Current Biology, 5: 944-955.

c) Ekker, S. C., McGrew, L. L., Lai, C. J., Lee, J. J., von Kessler, D. P., Moon, R. T. and Beachy, P. A. 

(1995). Distinct expression and shared activities of members of the hedgehog gene family of 

Xenopus laevis. Development 121, 2337-2347.

d) Porter, J. A., Ekker, S. C., Park, W. J., von Kessler, D. P., Young, K. E., Chen, C. H., Ma, Y., 

Woods, A. S., Cotter, R. J., Koonin, E. V., et al. (1996). Hedgehog patterning activity: role of a 

lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21-34.
Contact PD/PI: Faubion, William A

Biosketches Page 24

2) Development of morpholino antisense technology for vertebrate genomics applications using the 

zebrafish.

a) Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene 'knockdown' in zebrafish. Nature 

genetics 26, 216-220.

b) Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S. A. and Ekker, S. C.

(2007). p53 activation by knockdown technologies. PLoS genetics 3, e78.

c) Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A. and Ekker, S. C. (2009). A primer for 

morpholino use in zebrafish. Zebrafish 6, 69-77. PMCID: PMC2776066

d) Patent awarded: Ekker,S.C. et al. Inhibition of Gene Expression using PolynucleotideAnalogues. 

US Patent 6,867,349.

3) Development of transposons for vertebrate genomics applications in zebrafish. We deployed the first 

transposon system, Sleeping Beauty, for transgenesis and enhancer trapping, and then harnessed the 

endogenous Tol2 transposon for conditional mutagenesis and protein trapping applications.

a) Davidson, A. E., Balciunas, D., Mohn, D., Shaffer, J., Hermanson, S., Sivasubbu, S., Cliff, M. P., 

Hackett, P. B. and Ekker, S. C. (2003). Efficient gene delivery and gene expression in zebrafish 

using the Sleeping Beauty transposon. Developmental biology 263, 191-202.

b) Balciunas, D., Davidson, A. E., Sivasubbu, S., Hermanson, S. B., Welle, Z. and Ekker, S. C. 

(2004). Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC genomics 5, 

62.

c) Balciunas, D., Wangensteen, K. J., Wilber, A., Bell, J., Geurts, A., Sivasubbu, S., Wang, X., 

Hackett, P. B., Largaespada, D. A., McIvor, R. S., et al. Ekker, S. C. (2006). Harnessing a high 

cargo-capacity transposon for genetic applications in vertebrates. PLoS genetics 2, e169.

d) Clark, K. J., Balciunas, D., Pogoda, H. M., Ding, Y., Westcot, S. E., Bedell, V. M., Greenwood, T. 

M., Urban, M. D., Skuster, K. J., Petzold, A. M., et al. Ekker, S. C. (2011). In vivo protein trapping 

produces a functional expression codex of the vertebrate proteome. Nature methods 8, 506-515. 

PMCID: PMC3306164

4) In vivo genome editing. We developed new tools and approaches for genome editing including the first 

targeted knockins in zebrafish. This approach also works well in in vitro systems. 

a) Bedell, V. M., Wang, Y., Campbell, J. M., Poshusta, T. L., Starker, C. G., Krug, R. G., 2nd, Tan, W., 

Penheiter, S. G., Ma, A. C., Leung, A. Y., et al. Ekker, S. C. (2012). In vivo genome editing using a 

high-efficiency TALEN system. Nature 491, 114-118. PMCID: PMC3491146

b) Neff, K. L., Argue, D. P., Ma, A. C., Lee, H. B., Clark, K. J. and Ekker, S. C. (2013). Mojo Hand, a 

TALEN design tool for genome editing applications. BMC bioinformatics 14, 1. PMCID:

PMC3575288

c) Ma, A. C., Lee, H. B., Clark, K. J. and Ekker, S. C. (2013). High efficiency In vivo genome 

engineering with a simplified 15-RVD GoldyTALEN design. PloS ONE 8, e65259. PMCID:

PMC3667041

d) Peng, Y., Clark, K. J., Campbell, J. M., Panetta, M. R., Guo, Y. and Ekker, S. C. (2014). Making 

designer mutants in model organisms. Development 141, 4042-4054. PMCID: PMC4302887

5) Establishment of Integrated Science Education Outreach (InSciEd Out), a new approach for excellence 

in science education for the betterment of human health for all (http://insciedout.org/). We are 

operational on three continents (North America, Africa and India). Our highly collaborative program 

focuses on core cultural change, implementing science education within the context and needs of each 

partner community.

a) Pierret,C., Sonju,J., Leicester,J. Hoody,M., LaBounty,T., Frimannsdottir,K.,R., Ekker,S.C. (2012) 

Improvement in Student Science Proficiency Through InSciEd Out, Zebrafish, 9, 155-68. PMCID:

PMC3529492

b) Clark, K. J. & Ekker, S. C. (2015)  How Zebrafish Genetics Informs Human Biology. Nature 

Education 8(4):3.

URL for bibliography: 

http://www.ncbi.nlm.nih.gov/pubmed?Db=pubmed&Cmd=DetailsSearch&Term=ekker+sc%5BAll+Fields%5D&

WebEnv=0SoUqK-GD_T4YuzbaQadqM8hNqScpHAFyiFvTbm7gSa2SDumUj4r3YHA2epX7EHqWGGoT1pliY_npV%40255F54F3705678F0_0091SID&WebEnvRq=1
Contact PD/PI: Faubion, William A

Biosketches Page 25

D. Research Support

Ongoing Research Support

Genome Biology

5R01/56-GM63904-11 (Ekker, PI; Farber, Hammerschmidt, Xu and Clark as Co-I) 09/01/01 – 08/31/16 

"Systematic Vertebrate Functional Genomics"

This grant funds a transposon-based phenotypic screen in the zebrafish to assess the role of 500 genes in 

development, skin, heart and digestive system biology.

1R01 HG006431-04 (Ekker, PI) 08/01/11 – 07/30/17

“International Zebrafish Mutagenic Protein Trap”

This resource grant funds the generation of 1000 new gene-break transposon lines for the zebrafish 

community. There is no support for biological annotation in this grant.

Supporting Grants

1P30 DK84567-06 (PI: LaRusso, N/Genetics Core PI: Ekker) 09/01/09 – 08/31/19 

“Mayo Center for Cell Signaling in Gastroenterology”

SCE is PI of the Genetics and Model Organism Core that is designed to bring consultation, training and stateof-the-art genetics tools and model organisms to local scientists.

P30 DK090728-5 (PI:Torres,V.E.; Ekker, Model Organism Core PI) 09/30/10 – 06/30/15 

“Mayo Translational PKD Center (MTPC)”

Dr. Ekker is Director of the Model Systems Core. [The competitive renewal received a score of 11 and will 

likely be continued.]

Education Grants

2UL1TR000135-9 (PI: Khosla, S) 07/01/11 – 06/30/16

“Mayo Clinic Center for Translational Science Activities”

Dr. Ekker is Associate Director for the Predoctoral training program in translational sciences, including the K-8 

InSciEd Out science education reform initiative for the healthy community program.

Completed Research Support

5R01 DA14546-10 (Ekker, PI) 05/01/01 – 06/30/14

“Intron-based Mutagenic Transposons for Zebrafish”

The grant funded the development of the gene-break transposon technology for zebrafish functional genomics 

including the use of GBTs for behavioral genetics of the nicotine response.
Contact PD/PI: Faubion, William A

Biosketches Page 26

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Thomas C. Smyrk, M. D.

POSITION TITLE: Professor of Pathology

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date 

MM/YYYY

FIELD OF STUDY

University of Minnesota, Minneapolis, MN BA 07/78 English

University of Minnesota, Minneapolis, MN MD 07/82

Mayo Clinic, Rochester, MN 07/86

Please refer to the Biographical Sketch sample in order to complete sections A, B, C, and D of the 

Biographical Sketch.

A. Personal Statement:

I have twenty-nine years of experience as a surgical pathologist with interest in gastrointestinal pathology. 

Colitis and colitis-associated dysplasia/carcinoma have been particular interests of mine. Dr. Faubion and I 

have successfully collaborated on several previous projects and I look forward to collaborating with him and his 

excellent colleagues in the near future.

1) Garrity-Park MM, Loftus EV, Bryant SC, Sandborn WJ, Smyrk TC. Tumor necrosis factor-alpha 

polymorphisms in ulcerative colitis-associated colorectal cancer. Am J Gastroenterol. 2008 Feb; 

103(2):407-15. PMID:18289203. DOI:10.1111/j.1572-0241.2007.01572.x.

2)) Kisiel JB, Garrity-Park MM, Taylor WR, Smyrk TC, Ahlquist DA. Methylated eyes absent 4 (EYA4) 

gene promotor in non-neoplastic mucosa of ulcerative colitis patients with colorectal cancer: evidence 

for a field effect. Inflamm Bowel Dis. 2013 Sep;19(10):2079-83. PMID:23867875. PMCID:4119944.

DOI:10.1097/MIB.0b013e31829b3f4d.

3) Sarmento OF, Svingen PA, Xiong Y, Xavier RJ, McGovern D, Smyrk TC, Papadakis KA, Urrutia RA, 

Faubion WA. A novel role for KLF14 in T regulatory cell differentiation. Cell Mol Gastroenterol Hepatol. 

2015 Mar 1; 1(2):188-202.e4. PMID:25750932. PMCID:4349492. DOI:10.1016/j.jcmgh.2014.12.007.

4) Dave M, Hayashi Y, Gajdos GB, Smyrk TC, Svingen PA, Kvasha SM, Lorincz A, Dong H, Faubion WA 

Jr, Ordog T. Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via 

prostaglandin e2 secretion. Gastroenterology. 2015 May;148(5):978-90. Epub 2015 Jan 28. 

PMID:25637652. PMCID:4409492.

B. Positions and Honors

Positions and Employment

eRA COMMONS USER NAME (credential, e.g., agency login):

1986-1990 Assistant Professor of Pathology, Creighton University, Omaha, NE

1990-1997 Associate Pathologist, Clarkson Hospital, Omaha, NE

1997-2000 Associate Professor of Pathology, University of Nebraska, Omaha, NE
Contact PD/PI: Faubion, William A

Biosketches Page 27

2000-2012 Associate Professor of Pathology, Mayo Clinic, Rochester, MN 

2013-present Professor of Pathology, Mayo Clinic, Rochester, MN

C. Contribution to Science

Lynch Syndrome: I collaborated with Henry Lynch on multiple descriptive studies covering clinical and 

pathologic features of Lynch syndrome, written before the genetics of that condition were known. One review 

has been cited more than 1000 times. (1) Jeremy Jass and I collaborated on a histology paper that got the 

pathology of Lynch Syndrome largely correct. (2) I attended the Bethesda conference in 1996 and 

collaborated on the publication of the Bethesda criteria. (3) I designed, performed and wrote the study 

characterizing tumor infiltrating lymphocytes as a robust predictor of microsatellite instability status. (4)

1) Lynch HT, Smyrk TC, Watson P et al. Genetics, natural history, tumor spectrum and pathology of 

hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology 1993;104:1535-1549. 

PMID: 8482467

2) Jass JR, Smyrk TC, Stewart SM, Lane MR, Lanspa SJ, Lynch HT. Pathology of hereditary nonpolyposis colorectal cancer. Anticancer Res 1994;14:1631-1634. PMID: 7979198

3) Rodriguez-Bigas MA, Boland CR, Hamilton SR et al. A National Cancer Institute workshop on 

hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines. J 

Natl Cancer Inst 1997;89:1758-1762. PMID: 9392616

4) Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor-infiltrating lymphocytes are a marker for microsatellite 

instability in colorectal carcinoma. Cancer 2001;91:2417-2422. PMID: 11413533

Eosinophilic esophagitis: We published the first description of eosinophilic esophagitis as a syndrome involving 

dysphagia. (1) My interest in that condition has continued over the years, resulting in contributions addressing 

whether diagnostic criteria are too strict (2), evaluating new diagnostic techniques (3) and elucidating the 

pathophysiology. (4)

1) Attwood SE, Smyrk TC, DeMeester TR, Jones JB. Esophageal eosinophilia with dysphagia. A distinct 

clinicopathologic syndrome. Dig Dis Sci 1993;38:109-116. PMID: 8420741

2) Ravi K, Talley NJ, Smyrk TC et al. Low grade esophageal eosinophilia in adults: an unrecognized part 

of the spectrum of eosinophilic esophagitis? Dis Dis Sci 2011;56:1981-1986. PMID: 21298480

3) Katzka DA, Geno DM, Ravi K et al. Accuracy, safety and tolerability of tissue collection by Cytosponge 

vs endoscopy for evaluation of eosinophilic esophagitis. Clin Gastroenterol Hepatol 2015;13:77-83. 

PMID: 24997328

4) Katzka DA, Ravi K, Geno DM et al. Endoscopic mucosal impedance measurements correlate with 

eosinophilia and dilation of intercellular spaces in patients with eosinophilic esophagitis. Clin 

Gastroenterol Hepatol 2015. PMID: 25592662

IgG4-related disease: Our 2003 paper was the first to clearly separate two forms of autoimmune pancreatitis, 

one IgG4-related and one not. (1) That separation is now widely accepted. We also had an early paper

described the role of tissue IgG4 in the diagnosis. (2) I participated in the first international consensus 

conference on IgG4-related disease and contributed to the publication of its findings. (3, 4)

1) Notohara K, Burgart LJ, Yadav D, Chari S, Smyrk TC. Idiopathic chronic pancreatitis with periductal 

lymphoplasmacytic infiltration: clinicopathologic features of 35 cases. Am J Surg Pathol 2003;8:1119- 

1127. PMID: 12883244

2) Zhang L, Notohara K, Levy MJ, Chari ST, Smyrk TC. IgG4-positive plasma cell infiltration in the 

diagnosis of autoimmune pancreatitis. Mod Pathol 2007;20:23-28. PMID: 16980948

3) Stone JH, Khosroshahi A, Despande V et al. Recommendations for the nomenclature of IgG4-related 

disease and its individual organ system manifestations. Arthritis Rheum 2012;64:3061-3067. PMID: 

22736240

4) Deshpande V, Zen Y, Chan JK et al. Consensus statement on the pathology of IgG4-related disease. 

Mod Pathol 2012;25:1181-1192. PMID: 22596100

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/thomas.smyrk.1/bibliography/47997356/public/?sort=date&direction=

ascending
Contact PD/PI: Faubion, William A

Biosketches Page 28

D. Research Support

Ongoing Research Support

8702.02-A1 P50CA 102701 Petersen (PD/PI) 9/18/14-8/31/19 

NCI

Mayo Clinic SPORE in Pancreatic Cancer – Core 2: Tissue Core 

Build and administrate the pancreatic tissue core.

Role: Co-Investigator

FP00051409 P30DK 84567 LaRusso (OP) 9/1/14-8/31/19 

NIDDK

Mayo Center for Cell Signaling in Gastroenterology

This center core grant correlates research requests with appropriate tissue sources. 

Role: Other Professional

FP00062460 CA 163059 Wang (CI) 9/21/11-8/31/16 

NIH

Validation Core C ? U MI Betmet Core

This study assesses new imaging techniques for dysplastic Barrett’s esophagus. 

Role: Co-Investigator

18248.02 U01DK 74008 Farrugia (CI) 9/1/12-8/31/16 

NIDDK

Gastroparesis Clinical Research Consortium – Data Coordinating 

This major goal is to study the pathology gastroparesis.

Role: Co-Investigator

FP00064454 

HHSN2612012000421 

NIH

Limburg (CI) 9/24/12-9/23/19

Cancer Prevention Agent Development Program: Early Phase Clinical Research 

The major goal is to use pathology to assess response to chemoprevention.

Role: Co-Investigator

FP00067546 U01AA 21788 Shah (CI) 9/20/12-6/30/17 

NIH

TREAT – Mayo

This major goal is to determine prognostic markers for alcoholic liver disease. 

Role: Co-Investigator

FP00062052; CA 163004 Wang (CI) 9/26/11-8/31/16 

NIH

Stem Cells and the Origins of Barrett’s Esophagus

This major goal is to identify esophageal stem cells and delineate their role in Barrett’s esophagus. 

Role: Co-Investigator

Completed Research Support

n/a
Contact PD/PI: Faubion, William A

Biosketches Page 29

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Urrutia, Raul

POSITION TITLE: Professor of Medicine, Biophysics, and Biochemistry & Molecular Biology

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date 

MM/YYYY

FIELD OF STUDY

University of Cordoba, Cordoba, Argentina Fellowship 05/1984 Biochemistry

University of Cordoba, Cordoba, Argentina M.D. 05/1987 Tumor Biology

A. Personal Statement: 

I received my basic science training at the University of Cordoba, Argentina where I performed my first studies 

on pancreatic cancer. Subsequently, I came to the United States to work in the area of cell motility and 

migration at the National Institute of Health (NIH), Bethesda, MD. Subsequently, I joined the faculty of the 

Mayo Clinic where I still continue my research in pancreatic cancer. Thus, my scientific dedication to cancer 

has remained a continuum for almost 30 years. Our laboratory has pioneered basic science studies in the area 

of transcription, chromatin, and epigenetics in Gastrointestinal cancer and other human diseases, including 

metabolic, neuropsychiatric, immunological, vascular, and auditory. Relevant to the current application, our 

work on KLF transcription factors extended into the characterization of many chromatin co-factors, which 

function as writer, reader, and erasers of the histone code. In particular, I have an active line of investigation 

on EZH2, because of which I have become collaborator of the current proposal. I have mentored more than 30 

graduate and postgraduate fellows who have gone on to have independent laboratories. Over the years at 

Mayo, I have served as Director of the GI Research Unit, Director of the Ph.D. Program in Tumor Biology, 

Associate Director for Genomics at the Mayo Clinic General Clinical Research Center (GCRC), and Director of 

the GI Cancer Research Program at the Mayo Cancer Center and member of the NIDDK GI Center, the Mayo 

Pancreatic Cancer Spore, and the Mayo Epigenetic Interest group. Currently, I am part of the Translational 

Epigenomic Program in the Center for Individualized Medicine, for which I serve as Director for Education and 

Academic Relationships. Our mission is to promulgate the study and application of epigenetics and 

epigenomics to medicine, mechanistically and translationally. Regarding interactions with the extramural GI 

and pancreatic community, I have been council of the American Pancreatic Association, serving also as its 

President in 2008. I have also been on the council of the International Association of Pancreatology (IAP) and 

American Gastroenterology Association (AGA). Furthermore, I have served as the Vice-Chair and later chair of 

the pancreatic section of American Gastroenterological Association. I have served as the Editor-in-Chief for 

three journals in the field, including Pancreatology, International Journal of Gastrointestinal Cancer, and Case 

Reports in Gastroenterology. In summary, I have an established record of successful and productive research 

projects regarding transcriptional regulation, epigenetics, and chromatin dynamics. My laboratory is located in 

the highly collaborative environment of the GI Research Unit at Mayo in adjacency to the PI of the current 

grant, with whom I have the pleasure to collaborate intensively. Thus, I will be helpful for planning and 

interpreting experiments included in the current grant.

1. Grzenda A, Lomberk G, Svingen P, Mathison A, Calvo E, Iovanna J, Xiong Y, Faubion W, Urrutia R. 

Functional Characterization of EZH2beta reveals the increased complexity of EZH2 isoforms involved 

in the regulation of mammalian gene expression. Epigenetics Chromatin. 2013; 6(1):3. PMCID: 

PMC3606351

eRA COMMONS USER NAME (credential, e.g., agency login):
Contact PD/PI: Faubion, William A

Biosketches Page 30

2. Xiong Y, Khanna S, Grzenda AL, Sarmento OF, Svingen PA, Lomberk GA, Urrutia RA, Faubion WA

Jr. Polycomb Antagonizes p300/CREB-binding Protein-associated Factor to Silence FOXP3 in a 

Kruppel-like Factor-dependent Manner. J Biol Chem. 2012 Oct 5;287(41):34372-85.

3. Mitic T, Caporali A, Floris I, Meloni M, Marchetti M, Urrutia R, Angelini GD, and Emanueli C. EZH2 

modulates angiogenesis in vitro and in a mouse model of limb ischemia. Mol Ther. 2015 Jan;23(1):32- 

42

4. Floris I, Descamps B, Vardeu A, Miticc T, Posadino AM, Shantikumar S, Sala-Newby G, Capobianco G, 

Mangialardy G, Howard L, Dessole S, Urrutia R, Pintus G, Emanueli C. Gestational diabetes mellitus 

impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone 

methyltransferase enhancer of zester homolog-2. Arterioscler Thromb Vasc Biol. 2015 Mar;35(3):664- 

74.

B. Positions and Honors

Positions and Employment

Honors and Awards

1987 Magna Cum Laude, Medical Doctor

1995 Cancer Award from Fraternal Order of Eagles

1987-1989 Visiting Fellow, Laboratory of Molecular Otology, NIDCD, National Institutes of Health,

Bethesda, MD

1989-1990 Visiting Fellow, Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, MD

1990-1991 Visiting Associate at the Laboratory of Cell Biology, NHLBI, National Institutes of Health,

Bethesda, MD

1991-1992 Visiting Associate, Laboratory of Cellular Biology, NIDCD National Institutes of Health,

Bethesda, MD

1992-1994 Research Associate, Center for Basic Research in Digestive Diseases, Mayo Clinic,

Rochester, MN

1994-2000 Assistant Professor of Medicine, College of Medicine, Mayo Clinic, Rochester, MN

1995- Assistant Professor, Tumor Biology, Mayo Graduate School, Mayo, Clinic, Rochester, MN

1996- Assistant Professor of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic,

Rochester, MN

1997-2004 Council, International Association of Pancreatology

1998-2002 Senior Associate Consultant, Mayo Clinic Cancer Center and GI Research Unit, Mayo Clinic,

Rochester, MN

1998-2007 Council, American Pancreatic Association

2000-2011 Chair, GI Research Unit, Mayo Clinic, Rochester, MN

2000-2004 Education Coordinator, Tumor Biology Program, Mayo Graduate School, Mayo Clinic,

Rochester, MN

2000- Professor of Medicine, College of Medicine, Mayo Clinic, Rochester, MN

2001-2002 Genomics Program Director, General Clinical Research Center, Mayo Clinic, Rochester, MN

2002- Consultant, Mayo Clinic Cancer Center and GI Research Unit, Mayo Clinic, Rochester, MN

2002-2005 Director, GI Cancer Group, Mayo Clinic Cancer Center, Mayo Clinic, Rochester, MN

2003-2004 Vice-chair, Pancreatic Diseases Section, American Gastroenterological Association

2005-2010 Editor-in-Chief, Pancreatology

2005- Professor of Biophysics, College of Medicine, Mayo Clinic, Rochester, MN

2004-2006 Chair, Pancreatic Diseases Section, American Gastroenterological Association

2006-2007 President, American Pancreatic Association

2006-2009 Editor-in-Chief, Journal of Gastrointestinal Cancer

2007- Editor-in-Chief, Case Reports in Gastroenterology

2008-2011 Director, ACS/IRG, Mayo Clinic Cancer Center, Mayo Clinic, Rochester, MN

2012- Director for Education and Academic Relationships, Translational Epigenomic Program, Center

for Individualized Medicine, Mayo Clinic, Rochester, MN
Contact PD/PI: Faubion, William A

Biosketches Page 31

1996-1997 Cancer Award from Fraternal Order of Eagles

1994-1999 NIH First Award, R-29, National Institutes of Health

1998-2002 NIDDK R01 Award

1999 Charlotte Geyer Foundation Award

2000 Charlotte Geyer Foundation Award

2000 NIH R01, National Institutes of Health

2002 The Lustgarten Foundation for Pancreatic Cancer Research

2002-2008 NIDDK R01 Renewal, National Institutes of Health

2009-2014 NIDDK R01 Renewal, National Institutes of Health

2014-2019 NIDDK R01 Renewal, National Institutes of Health

C. Contribution to Science

1. I graduated as an M.D. Magna Cum Laude in 1987 from the University of Cordoba Medical School in 

Cordoba, Argentina, where I initiated my studies in the area of pancreatic cancer. To pursue training in this 

field, I joined the National Institutes of Health from where I published work that lead to seminal discoveries 

in the area of mechanochemical translocators. My basic science studies on the molecular properties of 

these proteins demonstrated for the first time that kinesin transports organelles along microtubules. 

Subsequently, I co-discovered dynamin II and demonstrated that this protein, which was thought to be only 

a neuronal microtubule motor, was indeed an endocytic GTPase universally conserved form yeast to 

vertebrates. I also discovered myosin I E, which today is dubbed as “super myosin” due to its ability to 

support rapid actin-based movement. Together, these proteins are the best known ATP and GTP 

hydrolyzing enzymes that support cancer-associated processes by critically regulating cell motility, 

migration, organelle transport, chromosome movements, and signaling.

a. Urrutia R, McNiven MA, Albanesi JP, Murphy DB, Kachar B. Purified kinesin promotes vesicle 

motility and induces active sliding between microtubules in vitro. Proc Natl Acad Sci USA. 1991; 

88:6701-6705. PMCID: PMC52156

b. Torre E, McNiven MA, Urrutia R. Dynamin 1 antisense oligonucleotide treatment prevents neurite 

formation in cultured hippocampal neurons. J Biol Chem. 1994; 269(51):32411-7. PMID:7798241

c. Cook TA, Urrutia R, McNiven MA. Identification of Dynamin 2, an isoform ubiquitously expressed 

in rat tissues. Proc Natl Acad Sci USA. 1994; 91:644-648. PMCID: PMC43005

d. Urrutia R, Jung G, Hammer JA III. The Dictyostelium myosin IE heavy chain gene encodes a 

truncated isoform that lacks sequences corresponding to the actin binding site in the tail. Biochim 

Biophys Acta. 1993; 1173:225-229. PMID: 8504170

2. With the initial goal of defining how these proteins regulate cancer processes, I began my studies on 

transcriptional regulation, which led us to change gears in our research and pioneer the field of KLF 

proteins. While KLFs have gained significant attention as transcription factor proteins that play a significant 

role in nuclear reprogramming (KLF4 is one of the best known Yamanaka factors for iPS cells), these 

proteins play a pleotropic role in the pathobiology of many diseases. My laboratory discovered a family of 

KLF proteins that work at the intersection of metabolism and cancer. Specifically, we cloned KLF10, 

KLF11, KLF13, KLF14, and KLF16, which function as metabolic regulators and tumor suppressor proteins. 

We demonstrated that while germ line mutation of these proteins cause metabolic diseases, such as 

diabetes, their epigenetic silencing contributes to the process of pancreatic carcinogenesis.

a. Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, Miller L, Urrutia R. An mSin3A 

interaction domain links the transcriptional activity of KLF11 with its role in growth regulation. EMBO 

J. 2003; 22(18):4748-58. PMCID: PMC212736

b. Neve B, Fernandez-Zapico ME, Ashkenazi-Katalan V, Dina C, Hamid YH, Joly E, Vaillant E, 

Benmezroua Y, Durand E, Bakaher N, Delannoy V, Vaxillaire M, Cook T, Dallinga-Thie GM, Jansen 

H, Charles MA, Clement K, Galan P, Hercberg S, Helbecque N, Charpentier G, Prentki M, Hansen 

T, Pedersen O, Urrutia R, Froguel P. Role of transcription factor KLF11 and its diabetes-associated 

gene variants in pancreatic beta cell function. Proc Natl Acad Sci USA. 2005; 102(13):4807-12. 

PMCID: PMC554843
Contact PD/PI: Faubion, William A

Biosketches Page 32

c. Bonnefond A, Lomberk G, Buttar N, Busiah K, Vaillant E, Lobbens S, Yengo L, Dechaume A, 

Mignot B, Simon A, Scharfmann R, Neve B, Tanyolac S, Hodoglugil U, Pattou F, Cave H,Iovanna 

J, Stein R, Polak M, Vaxillaire M, Froguel P, Urrutia R. Disruption of a novel KLF-p300-regulated 

pathway for insulin biosynthesis revealed by studies of the c.-331 INS mutation found in neonatal 

diabetes mellitus. J Biol Chem. 2011; 286(32):28414-24. PMCID: PMC3151084

d. Lomberk G, Grzenda A, Mathison A, Escande C, Zhang JS, Calvo E, Miller LJ, Iovanna J, Chini EN, 

Fernandez-Zapico ME, Urrutia R. Krüppel-like factor 11 regulates the expression of metabolic 

genes via an evolutionarily conserved protein interaction domain functionally disrupted in maturity 

onset diabetes of the young. J Biol Chem. 2013; 288(24): 17745-58. PMCID: PMC3682574

3. Attempts to better understand how KLF proteins work, my research began to characterize on how they 

couple to chromatin and mediate epigenetic signaling. We focused on testing the histone code hypothesis 

postulated by David Allis, which proposed that the type and combination of posttranslational modifications, 

known as histone marks, were sufficient to function as a predictive epigenetic code. As postulated, this 

hypothesis did not take into consideration the impact that the regulation of writers, readers, and erasers of 

these marks by themselves had on epigenetic inheritance. However, my studies demonstrated that 

posttranslational modifications in the readers of these marks acted as additional “subcodes”, which were 

necessary for gene silencing. By working on histone code erasers, we discovered Sin3-Interacting 

Domains, which are protein motifs that function as a link between DNA-bound transcription factors and 

histone deacetylases (HDACs). Subsequent work established that these pathways are antagonized by 

writer enzymes such as HATs and HMTs, including the identification of novel EZH2 proteins, which are 

among the best epigenetic oncogenes known to date.

a. Lomberk G, Bensi DE, Fernandez-Zapico ME, Urrutia R. Evidence for the existence of an HP1- 

mediated subcode within the histone code. Nat Cell Biol. 2006; 8: 407-15. PMID: 16531993

b. Zhang J-S, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. A conserved αhelical motif mediates the interaction of Sp1-like transcriptional repressors with the corepressor 

mSin3A. Mol Cell Biol. 2001; 21:5041-5049. PMCID: PMC87230

c. Grzenda A, Lomberk G, Svingen P, Mathison A, Calvo E, Iovanna J, Xiong Y, Faubion W, Urrutia

R. Functional Characterization of EZH2beta reveals the increased complexity of EZH2 isoforms 

involved in the regulation of mammalian gene expression. Epigenetics Chromatin. 2013; 6(1):3.

PMCID: PMC3606351

d. Lomberk G, Mathison AJ, Grzenda A, Seo S, DeMars CJ, Rizvi S, Bonilla-Velez J, Calvo E, 

Fernandez-Zapico ME, Iovanna J, Buttar NS, Urrutia R. Sequence-specific recruitment of 

heterochromatin protein 1 via interaction with Krüppel-like factor 11, a human transcription factor 

involved in tumor suppression and metabolic diseases. J Biol Chem. 2012; 287(16):13026-39. 

PMCID: PMC3339955

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/raul.urrutia.1/bibliography/40845204/public/?sort=da

te&direction=ascending

D. Research Support

Ongoing Research Support

R01 DK052913 (Urrutia, PI) 09/01/2014-08/31/2019 

NIDDK

The Role of Zinc Finger Co-Factors in Pancreatic Cell Growth

The major goal of this project is to determine the role of novel zinc finger transcription factors, KLFs, and their 

co-factors in pancreatic cell growth.

Role: Principal Investigator
Contact PD/PI: Faubion, William A

Biosketches Page 33

P50 CA102701 (Petersen, PI) 09/01/2014-08/31/2015

NIH-NCI – Mayo Clinic Pancreatic Cancer SPORE

Pilot Project Award: EZH2-Based Experimental Therapeutics in Pancreatic Cancer

The major goal of this project is to characterize the effect of EZH2 downstream of oncogenic pathways on 

growth-promoting gene networks to facilitate PDAC initiation and promotion in a manner that is amenable to 

pharmacological inhibition.

Role: Principal Investigator of Pilot Project
Contact PD/PI: Faubion, William A

Page 34

Funding Opportunity Number: PA-13-302. Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

PHS 398 Cover Page Supplement OMB Number: 0925-0001

1. Project Director / Principal Investigator (PD/PI)

Prefix:

First Name*: William

Middle Name: A

LastName*: Faubion

Suffix: MD

2. Human Subjects

Clinical Trial? ● No ❍ Yes

Agency-Defined PhaseIII Clinical Trial?* ● No ❍ Yes

3. Permission Statement*

If thisapplicationdoesnot resultinanaward,isthe Government permitted to disclosethetitleofyourproposedproject,andthename, 

address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be 

interested in contacting you for further information (e.g., possible collaborations, investment)?

❍ Yes ● No

4. Program Income*

Isprogramincomeanticipatedduring the periodsforwhichthegrantsupportisrequested? ❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and source(s). 

Otherwise, leave this section blank.

Budget Period* AnticipatedAmount ($)* Source(s)*
Contact PD/PI: Faubion, William A

Page 35

Funding Opportunity Number: PA-13-302. Received Date:

2015-07-02T14:04:51.000-04:00 Tracking Number: GRANT11954038

PHS 398 Cover Page Supplement

5. Human Embryonic Stem Cells

Does the proposedproject involvehumanembryonicstemcells?* ● No ❍ Yes

If theproposedproject involveshumanembryonicstemcells, list belowthe registration numberof thespecificcellline(s)fromthefollowing 

list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time, please check the box 

indicating that one from the registry will be used:

Cell Line(s): ❏ Specific stem cell line cannot be referenced at this time. One from the registry will be used.

6. Inventions and Patents (For renewal applications only)

Inventionsand Patents*: ❍ Yes ● No 

If the answeris"Yes" thenpleaseanswerthefollowing: 

Previously Reported*: ❍ Yes ❍ No

7. Change of Investigator / Change of Institution Questions

❑ Change of principal investigator / program director 

Name of former principal investigator / program director: 

Prefix:

First Name*:

Middle Name:

Last Name*:

Suffix:

❑ Change of Grantee Institution 

Name of former institution*:
Contact PD/PI: Faubion, William A

Page 36

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

OMB Number: 0925-0001

Budget Period: 1

Start Date: 07/01/2016 End Date: 06/30/2017

A. Direct Costs

Direct Cost less Consortium F&A*

Consortium F&A

Total DirectCosts*

Funds Requested($)

0.00

B. Indirect Costs

Indirect Cost Type Indirect CostRate (%) Indirect Cost Base ($) Funds Requested ($)

1. MTDC

2.

3.

4.

Cognizant Agency DHHS, Arif Karim and Narendra Gandhi,

(Agency Name, POC Name and Phone Number)

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C.TotalDirectandIndirectCosts(A + B) Funds Requested ($)
Contact PD/PI: Faubion, William A

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Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

Budget Period: 2

Start Date: 07/01/2017 End Date: 06/30/2018

A. Direct Costs

Direct Cost less Consortium F&A*

Consortium F&A

Total DirectCosts*

Funds Requested($)

0.00

B. Indirect Costs

Indirect Cost Type Indirect CostRate (%) Indirect Cost Base ($) Funds Requested ($)

1. MTDC

2.

3.

4.

Cognizant Agency DHHS, Arif Karim and Narendra Gandhi,

(Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date 01/20/2015 TotalIndirect Costs

C.TotalDirectandIndirectCosts(A + B) Funds Requested ($)
Contact PD/PI: Faubion, William A

Page 38

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

Budget Period: 3

Start Date: 07/01/2018 End Date: 06/30/2019

A. Direct Costs

Direct Cost less Consortium F&A*

Consortium F&A

Total DirectCosts*

Funds Requested($)

0.00

B. Indirect Costs

Indirect Cost Type Indirect CostRate (%) Indirect Cost Base ($) Funds Requested ($)

1. MTDC

2.

3.

4.

Cognizant Agency DHHS, Arif Karim and Narendra Gandhi,

(Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date 01/20/2015 TotalIndirect Costs

C.TotalDirectandIndirectCosts(A + B) Funds Requested ($)
Contact PD/PI: Faubion, William A

Page 39

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

Budget Period: 4

Start Date: 07/01/2019 End Date: 06/30/2020

A. Direct Costs

Direct Cost less Consortium F&A*

Consortium F&A

Total DirectCosts*

Funds Requested($)

0.00

B. Indirect Costs

Indirect Cost Type Indirect CostRate (%) Indirect Cost Base ($) Funds Requested ($)

1. MTDC

2.

3.

4.

Cognizant Agency DHHS, Arif Karim and Narendra Gandhi,

(Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date 01/20/2015 TotalIndirect Costs

C.TotalDirectandIndirectCosts(A + B) Funds Requested ($)
Contact PD/PI: Faubion, William A

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Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

Budget Period: 5

Start Date: 07/01/2020 End Date: 06/30/2021

A. Direct Costs

Direct Cost less Consortium F&A*

Consortium F&A

Total DirectCosts*

Funds Requested($)

0.00

B. Indirect Costs

Indirect Cost Type Indirect CostRate (%) Indirect Cost Base ($) Funds Requested ($)

1. MTDC

2.

3.

4.

Cognizant Agency DHHS, Arif Karim and Narendra Gandhi,

(Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date 01/20/2015 TotalIndirect Costs

C.TotalDirectandIndirectCosts(A + B) Funds Requested ($)
Contact PD/PI: Faubion, William A

Page 41

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date: 2015-07-02T14:04:51.000-04:00

PHS 398 Modular Budget

Cumulative Budget Information

1. Total Costs, Entire Project Period

Section A, Total Direct Cost less Consortium F&A for Entire Project Period ($)

Section A, Total Consortium F&A for Entire Project Period ($) 0.00

Section A, Total Direct Costs for Entire Project Period ($) 

SectionB,TotalIndirectCostsfor Entire ProjectPeriod($)

Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period ($)

2. Budget Justifications

Personnel Justification Personnel_Justification_1_.pdf

Consortium Justification 

Additional Narrative Justification
Contact PD/PI: Faubion, William A

PERSONNEL JUSTIFICATION:

Senior/Key Personnel:

Faubion, WA Jr. MD, PI, (30%) 3.6 person months/year over the life of the grant. As Principal Investigator, Dr. 

Faubion is responsible for the daily conduct of the proposed studies. Dr. Faubion received his medical degree 

from the University of Texas Health Science Center, Houston, Tx, and his GI subspecialty training at Mayo 

Clinic, Rochester, MN. He received basic immunology training in the laboratory of Dr. Cox Terhorst, Beth 

Israel, Harvard, MS in 2000-2003. Dr. Faubion is an expert in cellular immunology, murine models of colitis, 

and specifically T regulatory cell biology, and FOXP3 gene regulation. As a member of the Epigenetic and 

Chromatin Dynamics Laboratory, he is intensely focused on the epigenetic regulation of FOXP3 dependent 

gene networks. Dr. Faubion has the expertise and his laboratory has the appropriate methodology to support 

the feasibility of the current proposal.

Collaborators:

Urrutia, Raul A. MD, Collaborator, no measurable effort. Dr. Urrutia, as the director of the Chromatin and 

Epigenetics Laboratory at Mayo Clinic, already provides intellectual input. He has provided and will continue to 

provide a variety of unique and critical reagents such as the EZH2 SET domain mutant and EZH2 HMT 

pharmacologic inhibitors. His letter of support confirms his willingness to collaborate.

Smyrk, Thomas C. MD, Collaborator, no measurable effort. Dr. Smyrk has agreed to provide pathologic 

expertise in reviewing the histologic sections of mouse colon and to provide a histologic colitis score. His letter 

of support confirms his willingness to collaborate.

Pasare, Chandrashekhar, PhD, UTSW Collaborator, no measurable effort. Dr. Pasare is an Associate 

Professor in the Department of Immunology, UTSW. He has extensive experience in IL6 signaling pathways in 

relationship to Treg function. He has already provided intellectual input and the IL6R signaling mutant mouse 

line. His letter of support confirms his willingness to collaborate.

Ekker, Stephen PhD, Collaborator, no measurable effort. Dr. Ekker is a Professor of BMB and an expert in 

gene editing technology. He has already provided intellectual input and TALEN constructs. His willingness to 

collaborate on Aim 3 is indicated in his letter of support.

Other Personnel:

Svingen, P, Technician, 12 person months/year for years 1-5. Mrs. Svingen is a senior laboratory technician 

with over 30 years experience at Mayo Clinic. She has accomplished in areas of cellular immunology including 

T cell subset isolation, culture, cell transfection techniques, and flow cytometry. She also is capable of genomic 

DNA isolation and ChIP assay. Furthermore, she is proficient with animal colitis models such as bone marrow 

transfer, adoptive T cell transfer, DSS colitis, and TNBS colitis.

Xiong, Y, Technician, 3.78 person months/year for years 1-5. Dr. Xiong is an MD/PhD with experience in 

molecular biology, ChIP assay, and DNA and RNA isolation from colonic T cell subsets.

His efforts will be focused on the maintenance of the genome integrated FLP cell lines and the generation of 

new constructs and recombinant fusion proteins.

Lastly, our laboratory is highly interactive and these individuals will collaborate on each of the specific aims. 

Their data is presented once a week at lab meetings and periodically at retreats as well as national and 

international meetings. Therefore, the high coherence of this group and their exquisite training make them 

among the most qualified researchers possible to participate in this grant.

Personnel Justification Page 42
Contact PD/PI: Faubion, William A

PHS 398 Research Plan

Pleaseattachapplicablesectionsof the researchplan,below. OMB Number: 0925-0001

1. Introduction to Application Introduction_Final.pdf (for RESUBMISSION or REVISION only)

2. Specific Aims HYPOTHESIS_AND_SPECIFIC_AIMS.pdf

3. Research Strategy* Research_Plan.pdf

4. ProgressReportPublicationList Publication_List3.pdf

Human Subjects Sections

5. Protection of Human Subjects

6. Inclusion of Women and Minorities

7. Inclusion of Children

Other Research Plan Sections

8. VertebrateAnimals Vertebrate_Animals.pdf

9. Select Agent Research

10. Multiple PD/PI Leadership Plan

11. Consortium/Contractual Arrangements

12. Letters of Support letters_combined.pdf

13. Resource Sharing Plan(s) Resource_Sharing_Plan.pdf

Appendix (if applicable)

14. Appendix

Page 43

Tracking Number: GRANT11954038 Funding Opportunity Number: PA-13-302. Received Date:

2015-07-02T14:04:51.000-04:00
Contact PD/PI: Faubion, William A

Introduction Page44

INTRODUCTION TO APPLICATION

We appreciate the highly laudatory review on 2/19/2015 of our proposal (21st percentile). The 

constructive critique has resulted in the addition of extensive new preliminary data and the 

development of new reagents and animal models, which have improved the proposal in novelty, 

relevance, feasibility, design, and methodology. Below, we address both convergent suggestions as 

well as individual points not shared by all reviewers (revisions highlighted in yellow in newproposal).

Productivity: Since the last review, 3 additional senior authored manuscripts have been published 

totaling 10 peer reviewed publications generated through the experiments defined in the first four years 

of this award. Regarding the concern that “this new proposal is a major departure from the directions 

proposed in the previous proposal,” we have provided in detail (progress report) the continuum of logic 

flowing from the previous aims (KLF binding to DNA, coupling to epigenetic machinery, and with 

relevance to colitis) to the current hypothesis. In fact, this grant is focused on the epigenetic machinery 

(EZH2) identified in previous Aim3 and the relevance to Treg biology and colitis.

Key preliminary data: There was consensus among reviewers on the need for additional data.

(i) It was noted that, “Although the colitis T cell transfer model is primarily used in the proposal, the 

preliminary results use the DSS model.” We have developed a new tamoxifen-inducible CRE-EZH2 KO 

animal and demonstrated a requirement for EZH2 in FOXP3+ cells for prevention of Rbhi mediated 

colitis, a critical control for the in vivo mutational analysis (Aim3).

(ii) We were queried whether the “FOXP3dEZH2 mice show spontaneous colitis?” We have yet to age 

mice beyond 8 weeks; yet we show the susceptibility to subclinical (1%DSS) colitis in newexperiments.

(iii) More data in support of A2 was required, as “The model that EZH2 phosphorylation ... is critical in 

disrupting Treg cell function is highly speculative.” We have developed an experimental pipeline that 

consists of mass spectrophotometry, linear motif analyses, molecular modeling, mutational screens, 

and functional assays to definitively test the hypothesis of Aim 2. First, mass spectrophotometry 

identified phosphorylated residues predicted by molecular modeling to regulate EZH2 HMT activity. 

Extensive linear motif analysis predicted 6 such residues to be regulated by MAPK and Jak kinases. 

We generated 12 mutant EZH2 constructs and a CRISPR/Cas9 EZH2 mutant cell line in which to test 

the effect of phosphorylated residues on EZH2 HMT activity. Exemplary of this pipeline, we discovered 

phosphorylation of Y641 by Jak2 to regulate EZH2 activity. As we were queried in several ways for the 

effect of IL10 in this mechanism, we have incorporated IL10 into the tightly focused dissection of the 

critical inflammatory pathways leading to reversible modifications of EZH2 HMT activity.

Individual points not shared by all reviewers and not addressed above: 

Reviewer 1 (R1) noted previous data exists regarding IL6 disruption of Treg function. Indeed, IL6 

dependent T effector cell resistance to Treg suppression is well reported (Pasare and Medzhitov 2003, 

Viglietta, Baecher-Allan et al. 2004, Haas, Hug et al. 2005, Venken, Hellings et al. 2006); however 

purported mechanisms involve intrinsic T effector cell pathways, not Treg cell biology (Schenten, Nish 

et al. 2014, Bhela, Kempsell et al. 2015). The conversion of FOXP3+ cells to TH17 cells downstream of 

IL6 signaling has been reported (Komatsu, Okamoto et al. 2014); yet again, the mechanism(s) 

leading to Treg cellular conversion is unknown, represents a critical gap in knowledge, and 

addressed in this proposal. R1 also requested a broader description of outcome variables to assess 

in vivo studies and a discussion regarding the role for EZH2 in Stat3 activity. Both are provided in the 

revised proposal. Reviewer 2 (R2) queried “whether EZH2 may repress cytokines in other 

lymphocytes,” and that we “examine cell surface molecules and cytokines, such as IL-10, ... to 

determine whether they are also affected by EZH2 blockade.” We demonstrate flow analysis of surface 

molecules and the results of multiplex ELISA for cytokine production. Furthermore, we have explored a 

role for EZH2 in T effector cells in alternative approaches. R2 noted A3 to be “wide ranging with 

superficial discussion of pitfalls and alternative approaches.” We have streamlined the aim by exclusion 

of the neonatal transfer experiment (feasibility was questioned) and have greatly enhanced the Pitfalls 

and Alternative Approaches. Summary: In response to this excellent critique, we have provided 

evidence of continued productivity, preliminary data from three new major lines of experimentation, 

developed two new mouse models, and addressed each review point by point with modifications to the 

proposal.
Contact PD/PI: Faubion, William A

Specific Aims Page45

HYPOTHESIS AND SPECIFIC AIMS:

The transcription factor FOXP3 is critical to the regulation of numerous debilitating human 

immune-mediated diseases, the prevalence of which together affect over 8.5 million people (1 in 31

U.S. residents). In Inflammatory Bowel Disease (IBD) chronic intestinal inflammation indicates 

aberrant in vivo FOXP3+ T regulatory (Treg) cell function (1). Similarly, proinflammatory signals in 

vitro impair Treg function (2). Our lab was the first to characterize the essential role for the histone 

methyltransferase (HMT) EZH2 in the epigenetic regulation of FOXP3 (3). Recent published work 

extended our observations indicating a key role for EZH2 in FOXP3 repressor function (4); however 

the regulation and biological impact of the FOXP3-EZH2 pathway to IBD is unknown. This knowledge 

is important given the apparent loss of function of Treg cells in inflammation.

Our long-term goal is to dissect epigenetic mechanisms regulating Treg cellular differentiation 

and function, particularly within the setting of GI inflammatory diseases; as these discoveries will 

facilitate design of human cell therapy trials for IBD. Consequently, the objective of this grant is to 

characterize the role for the epigenetic regulator EZH2 in Treg suppressive function. These 

investigations are strongly supported by preliminary data demonstrating that: 1) EZH2 is required for 

Treg suppressive function; 2) IL6 signaling leads to phosphorylation and inhibition of EZH2; 3) 

lymphocytes isolated from the intestine of IBD patients demonstrate activation of IL6-induced gene 

networks and loss of EZH2 HMT function; and 4) conditional knockout of EZH2 in FOXP3+ T cells 

leads to in vivo immune dysfunction. Based upon these compelling data we propose the CENTRAL 

HYPOTHESIS that EZH2 plays a critical role in the homeostasis of Treg cells, and the 

disruption of EZH2 function by inflammatory signaling pathways contributes to IBD. Our 

rationale is that identification of the mechanism(s) to restore Treg suppressive function in the setting 

of intestinal inflammation will offer new therapeutic opportunities within the field of IBD. Our specific 

aims will test the following hypotheses:

Aim 1: Repression of immunoregulatory 

gene networks by FOXP3 requires the formation 

of a complex between this transcription factor 

and EZH2.

Aim 2: Inflammatory stimuli, such as IL6 

lead to EZH2 phosphorylation and thereby 

disrupt the enzymatic activity of this epigenomic 

regulator.

Aim 3: Inhibition of the IL6 to EZH2 

signaling pathway permits sustained Treg 

suppressive function in the setting of intestinal

inflammation.

Upon conclusion, we will understand the 

role for EZH2 in Treg loss of function in the 

setting of active inflammation. This discovery will 

stimulate new areas for experimental 

therapeutics in human chronic inflammatory 

diseases. Our environment in the Epigenetic and 

Chromatin Dynamics Laboratory combined with 

the Department of Immunology at the Mayo 

Clinic makes us uniquely qualified to pursue this

objective given the extensive collective 

experience of histone methyltransferase biology,

proinflammatory signaling networks, and FOXP3 gene regulation.

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Figure 1: Conceptual framework. Through the mechanistic 

experiments designed in the following aims we will identify the 

role for FOXP3 in the recruitment of EZH2 to core target genes 

required for Treg function (Aim 1). We will define the signaling 

network responsible for phosphorylation of EZH2 and disrupted 

HMT function (Aim 2). Finally, we will perform pre-clinical 

studies of innovative therapy designed to generate Treg cells 

resistant to disruptive modifications in the setting of inflammation 

(Aim 3).

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Contact PD/PI: Faubion, William A

Research Strategy Page 46

RESEARCH STRATEGY

Significance: FOXP3 is a transcription factor critical to the development, maintenance, and 

function of Treg cells. FOXP3 repression of key target gene networks is required for maintenance of 

Treg function (4,5). Key observations performed during the last funding cycle of this grant led us to 

identify for the first time a key role for EZH2 in the regulation of FOXP3 expression (3). The

significance of this discovery is evident in the fact that subsequently three groups have confirmed and 

extended our observations on the import of EZH2 in the differentiation and maintenance of Treg cells 

(4,6,7). Yet, actionable mechanistic insight into 1) the FOXP3 EZH2 protein complex, 2) inflammatory 

signaling regulating EZH2 activity, and 3) the potential for EZH2-based therapy in IBD represents a 

critical gap in knowledge addressed in this application.

The canonical function of EZH2 is trimethylation of lysine 27 (K27) of histone 3 (H3) 

(H3K27me3). This “histone mark” leads to chromatin inaccessibility and gene silencing (8). EZH2 

undergoes post-translational modifications downstream of metabolic, cell proliferative, and 

inflammatory signaling pathways (9-13). We have now discovered that proinflammatory pathways 

lead to EZH2 phosphorylation and inhibit EZH2 histone methyltransferase (HMT) activity in primary 

lymphocytes. This discovery is significant, as inflammatory signals can disrupt Treg function (2,14-16), 

which is apparent within the inflamed environment of IBD and characterized by the co-expression of

the proinflammatory cytokines IFNγ and IL17 (17,18). However, little still is known about the 

mechanism(s) of inflammation-induced disruption of Treg function. Thus, the current study will

characterize the role for inflammation-induced modification of EZH2, the inhibition of HMT activity, and 

the subsequent disruption of Treg suppressive function. This contribution is significant since it will 

establish that several pathways targeted by available therapies (i.e. IL1β, IL6, TNFα) have the 

potential to regulate EZH2 HMT activity through post-translational modifications. Furthermore, current 

Treg cell therapy trials (19), while promising have not addressed the key issue of in vivo inflammationinduced disruption of Treg function. This proposal seeks to address this knowledge gap.

IBD, increasing in prevalence, represents a major national cost measured by both patient 

suffering and economic burden (as evidenced by over 600 original studies on cost of IBD). Despite 

significant advances in care (i.e. biologic therapeutics), clinical trial data demonstrate remission rates 

at best of 40%; thus there remains an enormous unmet medical need. The clinical significance of our 

study will be the opening of a new avenue in therapeutics directed at stimulation of autologous Treg 

cells to function within the inflammatory milieu. Our results will impact not only IBD, but also human 

immune mediated disease broadly.

Innovation: Previous work on epigenetic mechanisms in Treg function has largely focused on 

genome wide associations using ChIP-Seq methodology (4). The proposed research is innovative 

because we investigate the effect of inflammatory signaling pathways on epigenetic complexes in 

Treg cells, a heretofore-unexamined process. Our experimental approach dives more deeply, 

biochemically and biologically into the mechanistic impact of this pathway beyond the simple catalog 

of gene targets provided by the previous ChIP-Seq methodology. Insight into epigenetic mechanisms 

is impactful as T cell progenitor cells inherit the parent transcriptional profile and unlike genetic 

change, they are modifiable by currently available therapy. In fact, anti-EZH2 drugs are currently 

being tested in clinical trials and knowing the role of this HMT in T cells will become imperative as 

they become widely available in the clinical setting. Conceptual innovations include: 1) the novel role 

for proinflammatory signaling networks in the regulation of the HMT EZH2 pathway in lymphocytes 

and IBD; 2) the discovery of the required partnership between FOXP3 and EZH2 in Treg function;and

3) ex vivo genetic engineering of Treg cells to function in the setting of inflammation. 

Methodologically, there are four new animal models used in this application including the conditional 

EZH2 KO mouse line, two inducible EZH2 KO mouse lines, and the conditional IL6Rα knockout 

mouse line. The in vivo experimental techniques utilized in this proposal, particularly the cell transfer

experiments utilizing adenoviral replacement of EZH2 and mutant constructs are state-of-the-art. 

Additional state-of-the-art methodology is provided by the use of CAS/CRISPR mutant cell lines and 

proteomic experiments, molecular mechanics and molecular dynamic techniques for studying the 

effects of post-translational modifications on the EZH2 protein, which combined are highly innovative 

in the field of immunology. In summary, the hypotheses guiding the proposal are novel and will be 

tested using a comprehensive approach with sophisticated methodology also giving rise to reagents, 

including new cell and animal models which will be widely shared with the scientific community.
Contact PD/PI: Faubion, William A

Research Strategy Page 47

Approach:

PROGRESS REPORT

General Statement: The beginning and ending dates of the current period of funding are 

2/15/2011-1/31/2016. In this grant’s previous cycle, 10 relevant original articles have been published.

Progress Towards Prior Specific Aims: Published Results. Our original hypothesis was that KLF 

family members, through coupling with chromatin modifying complexes regulate FOXP3 expression, 

and ultimately T reg cell function and colitis. Our most salient accomplishments during the prior 

funding period relevant to our prior three Specific Aims are provided below. The resulting publications 

are listed within the specified section of the application (see progress report publication list).

Prior Aim #1: KLF10 expression in T cells mediates a novel mechanism of resistance to colitis. 

As KLF10 is regulated by TGFβ and required for inducible FOXP3+ Treg cell (iTreg) development, we 

postulated that KLF10 is required in T cells to prevent immune mediated disease. KLF10 deficient

animals are susceptible to colitis (#1 publication list) (3). Colitis susceptibility is transferred with the 

bone marrow indicating the requisite role for KLF10 in immune cells to prevent colitis (#2 publication 

list) (20). In lymphocytes, KLF10 regulates both FOXP3 and TGFβRII transcription generating a 

positive feedback loop in Treg development (3,21) (#3 publication list). This regulatory loop is relevant 

in murine models of colitis and encephalitis (20,21). Thus, KLF10 required for TGFβ induction of iTreg

cells, functions in a positive feedback loop between TGFβ signaling and the TGFβRII promoter and is

critical to immune regulatory function.

Prior Aim #2: KLF10 selectively regulates distinct genomic control regions of Foxp3. Because 

suspected KLF binding sites exist within the FOXP3 promoter locus, we postulated that distinct KLF 

binding domains regulate FOXP3 transcription. Using genome integrated cell lines, mutant mouse 

lines, and primary human cells, we identified novel KLF binding sites in both the core promoter and 

key enhancer domains. KLF10 binding to the core promoter recruits Sin3-HDAC or the HAT PCAF to 

repress or activate FOXP3 gene transcription, respectively (#1 and 2 publication list) (3,20). KLF14 

binding to an enhancer domain represses FOXP3 through the heterochromatin protein 1 genesilencing pathway (22)(#4 publication list), a relationship found to be generalizable to other model 

systems (#5 publication list) (23). FOXP3 domain functions discovered in the conduct of this aim led 

to the functional characterization of a private mutation causing familial IBD (#6 publication list) (24) 

and innovative methodology to quantify in vivo human Treg kinetics (#7 publication list) (25). 

Collectively, these observations identify distinct genomic control regions of FOXP3 through which KLF 

family members regulate FOXP3 transcription.

Prior Aim #3: KLF10 works via distinct chromatin remodeling required for Foxp3 activation. 

Because KLF family members function to couple DNA binding to chromatin remodeling complexes, 

we examined the potential for KLF10 to regulate FOXP3 in an epigenetic fashion. We observed that 

KLF10 binding to the core promoter blocks recruitment of the HMT, EZH2 thus preventing gene 

repression (#1 publication list) (3). We discovered two structurally and functionally distinct EZH2 

HMTs; and both are capable of permanently silencing FOXP3 (#8 publication list) (26). Thus, KLF10 

alternatively represses or activates FOXP3 through coupling with Sin3 or PCAF, and in the absence 

of KLF10, FOXP3 is permanently silenced by EZH2. In summary, we have successfully completed 

work on our previous specific aims demonstrating fidelity to the proposal. Additionally, our work into

TGFβ signaling upstream of the FOXP3 promoter locus resulted in novel insights into hedgehog 

signaling pathways (#9 publication list) (27) and the function of an epithelial derived stem cell in colitis

(28) (#10 publication list). In addition, by lending critical expertise gained from the funded studies, we 

were able to contribute to additional collaborative publications, further attesting to the success of our 

program during the past funding cycle (29-34).

This grant proposal represents the continuum of logic flowing from the previous aims (KLF

binding to DNA and coupling to epigenetic machinery to regulate colitis) to the current hypothesis. In

the process of characterizing the role for EZH2 isoforms at the FOXP3 promoter locus (previous

Aim3) we discovered a profound role for EZH2 in FOXP3 repressor function in both murine and

human T regulatory cells. This critical and modifiable function within Treg cells in the setting of IBD is

the subject of this grant proposal.
Contact PD/PI: Faubion, William A

Research Strategy Page 48

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Aim 1: Repression of immunoregulatory gene networks by FOXP3 requires the formation of a 

complex between this transcription factor and EZH2.

J.5. -.E0.==265

Figure 2 (AIM1): Our model to be tested is that EZH2, FOXP3 

complex formation leads to recruitment of EZH2 to target genes, 

resultant H3K27 trimethylation, gene silencing, and appropriate 

facilitation of Treg cell function.

Introduction: FOXP3 represses gene 

networks (4); however the mechanism is not 

well established. The objective of this aim is 

to define the role for EZH2 in FOXP3- 

mediated gene repression. We will test the 

working hypothesis that EZH2 is recruited to 

target genes in complex with FOXP3 (Figure 

2). Our approach will be domain mutational 

analyses of EZH2 and FOXP3. We will test 

the function of these mutations by both 

molecular (target gene repression) and 

cellular (Treg suppressor function) assays. 

The rationale for this aim is that 

understanding the mechanism of FOXP3 

repressor function will lead to therapy to 

enhance Treg cell function in vivo. Our

expectation is to identify the protein domains within EZH2 and FOXP3 required to repress target 

genes and enhance Treg function. This knowledge is important because EZH2 modifying agents 

(EPZ-6438 and GSK-126) are currently in clinical trials for lymphoma; thus insights derived from this 

grant into in vivo Treg function are significant and impactful.

Justification, feasibility and preliminary data: We have identified a critical role for EZH2 HMT 

activity in mediating Treg suppressor function. We have generated a conditional, functional 

knockout of EZH2 through deletion of the enzymatically active SET domain under the control of the 

FOXP3 promoter (FOXP3$EZH2). By 35 days, 80% of FOXP3$EZH2 animals succumb to a lethal 

inflammatory disorder similar to the scurfy mouse characterized by intense inflammatory infiltrate of 

multiple organs (survival curve, n=107 total mice, Figure 3A). FOXP3$EZH2 animals surviving to 8 

weeks exhibit significantly increased susceptibility to colitis upon exposure to subclinical (1%) DSS

challenge (histology, n=16 total mice, Figure 3B, C). The histologic activity index represents blinded

scoring for 9 criteria as previously published (28). Note lamina propria expansion, goblet cell loss, 

crypt destruction, inflammatory cell infiltration and ulcer in severely affected FOXP3$EZH2 mice (Figure 

3C). FOXP3$EZH2 lymphocytes develop at expected frequency, and upon isolation, the cells express

the typical cell surface repertoire of regulatory proteins (Figure 4); however they lack IL10 and 

produce proinflammatory cytokines including significant upregulation of IFN! and IL17 (Figure 5A). 

FOXP3$EZH2 cells function poorly in vitro (Figure 5B). Furthermore, we discovered a direct 

interaction between EZH2 and FOXP3 by proximity ligation assay (PLA) in the nucleus of WT Treg 

cells (Figure 6). Thus, we conclude that FOXP3 and EZH2 co-localize in the nucleus and that Treg 

specific EZH2 HMT activity is required for normal immune homeostasis and in vitro FOXP3+ 

Treg function. This discovery is significant as the regulation of this complex through cell signaling 

networks will be an important modifiable mechanism to target for the treatment of immunological 

disorders.

!

100

# $ Figure 3: FOXP3!EZH2

*p<0.05

8

6

50 4

2

mouse. (A) Survival curve of 

WT (black), Heterozygous 

(blue), and Homozygous 

(green) littermates for EZH2 

deletion. (B, C) Colitis index 

and representative histology 

demonstrating severe colitis 

in 8 week old FOXP3!EZH2

0 mice in

0 50

Days

100 0 response to 

subclinical (1%) DSS

challenge (n=16 total mice).

Percent survival

Histologic Index
Contact PD/PI: Faubion, William A

Research Strategy Page 49

Figure 7: N and C terminal mutant constructs for FOXP3 (green) and EZH2 (blue) proteins. Regions 

of interest are the Zinc finger and forkhead (FH) domains of FOXP3. Disease inducing variants 

(human IPEX) are present in the FH domain. Similarly, the WD-binding domain and the enzymatic 

SET domain are shown in EZH2. Lymphoma associated variants are demonstrated.

Research Design: The general strategy of Aim 1 is to map interaction domains (1.1) and test 

domain function through assays of gene repression (1.2) and Treg cell suppressor function (1.3). The 

laboratory has published experience with co-immunoprecipitation (Co-IP) assays, luciferase reporter 

systems, ChIP assay, in vitro suppression assays, and in vivo colitis models (3,20,26). 

Overexpression of mutant constructs will be performed in cell lines by nucleofection and primary cells 

by adenoviral transduction. In our published experience, nucleofection efficiency in cell lines 

approximates 20% and adenoviral transduction efficiency is greater than 75%.

1.1 Biochemical approach to define the FOXP3-EZH2 complex

We will determine the interacting domains 

between EZH2 and FOXP3 using Co-IP with 

validation by PLA. We will transfect FLAG-EZH2 

and HIS-FOXP3 domain mutants in primary cells 

and T cell lines. The deletion mutations (Figure 7) 

were selected on the basis of protein interacting 

domains suggesting direct EZH2, FOXP3 

interaction (35-37). Results will be validated by 

PLA in primary Treg cells (Figure 6). In this assay, 

antibodies directed to either EZH2 or FOXP3 are 

tagged with oligonucleotide probes that upon

binding in proximity 

(30-40nm) hybridize to 

form circular DNA 

strands (38). DNA 

circles serve as 

template to amplify 

fluorescent probes 

indicating proximity. 

We expect EZH2 to 

complex with FOXP3 in 

a direct fashion, and 

the PLA results support 

this expectation. We 

expect to identify the 

specific domains

relevant to this 

interaction. As both EZH2 and FOXP3 have additional functions independent of each other, only with 

knowledge of precise interaction domains can we separate the biological effects of FOXP3-associated 

EZH2 function from EZH2 function broadly in lymphocytes.

Figure 6: FOXP3 and 

EZH2 co-localize in 

primary Treg cells. 

Proximity ligation assay 

showing direct interaction 

of EZH2 and FOXP3 (red

punctate 

FOXP3+

staining) in

cells (green

nuclei). No hybridization 

was evident upon addition 

of ligation mix, probe, and 

control Ab.

!"#$ !&'()* +!,-

!"01 .+&/ !"20

Figure 4: Cell surface staining of FOXP3!EZH2 

Treg cells (blue histogram) demonstrates 

upregulation of established immunoregulatory 

proteins. Marked upregulation over WT Treg cells 

(red histogram) suggests an activated Treg 

phenotype. Green histogram represents IgG 

control (representative of 3 independent 

experiments).

! # 8,000

800

* 600 6,000

p<0.05

400 * 4,000

200 *

2,000

0

0

Tresp WT Treg EZH2 KO Treg

Figure 5: FOXP3!EZH2 Treg cells express pro-inflammatory cytokines and 

function poorly in vitro. (A) ELISA of supernatants of FOXP3+ cells. (B) 

Proliferation assay shows that FOXP3!EZH2 Treg cells (closed histogram) fail 

to suppress WT T responder (WT Tresp, 1:1 ratio) when compared to WT 

Treg cells (*p<0.05, n=6 mice). pg/ml

Thymidine Counts
Contact PD/PI: Faubion, William A

Research Strategy Page 50

1.2 The functional relevance of EZH2 in target gene repression. We have performed RNA-Seq 

using CD4+ cells isolated from resection specimens in patients with active Crohn’s disease and ileal

biopsies of healthy age and gender matched 

control subjects. We analyzed existing ChIP-Seq 

datasets and have discovered 260 overlapping 

targets of FOXP3 and EZH2 (39-41). We then 

compared gene expression of these overlapping 

targets between samples from patients with CD 

and controls. From this broad dataset we have 

chosen RORA (RAR Orphan Receptor A) and IRF4 

(Interferon Regulatory Factor-4) as models to study 

the functional relevance of EZH2 in Treg cells. 

RORA and IRF4 are established targets of EZH2 

(42,43) and both are significantly upregulated in our 

Crohn’s disease sample (Figure 8). Moreover, both 

RORA and IRF4 are key transcription factors in the

development of Th17 cells, an immune subset responsible for human and murine colitis (44-49). 

Thus, we will use transcriptional regulation of these genes to test in vitro functional relevance of EZH2 

and EZH2, FOXP3 complex formation using reporter assays (1.21) and ChIP assay (1.22).

1.21 : Luciferase reporter assay: We will use luciferase reporter assays of the RORA and IRF4 

promoters in T cell lines. We will nucleofect EZH2 with titrated quantity of FOXP3 into reporter cell 

lines. We will determine the relevance of FOXP3 to the function of EZH2 through quantitative 

luciferase assay. We will perform identical assays utilizing EZH2 and FOXP3 domain mutants (Figure

7) to test the requirement of EZH2 and FOXP3 physical interaction for repressor function.

1.22 : ChIP assay: The mechanism of EZH2-mediated silencing is H3K27 trimethylation 

(H3K27me3) and chromatin compaction at target promoter loci. The presence and function of EZH2 at 

a particular promoter locus can be assayed using Chromatin Immunoprecipitation (ChIP) for the 

H3K27me3 histone “mark.” Utilizing FOXP3$EZH2 Treg cells, we will transduce WT or EZH2 mutant 

constructs and perform ChIP assay of the RORA and IRF4 promoter for the presence of H3K27me3 

marks, EZH2, and FOXP3. This animal line has been crossed to a mutant transgenic mouse line 

expressing the coxsackie adenovirus receptor (CAR) to allow efficient gene transduction in resting 

lymphocytes (26). Essential reagents include FOXP3$EZH2 mouse colony and adenoviral WT or EZH2 

mutant constructs (Figure 7). Our laboratory has published experience in ChIP and adenoviral 

transduction (1,20,26). We expect WT EZH2 but not mutants predicted to disrupt FOXP3-mediated 

recruitment to repress RORA and IRF4 through deposition of H3K27me3 histone marks.

1.3 The in vitro and in vivo functional relevance of EZH2 target gene repression.

Finally, we will test the significance of EZH2 target gene repression on Treg suppressor function

in vitro (1.31) and in vivo (1.32).

1.31 : The in vitro assay consists of co-culture of naïve FOXP3- CD4+ T cells (T responder cells)

with titrated amounts of Treg cells in 

the presence of conditions to stimulate 

T responder cell proliferation (anti-CD3, 

anti-CD28) (1). We will transduce 

FOXP3$EZH2 Treg cells with WT EZH2 

and EZH2 mutant constructs (Figure 7) 

to test dependence of in vitro 

suppressor function upon EZH2 and 

FOXP3 complex formation.

1.32 In vivo colitis assay: We will 

test in vivo function of FOXP3!EZH2

Treg cells in colitis prevention. We will Rbhi

utilize the CD45 transfer model of

 p<0.001 p<0.001

Figure 8: RORA and IRF4 are significantly upregulated in 

CD4+ lymphocytes isolated from Crohn’s lesions. The 

histograms represent reads per kilobase of transcript per 

million reads mapped (RPKM) for RORA and IRF4 in RNASeq analysis of CD4+ lymphocytes isolated from Crohn’s 

disease resection specimens or ileal biopsies of healthy age, 

sex match control subjects (n=12 subjects).

!

30

#

*p<0.05

20

10

0

Figure 9: FOXP3!EZH2 Treg cells do not suppress Rbhi mediated colitis. 

Recipient mice (n=4) of mutant Treg cells lost more weight (A, black 

histogram) and demonstrated severe colitis (B, representative histology from 

Rbhi transfer) when compared to recipients of WT Treg cells (n=4). Note goblet 

cell loss, lamina propria expansion and intense inflammatory cell infiltration. PercentWeight Loss
Contact PD/PI: Faubion, William A

Research Strategy Page 51

colitis, a model in which we have extensive experience, mimics well the phenotype of human IBD, and 

in which preclinical trials have translated well to human therapy (50-52). For the in vivo model, 

350,000 naïve CD4+ lymphocytes are transferred into immunodeficient RAGnull mice alone, or along 

with 100,000 FOXP3+ Treg cells. Our experience with this system is well published (52-55). We have

established that FOXP3!EZH2 Treg cells do not prevent colitis upon co-transfer with WT naïve CD4+ 

cells (Figure 9). We have developed a new model of inducible EZH2 deletion through a tamoxifeninducible CRE-mediated recombination system driven by the chicken beta actin promoter/enhancer. 

Upon cross with the EZH2 flox mouse, EZH2 deletion can be obtained in any mouse cell upon 

treatment with tamoxifen. We harvested Treg cells from this mutant animal (and WT control), and cotransferred mutant (or WT) Treg cells along with WT CD45Rbhi cells into RAGnull mice. All animals 

were treated with a 5-day tamoxifen injection protocol. At 6 weeks, RAGnull mice injected with mutant 

Treg cells exhibited extreme weight loss and colitis while RAGnull mice treated with control Treg cells 

appeared healthy (Figure 9). We will now transduce WT EZH2 or mutant constructs into FOXP3!EZH2

Treg cells to test the requirement for EZH2 to associate with FOXP3 for the prevention of murine IBD. 

Colitis severity will be assessed by disease activity index (DAI), blinded histology assessment, 

cytokine expression profile of T cells isolated from mesenteric lymph nodes, and cytokine assessment 

of colonic supernatants (28). We expect adenoviral delivery of the WT EZH2 to rescue in vivo function 

of FOXP3!EZH2 Treg cells. Furthermore, we expect that EZH2 domain mutants, disrupted in their 

capacity to complex with FOXP3 will not prevent colitis. This finding would indicate the critical nature 

of this complex to prevent inflammation.

Anticipated Results, potential pitfalls, and alternative approaches: The objective of this aim 

is to define the role for EZH2 in FOXP3-induced gene repression and subsequent Treg function. We 

expect EZH2 to complex with FOXP3 in a direct fashion (1.1). We expect WT EZH2 to repress RORA 

and IRF4, but only in the presence of FOXP3 (1.2). We expect the functional relevance of the EZH2 

and FOXP3 interaction to be in vitro suppression of T responder cells and colitis prevention (1.3). 

These exciting experiments will be the first to test a modifiable epigenetic mechanism in the treatment 

of IBD.

The first alternative hypothesis considered is an indirect rather than direct EZH2, FOXP3 

interaction through obligate EZH2 binding partners EED and Suz12 (35,36). To directly test this 

possibility we could perform siRNA knockdown of EED and Suz12, and repeat Co-IP assay for WT 

EZH2 and FOXP3. A second alternative hypothesis is that rather than a requirement for FOXP3, 

EZH2 utilizes additional mechanisms for target gene localization. Non coding RNAs are increasingly 

recognized to directly bind with chromatin modifying complexes and play a role in altered gene 

expression in many tissues and particularly cancer (56). Should EZH2 not require FOXP3 for target 

gene localization we will perform cross-linking and immunoprecipitation methodology combined with 

RNAseq to identify EZH2-interacting RNA. Functional knockdown of long noncoding RNA could reveal 

this potential function.

Figure 10 (AIM2): Our model is that a 

kinase (JAK2) downstream of an 

inflammatory signal (IL6) modifies 

EZH2 (Y641 phosphorylation) resulting 

in impaired HMT activity at FOXP3 

target genes (RORA, IRF4) and 

subsequent Treg cell dysfunction. We 

will use mutant mouse/cell lines and 

kinase inhibition to identify the 

mechanism(s) of EZH2 

phosphorylation (by Mass Spec, MS 

and WB) and resultant HMT function 

(HMT assay and in vitro/vivo assays of 

suppressor function).

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Aim 2: Inflammatory stimuli, such as IL6 lead to EZH2 phosphorylation and thereby disrupt the 

enzymatic activity of this epigenomic regulator.
Contact PD/PI: Faubion, William A

Introduction: In CD4+ T cells, inflammatory signals such as IL1", TNF#, and IL6 abrogate Treg 

cell development and function (2,16,57-61), while IL10 in a paracrine fashion enhances Treg function

(62). Indeed in support of our hypothesis, the requirement for EZH2 HMT activity to the maintenance 

of Treg cellular phenotype and the conversion of Treg cells to IL17-like T effector cells through IL6 

signaling have both recently been reported (6,63); yet the precise mechanisms governing this 

cellular conversion are lacking. The objective of this aim is to identify the mechanism(s) by which 

cytokine signaling pathways regulate EZH2 function in Treg cells using IL6 as the relevant model 

system. We will test the working hypothesis that IL6 signaling modulates Treg function through 

regulation of EZH2 HMT activity by phosphorylation. Our approach is an experimental pipeline that

consists of identifying potential posttranslational modifications through bioinformatics with confirmation 

from MS experiments, protein structure analyses using molecular modeling with validation through 

mutational screens, and functional assays to definitively test the hypothesis of Aim 2. The rationale for 

this aim is that we will fill a critical gap in our understanding of inflammation mediated modulation of

Treg function. The acquisition of such knowledge is crucial to the development of improved cell based 

strategies for human IBD based upon ex vivo manipulation of Treg responsiveness to inflammatory 

signals. We expect that the disruptive IL6 signaling pathway will entail phosphorylation of EZH2 by 

JAK2 inhibiting EZH2 enzymatic function. This finding is important, because it will allow for the first 

time, the development of novel approaches to Treg based therapy in IBD.

Justification, feasibility, and preliminary data: IL6 signaling disrupts Treg cell function. 

Previous work identifying the disruptive role of IL6 to Treg function has focused on an effect on T 

responder cells (2). We demonstrate that disrupted function can be dependent upon Treg cell intrinsic 

IL6 signaling. We utilized a mutant mouse strain with a spontaneous point mutation in the gene 

encoding the IL6R# resulting in a complete lack of protein (in collaboration with Dr. Chandrashekhar 

Pasare, UTSW, see letter). Using IL6R-deficient T responder cells and WT Treg cells, we are able to 

isolate the effect of IL6 signaling to Treg cells in the suppression assay. In Figure 11A, we

demonstrate significant disruption of the in vitro suppression assay attributed to IL6 signaling uniquely 

in Treg cells. Using an ELISA based assay specific for EZH2 (H3K27) HMT activity, we discovered 

that IL6 signaling significantly represses EZH2 HMT activity in both primary WT CD4+ 

lymphocytes and cell lines (Figure 11B). This repressive effect was only consistently inhibited by 

specific inhibition of JAK2, and only minimally affected by AKT or MAPK pharmacologic inhibition 

(Figure 11B, AZD1480 and data not shown). To address potential phosphorylation sites attributable to

IL6, we performed a bioinformatic analysis to predict posttranslational modification sites within 

EZH2 by compiling and statistically scoring results obtained using NetPhosk 2.0 (64), PhosphoSVM

Seq# pos NetPhos2.0 PhosphoSVM GPS3.0

Context Score Score Kinase Score Kinase

92;)

!"#$

&'()$

!!*

Figure 12: Results of linear motif analysis of EZH2 for predicted phosphorylation 

sites. The score represents the potential for phosphorylation. The higher the 

value, the more potential the residue is phosphorylated. Predicted site-specific 

kinase is listed in column right.

+3+4*56748!3+&!9.-!++&7*!:*

Figure 13: IP of EZH2 and complex 

partners Suz12 and EED (top panel). 

MS analysis of lysates obtained from 

cells treated with IL6 confirmed 

phosphorylation of Y641.

Research Strategy Page 52

! # 3000

*p<0.05 2000

1000

* *

0

Figure 11: (A) WT Treg cells were plated 

alone (Treg) or 1:1 with Tresp cells. The 

Tresp cells were from the IL6R mt mouse. 

Closed histograms show failure of WT 

Treg cells to suppress Tresp in the 

presence of 10ng IL6 (B) Treatment with 

IL6 (closed histograms) in primary T cells 

and cell lines inhibits HMT activity. HMT 

inhibition with 3-deazaneplanocin (DNZ) 

serves as a control. Inhibition of HMT 

activity by IL6 is completely abrogated 

upon treatment with the JAK2 inhibitor 

AZD1480 (*p<0.05).

HMT (units)

610-S IQRGSKKHL 0.992 0.75 MAPK 8 MAPK

639-S NEFISEYCG 0.942 0.75 MAPK 10 MAPK

664-S

641-Y 

KYMCSFLFN 0.002 0.62 MAPK

FISEYCGEI 0.014 0.53 JAK2

3 MAPK

1 JAK2

658-Y RGKVYDKYM 0.037 0.53 JAK2 7 JAK2

726-Y LFFDYRYSQ 0.026 0.53 JAK2 3 JAK2
Contact PD/PI: Faubion, William A

Research Strategy Page 53

well-characterized

phosphomimetics (Y to E) and

non-phosphorylatable

phosphorylatable Y641F mutant. Thus, given this strong preliminary 

data, in Aim 2 we will confirm the key modifications relevant to 

cytokine signaling and test the functional relevance of the 

biochemical assays through cellular assays and gene expression.

Research Design:

The strategy of Aim 2 is to identify functionally relevant post-translational modifications of EZH2 

downstream of IL6 through a mass spectrophotometry screen (2.1) followed by a point mutational 

analysis consisting of functional assays of enzymatic activity (2.2), gene regulation and Treg 

suppressor function (2.3). We use Y641 as a powerful example of feasibility and significance;

however we will systematically assess all 6 phosphorylation sites of the SET domain identified by the

mass spectrophotometry screen.

2.1 Establish the mechanism of modifications to the EZH2 SET domain.

The preliminary data (bioinformatic analysis and pharmacologic inhibitor screens) are supportive 

of the substantial role for JAK2 in regulation of EZH2 enzymatic activity. To confirm this mechanism,

we will stimulate primary Treg cells and cell lines (Jurkat and Hap-1) with IL6 (10ng/ml) in the

(65), and GPS 3.0 (66). We focused on putative phosphorylation sites within the enzymatically active 

SET domain (AA 605-729), as they should have clear impact on the function of this HMT. The 

demonstrated motifs are predicted MAPK and JAK2 phosphorylation sites (Figure 12) and 

confirmed by MS to be phosphorylated upon pull down of native EZH2 (Figure 13). One of the key 

residues modified by phosphorylation is Y641, which mutations have previously shown to alter human 

B cell function (67). We gained useful insight into how Y641 phosphorylation may affect the activity of 

EZH2 through molecular modeling. Briefly, we developed a homology-based model using the SET 

domain of EZH2, which had been previously crystallized in the absence of both the SAM cofactors 

and the target histone peptide. These two molecules, which are key for the full HMT activity were 

obtained by homology superimposition with the SET domain of EHMT1/G9a and SETD2; for both are 

known to have an EZH2-like enzymatic mechanism (60). Figure 14 demonstrates the modeled

aforementioned hydrogen bond (Figure 14, right panel). 

Combined these studies suggest that phosphorylation of this Y 

residue may regulate the histone methyltransferase activity of 

EZH2. We next performed biochemical experiments to test the 

validity of these data. We developed a novel system based on 

using a CRISPR/CAS-mediated EZH2KO cell in which the 

enzymatic activity of transfected WT EZH2 was directly compared 

with both the phosphomimetic (Y641E) and nonphosphorylated 

(Y641F) Y641 mutants. As shown in Figure 15, the phosphomimetic 

mutant Y641E is inactive when compared to WT or the nonmethodology. We find that the

Y641E mutation disrupts the

Figure 14: Molecular Modeling of EZH2 SET domain (blue), SAM cofactor (green), and the mutations (Y to F)

dimethylated K27 residue of histone protein (yellow). Note Y641E mutation, right.

150

* p<0.05

100

50

0

Figure 15: Using ELISA, we show 

reduction in HMT activity upon 

mutation of Y641 to the 

phosphomimetic, Y641E.

structure of the EZH2 

methyltransferase domain. 

Note Y641 engages in a 

hydrogen bond with the deprotonated $-Nitrogen of the 

dimethylated lysine that will be 

tri-methylated, (dash line and 

yellow arrow, Figure 14 left 

panel). Using in silico 

mutagenesis, we investigated 

the potential effects of 

phosphorylation, by using the

OD/hr/mg
Contact PD/PI: Faubion, William A

Research Strategy Page 54

presence of JAK2 modulation and measure phosphorylation of the EZH2 SET domain by mass 

spectrophotometry and western blot. The rationale for both outcome assessments is the enhanced 

data acquisition of MS balanced with the speed and low cost of western blot analysis. We have 

developed and optimized a rabbit polyclonal Phospho-Y641 antibody.

We will inhibit JAK2 in three ways: pharmacologic JAK2 inhibition (AZD1480), JAK2 siRNA 

treatment, and overexpression of a JAK2 dominant negative construct (Y792F) (68). Constitutively 

active JAK2 V617F (69) will be used as an important gain-of-function control. We will use primary 

cells from our recently developed FOXP3-specific, IL6R conditional KO (FOXP3ΔIL6Rα) as a relevant 

negative control. We expect IL6 to lead to phosphorylation of EZH2 and subsequent impairment of 

HMT activity through activation of JAK2. Furthermore, we expect the FOXP3ΔIL6Rα Treg cells to be 

resistant to EZH2 phosphorylation.

2.2 Test the functional significance of EZH2 SET domain phosphorylation.

IL6 signaling leads to both EZH2 phosphorylation and reduced HMT activity in our system 

(Figure 11 and 13); thus we will now establish causality of impairment of EZH2 HMT activity to EZH2 

phosphorylation. The outcome variable for the subsequent experimental set is IL6-induced impairment 

of HMT activity in nuclear extracts of test cells. We have generated a library of EZH2 mutant

constructs designed to mimic (S to D; Y to E) or prevent (S to A; Y to F) phosphorylation. We have 

established an ideal in vitro cell system in which to screen the HMT activity of EZH2 mutant 

constructs. Utilizing a haploid fibroblast cell line (HAP-1) (70) in which the CAS9 system has induced 

a frameshift mutation in exon 7 of human EZH2, we demonstrate significant reduction of HMT activity 

in the Y641E mutant (mimic phosphorylation) when compared to WT EZH2 control (Figure 15). We 

have produced our first candidate mutant (Y641E) adenovirus, which will be used to confirm functional 

relevance in primary T cells.

HMT assay will be run using nuclear extracts from FOXP3ΔEZH2 Treg cells upon adenoviral 

transduction of either WT-EZH2 or mutant EZH2. We expect that Y641F (unable to be phosphorylated

and widely noted to be hyperactive in human lymphoproliferative conditions) will function well in the 

presence of IL6 as measured by HMT activity. Conversely, we anticipate, the Y641E mutant to have 

an impaired HMT activity when compared to the WT construct due to the phosphomimetic properties 

of the glutamic acid substitution. Our team has extensive experience in mutational analyses to identify 

modifiable residues (20,71).

2.3 Establish the functional relevance of SET P-Y641-EZH2 within the whole cell system.

IL6 stimulation reduces HMT activity in nuclear extracts of primary lymphocytes (Figure 11B). 

Thus in this subaim we will extend our preliminary findings to whole cell assays of functional 

relevance including: 2.31) luciferase reporter and 2.32) in vitro suppression assays.

2.31 : Luciferase reporter assays using genomically integrated T cell lines. As we have done 

previously, we generated genomically integrated T cell lines to study EZH2-dependent events in a 

non-episomal system (3,20). Using the Jurkat FLP system we created two reporter cell lines with the 

RORA and IRF4 promoters genomically integrated in frame with a luciferase reporter. RORA and 

IRF4 are ideal targets as both are established targets of EZH2 (42,43) and upregulated in CD (Figure 

8). Using this system we will overexpress WT EZH2 or EZH2 Y641 mutants to test the dependence of 

IL6 induction of RORA/IRF4 on EZH2 Y641 phosphorylation.

2.32 : In vitro suppression assays. We will test the capacity of WT and mutant EZH2 constructs 

to rescue in vitro suppressor function in the setting of IL6. Utilizing adenoviral transduction of 

FOXP3ΔEZH2 Treg cells, we will introduce WT or EZH2 Y641 mutant constructs and perform 

suppression assays in the presence or absence of IL6. As EZH2 Y641E mimics phosphorylation 

downstream of IL6 signaling, we expect the Y641E mutant to be a loss of function mutation, even 

without IL6 stimulation. Loss of function would be evident in the de-repression of luciferase expression 

and the inability to suppress T responder cell proliferation. We expect the EZH2 Y641F mutant 

(unable to be phosphorylated) to be a gain of function mutation, evident in the presence of IL6. Gain 

of function in this context will be demonstrated through the repression of luciferase and retained 

capacity to suppress in the setting of IL6 stimulation.
Contact PD/PI: Faubion, William A

Research Strategy Page 55

Anticipated Results, potential pitfalls, and alternative approaches: We expect IL6 

stimulation to lead to phosphorylation of EZH2 through activation of JAK2 (2.1), and phosphorylation 

to lead to inactivation of HMT activity (2.2). We expect this disruption to be evident in the 

transcriptional activation of IRF4 and RORA (2.31) and impairment of Treg in vitro function (2.32). We 

consider here several alternative hypotheses. First, we recognize additional inflammatory pathways 

including the TNF superfamily receptors OX40, 4-1BB, TNFR1 and IL1 family receptors have been 

linked to Treg dysfunction (72). The experimental pipeline (MS screen and mutational analysis) is 

amenable to any cytokine signaling input and could be used in future mechanistic studies of cytokine 

regulation of EZH2. Furthermore, IL10 has been suggested to enhance Treg function; however the

mechanism of this effect is not well understood. We could explore the effect of IL10 on alternative

post-translational modifications and subsequent effect on EZH2 HMT activity. Secondly, while IL6 

stimulation clearly leads to SET domain phosphorylation there are other potentially important 

modifications and mechanisms to disrupt EZH2 HMT activity. Phosphorylation of EZH2 may lead to

alternative substrate specificity and complex formation for this HMT. In glioblastoma cell lines, EZH2

methylates Stat3 at the K180 position, and this methylation activity is enhanced through EZH2 serine

phosphorylation at S21 by AKT (11). To investigate this possible alternative outcome, we could isolate

the EZH2 complex upon treatment with IL6 to identify alternative binding partners and substrates. Our

collaborative team has extensive experience in the isolation and characterization of HMT complexes

should this be required (73).

Aim 3: Inhibition of the IL6 to EZH2 signaling pathway permits sustained Treg suppressive 

function in intestinal inflammation.

Introduction: It is established that FOXP3+ Treg 

cells prevent colitis more efficiently than they treat 

active colitis in animal model systems (74-76). 

Furthermore, loss of regulatory or even gain of proinflammatory function of FOXP3+ cells in human IBD 

is evident given the frequency and cytokine 

expression pattern of FOXP3+ cells in actively 

inflamed mucosa of IBD patients (1,77). The objective

of this aim is to perform a pre-clinical animal trial of 

Treg cells engineered to function in the inflamed 

intestine. We will test the working hypothesis that 

sustained EZH2 HMT activity in Treg cells permits 

Treg cellular function in the setting of active 

inflammation. Our approach will be in vivo assays of 

Treg suppression in the aforementioned T cell transfer

model of colitis. The rationale for this aim is that we 

will fill a gap in knowledge, without which we cannot 

understand the mechanism for sustained Treg function 

in the setting of active inflammation. This knowledge is 

critical to the development of improved cell based 

strategies for human IBD. When the proposed studies 

for Aim 3 have been completed, it is our expectation

that disruption of the IL6 to EZH2 signaling pathway in

Treg cells will lead to successful treatment of active colitis. Such a finding would be of importance, 

because current non-targeted, systemic anti-IL6 therapy has a poor safety profile related to 

impairment of epithelial cell homeostasis (78,79).

Justification, feasibility and preliminary data: Antibody blockade of the IL6R is effective in 

adult Rheumatoid Arthritis (RA) (80) and has been used in very early onset IBD (81). Similarly, 

inhibition of Jak1/3 is effective in adult RA (82) and is undergoing advanced clinical trials in IBD (83). 

Infrequent occurrences of intestinal perforation in RA patients suggest a requirement for cell subtype 

specific therapy over the pan-inhibition of this pleiotropic cytokine (78,79). In this aim, we look to 

inhibit this pathway uniquely in Treg cells in vivo to treat active colitis. Through RNA-Seqmethodology

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induced phosphorylation) leads to colitis. We will test this 

model through adoptive transfer of WT and mutant Treg 

cells into the FOXP3ΔEZH2 mouse. Furthermore, we will 

test the capacity for Treg cells mutant in the IL6 signaling 

pathway or the EZH2 protein to function in the setting of 

active colitis through TREATMENT rather than prevention 

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Contact PD/PI: Faubion, William A

Research Strategy Page 56

using CD4+ T cells 

isolated from Crohn’s 

lesions, we have

identified marked 

upregulation of IL6 

induced signaling 

networks (Figure 17A). 

IL6 signaling leads to 

phosphorylation of EZH2 

and impaired HMT 

activity. Thus, it is 

important to note 

significant variation in the 

expression of EZH2 

targets between health 

and disease in the same 

cells (Figure 17B). 

Furthermore, the 

significant majority 

(379/457 target genes) is 

upregulated, a finding 

suggestive of impaired 

EZH2 HMT function 

(Figure 17B). Congruent 

with these observations, 

microarray data obtained

from FOXP3$EZH2 Treg

cells demonstrate marked

upregulation of

proinflammatory pathways regulated by IL6 signaling (Figure 17C). Finally, we demonstrate that 

inhibition of EZH2 (DZNep 1mg/kg ip) HMT activity significantly exacerbates DSS colitis (Figure 17D, 

E). Thus, we have observed IL6 to inhibit EZH2, a protein that itself is a negative regulator of IL6- 

induced proinflammatory pathways relevant to human IBD. Moreover, pharmacologic inhibition of 

EZH2 (mimicking the IL6 effect) worsens DSS induced colitis. We now propose to treat active colitis 

through genetically engineered Treg cells. Our model system will be the CD45Rbhi transfer model of 

colitis (discussed in Aim 1 and demonstrated in Figure 9), and Treg cell therapy will be applied to treat 

rather than prevent active inflammation. We have recently utilized this precise methodology to 

demonstrate the enhanced function of murine Treg cells engineered to maintain an active FOXP3 

promoter locus in the inflammatory setting (84). Focusing now on the IL6 to EZH2 pathway, we will 

perform identical experiments (genetic manipulation of Treg cells and co-transfer in the Rbhi mediated 

model of colitis) to utilize Treg cellular therapy in active colitis.

Research Design:

The general strategy of Aim 3 is to utilize adoptive transfer systems and genetically engineered 

Treg cells to demonstrate the capacity for Treg cells to function in the inflamed intestine and treat 

active colitis. We will use the naïve T cell into RAGnull colitis model as our model system, and we will 

genetically engineer Treg cells using adenoviral transduction (3.1) and TALEN methodology (3.2). Our 

efficient use of adenoviral constructs has been addressed above, and our genetic editing will be 

performed in collaboration with the Genetics and Model Systems Core with the Center for GI 

Signaling, Mayo Clinic (Dr. Stephen Ekker, see letter of collaboration).

3.1 Treatment of active colitis with EZH2 mutant cell lines.

We have demonstrated loss of Treg suppressor function in vitro upon treatment of co-culture 

assays with IL6. We now will test the effect of therapy directed to this pathway through in vivo assays 

of Treg function. We will use the IL6R"-deficient mice as the donor of Treg cells. We will harvest Treg

cells from the FOXP3$IL6R" mouse line and test the in vivo regulatory capacity of these cells to treat

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Figure 17: EZH2 controls IL6 inflammatory pathways. (A) Heat map of RNAseq of CD4+ cells 

isolated from Crohn’s lesions. The heat map is the IL6 pathway and shows marked variance 

between control subjects (CTR) and disease (Dz). (B) Volcano plot of relative expression of EZH2 

targets comparing DZ to CTR. Red dots show significant variance. (C) Heat map of microarray 

from FOXP3!EZH2 Treg cells vs control. Red cells show upregulation of genes. Note (arrow) genes of 

the Th17 and Th1 pathway. (D and E) 2% DSS colitis in WT mice treated with the EZH2 inhibitor 

DZNep (1mg/kg) at Day 2 and 5. Colon length and DAI show severe colitis upon EZH2 inhibition 

(n=8).

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Contact PD/PI: Faubion, William A

Research Strategy Page 57

colitis. As per our published data (84), we will inject titrations of 100-300,000 cells of WT or IL6R" 

mutant Treg cells into recipient RAGnull animals with established colitis (4 weeks post T effector cell 

transfer). We expect IL6R" mutant cells to robustly treat colitis, as compared to WT Treg cells. We 

will rescue FOXP3$EZH2 Treg cells with EZH2 WT, Y641E, or Y641F mutant constructs. We expect the

EZH2 Y641F (gain of function) but not the EZH2 Y641E (loss of function) to effectively treat 

established colitis.

3.2 Treatment of active colitis with genetically edited cells.

Finally, we will test ex vivo cellular therapy with clear translational potential to human IBD. Our 

department of Biochemistry and Molecular Biology has extensive experience with transcription 

activator-like effector nucleases (TALENs) and has recently published successful genetic editing of 

primary T cells (85,86). We will isolate WT Treg cells from C57/BL6 mice and perform genetic 

editing/deletion of IL6R" using TALENs (see letter of collaboration). As per previous study, 20 mcg of

TALEN construct (left arm and right arm) and pEGFP-N1 (Clontech, Mountain View, CA, USA) DNAs

will be transiently nucleofected by electroporation into primary Treg cells (RFP+). Forty-eight hours 

after nucleofection, GFP-expressing cells will be selected by fluorescence-activated cell sorting.

Deletion of the IL6R" will be confirmed by flow cytometry. The TALEN edited IL6R" null cells will be 

expanded using Treg ex vivo cell expansion methods we have optimized (87), and injected into 

recipient RAGnull animals with established colitis as above. We expect genetic editing ex vivo of WT

Treg cells to enhance suppressor function in the setting of active colitis, and this finding to thus 

provide a clear roadmap for translational cell therapy studies in patients with IBD.

Anticipated Results, potential pitfalls, and alternative approaches: We expect in 3.1 robust

treatment of active colitis with FOXP3+ Treg cells impaired in IL6R to EZH2 signaling either at the 

level of the receptor (IL6R" KO Treg cells) or EZH2 (EZH2 Y641F mutant) when compared to WT 

Treg cells. We expect in 3.2 genetic editing ex vivo of WT Treg cells to enhance suppressor function

in the setting of active colitis, and this finding to provide pre-clinical data for translational cell therapy 

studies in patients with IBD. As the Y641F mutant in whole lymphocyte populations has been 

associated with lymphoma, this particular mutation uniquely within the Treg subset will require 

extensive analysis. To test the long-term behavior of mutant Treg cells, we could perform rescue 

experiments of the scurfy mouse (no functional Treg cells) to study function and toxicity. A second 

alternative hypothesis is that impaired Treg function in the setting of inflammation results not from 

intrinsic Treg dysfunction but the resistance to Treg suppressive mechanisms by T effector cells. This 

hypothesis is readily testable using our current reagents through the treatment of colitis induced by

IL6R" mutant T effector cells with WT Treg cells. We can now dissect the role for EZH2 in any cell 

population using tamoxifen-inducible Cre-ER(T) mutant mouse under the promotional control of actin.

A third alternative hypothesis is that EZH1, not EZH2 is the primary target of IL6 signaling in Treg 

cells. Beyond initiation of the H3K27me3 mark (EZH2-containing Polycomb Repressor Complex 2 

function), maintenance of the mark by the EZH1 HMT may well be involved in maintained Treg 

function. Given the strength of the preliminary data and the phenotype of the conditional EZH2 KO 

mouse, we have necessarily chosen to focus on EZH2; however our laboratory and collaborative 

partners have the necessary tools and experience to dissect additional HMT pathways should these 

investigations become necessary.

Conclusion: We are studying an innovative membrane to nucleus signaling pathway connecting 

environmental inflammatory signals (IL6R) to cell differentiation machinery (EZH2) responsible for 

Treg cell fate and function. Our work has clear biomedical relevance to patients with IBD. Moreover, 

our work represents a continuum beginning with basic mechanisms of kinase regulation of EZH2 (this 

proposal) to the ex vivo manipulation of Treg cells for cell therapy of IBD. The work is significant as it 

should lead to first-in-man studies of engineered Treg cells in human IBD.

SA3

SA2

SA1

Aims Year 1 Year 2 Year 3 Year 4 Year 5
Contact PD/PI: Faubion, William A

PROGRESS REPORT PUBLICATION LIST:

AI89714 Competitive Renewal Publication List:

Original research publications emanating directly from experiments designed in previous aims:

1. Xiong, Y., Khanna, S., Grzenda, A.L., Sarmento, O.F., Svingen, P.A., Lomberk, G.A., Urrutia, R.A., 

Faubion, W.A., Jr. (2012). Polycomb antagonizes p300/CREB-binding protein-associated factor to 

silence FOXP3 in a Kruppel-like factor-dependent manner. Journal of Biological Chemisry, 

287(41):34372-85. PMCID: PMC3464543. (AIM1 and AIM3)

2. Xiong, Y., Svingen, P.A., Sarmento, O.O., Smyrk, T.C., Dave, M., Khanna, S., Lomberk, G.A., Urrutia, 

R.A., Faubion, W.A., Jr .(2014) Differential coupling of KLF10 to Sin3-HDAC and PCAF regulates the 

inducibility of the FOXP3 gene. American Journal of Physiology Regulatory, Integrative and 

Comparative Physiology, 307(6):R608-20. PMCID: PMC4166759 (AIM1)

3. Papadakis, K.A., Krempski, J., Reiter, J., Svingen, P., Xiong, Y., Sarmento, O.F., Huseby, A., Johnson, 

A.J., Lomberk, G.A., Urrutia, R.A., Faubion, W.A.. (2015) Krüppel-like factor KLF10 regulates 

transforming growth factor receptor II expression and TGF-β signaling in CD8+ T lymphocytes, 

American Journal of Physiology Cell Physiology, 308(5):C362-71. PMCID: PMC4346734

4. Sarmento, O.F., Svingen, P.A., Xiong, Y., Xavier, R.J., McGovern, D., Smyrk, T.C., Papadakis, K.A., 

Urrutia, R.A., Faubion, W.A..(2015). A novel role for KLF14 in T regulatory cell differentiation. Cellular 

and Molecular Gastroenterology and Hepatology, 1(2):188-202. PMCID: PMC4349492.

5. Mathison, A., Grzenda, A., Lomberk, G., Velez, G., Buttar, N., Tietz, P., Hendrickson, H., Liebl, A., 

Xiong, Y.Y., Gores, G., Fernandez-Zapico, M., Larusso, N.F., Faubion, W. , Shah, V.H., Urrutia, R.. 

(2013) Role for Kruppel-like transcription factor 11 in mesenchymal cell function and fibrosis. PLoS 

One. 8(9):e75311. PMCID: PMC3775729. (AIM2)

6. Okou, D.T., Mondal, K., Faubion, W.A. , Kobrynski, L.J., Denson, L.A., Mulle, J.G., Ramachandran, D., 

Xiong, Y.,Svingen, P., Patel, V., Bose, P., Waters, J.P., Prahalad, S., Cutler, D.J., Zwick, M.E., 

Kugathasan, S. (2014) Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family 

With Inflammatory Bowel Disease. Journal of Pediatric Gastroenterology and Nutrition, 58(5):561-8. 

PMCID: 4277865. (AIM2)

7. Rahman, M.K., Offord, C.P., Khanna, S., Ford, G.C., Perrson, X.M., Svingen, P.A., Xiong, Y., Bajzer, 

Z., Faubion, W.A. .(2013) Regulatory T cell kinetics in the peripheral blood of patients with Crohn's 

disease. Human Immunology, 74(2):145-50. PMCID: PMC4224946 (AIM2)

8. Grzenda, A., Lomberk, G., Svingen, P., Mathison, A., Calvo, E., Iovanna, J., Xiong, Y., Faubion, W. , 

Urrutia, R. (2013) Functional characterization of EZH2beta reveals the increased complexity of EZH2 

isoforms involved in the regulation of mammalian gene expression. Epigenetics and Chromatin, 

6(1):3. PMCID: PMC3606351. (AIM3)

9. Mills, L.D., Zhang, L., Marler, R., Svingen, P., Fernandez-Barrena, M.G., Dave, M., Bamlet, W., 

McWilliams, R.R., Petersen, G.M., Faubion, W, Fernandez-Zapico, M.E.. (2014) Inactivation ofthe

Transcription Factor GLI1 Accelerates Pancreatic Cancer Progression. Journal of Biological Chemistry, 

289(23):16516-25. PMCID: PMC4047418. (AIM1 and AIM3)

10. Dave, M., Hayashi, Y., Gajdos, G.B., Smyrk, T.C., Svingen, P.A., Kvasha, S.M., Lorincz, A., Dong, H.,

*Faubion, W.A., Jr, *Ordog, T. (2015) Stem cells for murine interstitial cells of cajal suppress cellular 

immunity and colitis via prostaglandin e2 secretion. Gastroenterology,148(5):978-90. PMCID: 

PMC4409492 (* Co-corresponding authors)

Collaborative projects not directly related to prior aims:

1. Li, H., Chan, L., Bartuzi, P., Melton, S.D,, Weber, A., Ben-Shlomo, S., Varol ,C., Raetz, M., Mao, X., 

StarokadomskyyP,roPg.

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Contact PD/PI: Faubion, William A

Progress Report Publications List Page59

Esko, T., Metspalu, A., Kumar, V., Faubion, W.A., Yarovinsky, F., Hofker, M.,Wijmenga, C., Kracht, 

M, Franke, L., Aguirre, V., Weersma, R.K., Gluck, N., van de Sluis, B.,Burstein, E.(2014). Copper 

metabolism domain-containing 1 represses genes that promote inflammation and protects mice from 

colitis and colitis-associated cancer. Gastroenterology, 147(1):184-195.e3.

Epub 2014 Apr 13. PubMed PMID: 24727021; PubMed Central PMCID: PMC4086320.

2. Kauder, S.E., Santell, L., Mai, E., Wright, L.Y., Luis, E., N'Diaye, E.N., Lutman, J., Ratti, N., Sa, 

S.M., Maun, H.R., Stefanich, E., Gonzalez, L.C., Graham, R.R., Diehl, L., Faubion, W.A.. Jr., Keir, 

M.E., Young, J., Chaudhuri, A., Lazarus, R.A., Egen, J.G. (2013). Functional consequences of the 

macrophage stimulating protein 689C inflammatory bowel disease risk allele. PLoS One, 

23;8(12):e83958. PMCID: PMC3884107.

3. Dave, M., Papadakis, K.A., Faubion, W.A., Jr. (2014). Immunology of inflammatory bowel

disease and molecular targets for biologics. Gastroenterology Clinics of North America., 43(3):405- 

24. PMID:25110250.

4. Faubion, W.A., Jr, Fletcher, J.G., O'Byrne, S., Feagan, B.G., de Villiers, W.J., Salzberg, B., Plevy, 

S., Proctor, D.D., Valentine, J.F., Higgins, P.D., Harris, J.M., Diehl, L., Wright, L., Tew, G.W., Luca,

D., Basu, K., Keir, M.E. (2014). EMerging BiomARKers in Inflammatory Bowel Disease (EMBARK) 

study identifies fecal calprotectin, serum MMP9, and serum IL-22 as a novel combination of 

biomarkers for Crohn's disease activity: role of cross-sectional imaging. American Journal of 

Gastroenterology,108(12):1891-900. Epub 2013 Oct 15. Erratum in: Am J Gastroenterol. 2014 

May;109(5):780. PubMed PMID: 24126633.

5. Cayatte, C., Joyce-Shaikh, B., Vega, F., Boniface, K., Grein, J., Murphy, E., Blumenschein, W.M., 

Chen, S.,Malinao, M.C., Basham, B., Pierce, R.H., Bowman, E.P., McKenzie, B.S., Elson, C.O., 

Faubion, W.A., Malefyt Rde, W., Kastelein, R.A., Cua, D., McClanahan, T.K., Beaumont, M. (2012). 

Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease. Clinical 

and translational Gastroenterology, ;3:e10. PubMed Central PMCID: PMC3365674.

6. van Driel, B,, Liao, G., Romero, X., O'Keeffe, M.S., Wang, G., Faubion, W.A., Berger, S.B., 

Magelky, E.M., Manocha, M., Azcutia, V., Grisham, M., Luscinskas, F.W., Mizoguchi, E., de

Waal Malefyt, R., Reinecker, H.C., Bhan, A.K., Wang, N., Trehorst, C. (2012) Signaling lymphocyte 

activation molecule regulates development of colitis in mice. Gastroenterology, 143(6):1544- 

1554.e7. PubMed Central: PMCID: PMC3578298.
Contact PD/PI: Faubion, William A

Vertebrate Animals Page60

VERTEBRATE ANIMALS:

The mice used in this proposal will be from protocols approved by the Mayo Clinic Institutional Animal Care 

and Use Committee (IACUC). Mayo Clinic assures strict compliance with all federal regulations and guidelines 

involving the use of laboratory animals in biomedical research. Animal use assurances have been filed with the 

IACUC and the NIH. These procedures are closely adhered to and monitored within the institution. All 

protocols presently being conducted and those proposed, regardless of funding sources, are reviewed and 

must be approved before their initiation in accordance with these assurance statements. All protocols are 

active for 3 years after the approval date and adhere to Mayo Clinic and NIH policies on the humane care of 

animals. All animals will be maintained in an Association for Assessment and Accreditation of Laboratory 

Animal Care–accredited and centralized facility located in the Guggenheim Building.

1. Provide a detailed description of the use of animals in the research. Identify species, strains, ages, sex, 

and numbers of animals to be used.

This proposal will use the following mouse lines: 1. FOXP3ΔEZH2 2. B6129S-Tg(Foxp3-EGFP/cre)1aJbs/J

3. Foxp3tm9(EGFP/cre/ERT2)Ayr/J 4. FOXP3ΔIL6R 5. C57/BL6CAR 6. C57BL/6-Foxp3tm1Flv/J (FOXP3 RFP mouse) 7.

B6.129S7-Rag1tm1Mom/J 8. C57/BL6 EZH2flox/flox

These animals are currently being bred in our lab under IACUC# A31013. Below we iterate the animal 

requirements by experimental protocol. Total anticipated animal requirements are listed below each protocol. 

1.1, 2.1, and 2.2: Co-IP, PLA, and HMT assay of WT and mutant constructs nucleofected or transduced into 

WT CD4+ lymphocytes, IL6Rα mutant Treg cells, and FOXP3ΔEZH2 Treg cells.

Each spleen provides 4x106 naïve CD4+ T cells and 1x105 Treg cells. In review of the recent conduct of similar 

experiments generating preliminary data, our laboratory utilizes approximately 3-5 WT donor mice per week 

(approximately 150 per year), 2 IL6Rα mutant mice (approximately 100 per year), and 2 FOXP3ΔEZH2 mice 

(approximately 100 per year). We have performed experiments on both male and female WT mice with similar 

results thus plan to use both. Age is 4-6 weeks.

Total WT=150/yr

Total IL6Rα mutant=100/yr 

Total FOXP3ΔEZH2=100/yr

1.2 : ChIP assay post viral transduction of mutant contructs into FOXP3ΔEZH2 mice. We perform ChIP assay on 

transduced cells approximately 2x/month, or approximately 25 mice per year. Gender and age asabove.

Total FOXP3ΔEZH2=25/yr

1.3 , 2.3, 3.1, and 3.2: In vitro suppression assay and CD45Rbhi transfer model. The in vitro suppression model 

requires approximately 1x105 Treg cells per assay, performed in triplicate (acquired from approximately 3 

spleens). One donor WT animal is all that’s required to provide the T responder cells. Given at least three 

mutant EZH2 constructs to test and the requirement to perform the experiment in triplicate to maintain scientific 

integrity, we anticipate using 27 FOXP3ΔEZH2 mice in protocol 1.3 (3 spleens*3 constructs*triplicate=27) and 9 

WT mice (1 spleen*3*triplicate=9).

Total FOXP3ΔEZH2=27

For the in vivo colitis experiments, one requires 4 B6.129S7-Rag1tm1Mom/J recipients per treatment group. This 

number of mice was calculated based upon a one-way ANOVA analysis to allow 90% power when the 

differences between colitis are 1.25 standard deviations more than the controls. Given rescue with at least 

three constructs to test and the requirement to perform the experiment in triplicate to maintain scientific 

integrity, we anticipate using 36 RAGnull recipients and 10-15 donor mice for a total of approximately 50 mice in 

protocol 1.3. Similarly for the active colitis treatment protocol of 3.2, we will rescue RAGnull recipients with IL6R 

mutant Treg cells or FOXP3ΔEZH2 Treg cells transduced with three EZH2 constructs. Using the same 

calculations and justification as above, each colitis permutation requires 4 RAGnull recipients, repeated 3 

times=12 total RAGnull animals. As protocol 3.2 tests 4 Treg variants, we will require 48 total RAGnull animals.

The addition of 10-15 donor mice to the 48 total brings the total anticipated animal requirements to 

approximately 65 animals. 

Total RAGnull mice: 84 mice

1 
Contact PD/PI: Faubion, William A

2

Vertebrate Animals Page61

Total IL6R mutant donor mice: 10 

Total FOXP3ΔEZH2 donor mice: 30

Neonatal transfer experiments of 3.1: Litters of mouse pairs predicted by genotype to produce FOXP3ΔEZH2

pups will be injected ip with 1x106 WT Treg cells labeled with RFP (C57BL/6-Foxp3tm1Flv/J donor mice).

Experience demonstrates 1-2 appropriate FOXP3ΔEZH2 pups per litter; thus we will need to perform this 

experiment on 3 litters, requiring 12 FOXP3 RFP donor mice.

We will also need to maintain breeding pairs to continuously generate the lines being used in the experiments. 

The breeding of the FOXP3ΔEZH2 mouse is intense, as CRE+, heterozygous EZH2WT/flox mice need to be bred to 

the C57/BL6 EZH2flox/flox mouse to produce the appropriate genotype (typically 20% of any litter). This is the 

primary rationale to the generation of the tamoxifen inducible FOXP3ΔEZH2 mouse line. These adult animals, 

upon tamoxifen induction of CRE, will serve as donors of EZH2-deficient Treg cells. For every 6 animals used 

in an experiment, typically 1 breeder pair is required.

Thus total mouse requirements by strain:

WT=150/yr * 5yr=750 

WT breeding=125 

IL6mt=100/yr*5=500 

IL6mt breeding=83

FOXP3ΔEZH2=100/yr*5=500

FOXP3ΔEZH2 breeding=83 

RAGnull=84

RAGnull breeding=14

TOTAL=2139 mice over the 5 year period of the grant (428 mice per year).

All newborn animals with the incorrect genotype will be euthanized before they are 3 weeks old, unless they 

can be used for breeding. The health of the animals will be monitored on a regular basis. Any animals showing 

signs of excessive stress, pain, or discomfort during the treatment period will be euthanized as described 

below. At the end of the treatment cycles, colons will be excised and embedded for histological analysis of 

inflammation.

2. Justify the use and number of animals and choice of species with additional justification if animals are 

in short supply or are costly or if you plan to use large numbers.

As principal investigator, I have determined, by means of the following sources, searches, or methods, that 

alternatives to the procedures which may cause animal pain or distress proposed in this protocol are not 

available and that this protocol does not unnecessarily duplicate previous experiments. Sources searched 

include: Medline Current Research Information Services (CRIS) Animal Welfare Information Center (AWIC - 

National Agriculture Library) PUBMED. The key words that were used in the search (e.g., MESH headings): 

animal model; colitis; FOXP3; IL6; EZH2. We covered the years 1980 – 2014. Mice are required for the above 

experiments as there is no safe human options or in vitro options. The complex questions regarding the 

development of Treg cells in the intestinal milieu cannot be answered in vitro. The justification of animal 

numbers is above with experimental procedures.

There is currently no other experimental system that looks at epigenetic changes resulting in colitis prevention 

that is comparable to the combination of animals brought together in these studies. Mice are needed because 

the focus of the project is to determine the role of EZH2 in the development of coitis. These studies cannot be 

performed without a few key animal models with selective gene deletions. We will limit the number of mice 

required. To maintain breeding of the various animals, as well as longitudinal studies, we estimate it will be 

necessary to carry approximately 85 cages of mice on a regular basis (5 adult, same gender mice/cage).

3. Provide information on veterinary care for the animals.

Animals are housed in a pathogen-free laminar flow facility located in the Mayo Clinic Animal Facility on the 

19th and 20th floors of the Guggenheim Research Building. The animal facility has controlled temperature, 

humidity and lighting. Three full-time veterinarians certified in laboratory animal medicine and veterinary 

pathology will oversee all aspects of animal care in this facility. Beyond the provision of food, water and
Contact PD/PI: Faubion, William A

3

Vertebrate Animals Page62

housing (that is inspected daily), no specific veterinary care is anticipated. The animal care facilities and use 

program meets all federal regulations and guidelines. Mayo Foundation is registered with the USDA (41-R006) as an animal research facility and maintains an NIH animal assurance statement (A3291-01) with the 

Office of Laboratory Animal Welfare.

4. Describe procedures for ensuring that discomfort, pain, and injury will be limited to what is unavoidable. 

Describe the use of analgesic, anesthetic, tranquilizing drugs, and restraining devices to minimize 

discomfort, distress, pain, and injury.

Mayo Clinic strives to ensure that the institutional facilities and procedures adhere in all respects to USDA 

regulations and NIH guidelines for care and use of laboratory animals. Investigators are required to administer 

appropriate analgesics to all animals associated with a procedure that would normally require pain medication 

in humans. The mice are cared for in a clean, climate-controlled environment, with a controlled light-dark cycle. 

They are fed and watered daily, and cages cleaned weekly. Experiments are ended at pre-defined time points 

OR when animals have lost more than 20% of the starting body weight or exhibit a moribund appearance.

Animals are weighed and examined by the study team daily while in study. Mice will be maintained in 

accordance with the applicable portions of the Animal Welfare Act and the DHHS Guide for the Care and Use 

of Laboratory Animals.

During adoptive cell transfer, mice are briefly restrained in a clean, warm plexiglass cylinder to allow safe 

transfer of cells by tail vein injection. Mice are treated with Tylenol overnight following adoptive transfer.

5. Describe any euthanasia method to be used and the reasons for its selection. State whether this 

method is consistent with the recommendations of the American Veterinary Medical Association 

Guidelines on Euthanasia. If not, justify not following the recommendations.

Requests to perform euthanasia are reviewed and approved by our IACUC before initiation. Euthanasia may 

be performed as required by protocol to terminate a study, or as a means to relieve pain or distress that cannot 

be alleviated by analgesics, or if deemed necessary by the consulting veterinarian. We utilize CO(2) inhalation 

for euthanasia for the following reasons. (1) The rapid depressant, analgesic, and anesthetic effects of CO2 

are well established. (2) Carbon dioxide is readily available and can be purchased in compressed gas 

cylinders. (3) Carbon dioxide is inexpensive, nonflammable, nonexplosive, and poses minimal hazard to 

personnel when used with properly designed equipment. This method is consistent with the recommendations 

of the Panel on Euthanasia of the American Veterinary Medical Association.
Contact PD/PI: Faubion, William A

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Contact PD/PI: Faubion, William A

Letters Of Support Page 68

William Faubion, M.D. 

Mayo Clinic

June 18, 2015 

Dear Bill,

I am happy to provide this letter of collaboration in support of your R01 proposal entitled “Inflammatory cascades disrupt Treg function through epigenetic mechanisms.” As the director of the Genetics 

and Model Systems Core, my passion has been to develop genetic editing tools as a service line for our 

investigators engaged in the NIH-funded Center for Cell Signaling, Mayo Clinic. As we discussed, we 

have deep experience in several tools that will be of use in your system including CRISPRs, TALENs, 

and the Sleeping Beauty and Tol2 transposon systems. Your choice to utilize TALENs to edit primary T 

cells makes sense given our published experience in the genetic editing of lymphoid cells lines with this 

system. We are also ready to help you try other tools such as the rapidly developing CRISPR/Cas9 systems as they become available. I look forward to our collaboration, particularly as it relates to the genetic editing of WT lymphocytes and the treatment of active colitis with Treg cellular therapy.

I wish you every success with your application and look forward to continuing collaboration on this interesting project.

Sincerely,

Stephen C. Ekker, Ph.D. 

Professor of Biochemistry and Molecular Biology 

Director, Mayo Addiction Research Center

Editor-in-chief, the Zebrafish journal 

Stephen C. Ekker, Ph.D.

Department of Biochemistry &Molecular Biology 

200 First St SW

1342C Guggenheim 

Rochester, MN 55905
Contact PD/PI: Faubion, William A

Letters Of Support Page 69

Chandras hekhar Pas are, P h. D.

Louise W. Kahn Scholar in Biomedical Research 

Associate Professor, Dept of Immunology

UT Southwestern Medical Center 

5323 Harry Hines Boulevard 

Dallas, TX 75930-9093

June 18, 2015

William A. Faubion, M.D.

Division of Gastroenterology and Hepatology 

Assistant Professor of Medicine

Mayo Clinic

200 First Street, SW 

Rochester, MN 55905

Dear Bill,

I am happy to provide this letter of collaboration in support of your R01 

proposal entitled “Inflammatory cascades disrupt Treg function through 

epigenetic mechanisms.” As we recently discussed in person, I am looking 

forward to our collaboration on the role of IL6 signaling and Treg function. As 

you know, we have developed an animal line with a spontaneous point 

mutation in the IL6 receptor leading to an absence in functional protein. I 

think this mouse line will be of interest to you, as you develop your assays of 

EZH2 post-translational modification downstream of IL6, and the treatment 

protocols of active colitis. We have been happy to provide fresh cells for 

your studies and look forward to the development of a breeding colony in 

your laboratory. Congratulations on your proposal and I look forward to our 

continued collaboration.

Sincerely,

Chandrashekhar Pasare, Ph.D.
Contact PD/PI: Faubion, William A

Letters Of Support Page 70

MAYO CLINIC

Mayo Clinic

200 First Street SW 

Rochester, MN 55905

Thomas C. Smyrk, M. D.

Division of Anatomic Pathology

June 18, 2015

William A. Faubion, M. D. 

Gastroenterology and Hepatology 

Mayo 9East

Dear Bill:

I am happy to provide this letter of collaboration in support of your ROl proposal entitled 

"Inflammatory cascades disrupt Treg function through epigenetic mechanisms." In my role as an 

expert GI pathologist, I look forward to our continued collaboration as it relates to interpretation of colitis 

in your murine models. We have used our blinded histologic scoring assay successfully in our recent 

collaborative papers. I look forward to our continued work in your new studies ofthe treatment of active 

colitis with Treg cellular therapy. Congratulations on your proposal and I look forward to our continued 

work together,

Sincerely,

Thomas C. Smyrk, M.D.

TCS/cjh
Contact PD/PI: Faubion, William A

Letters Of Support Page 71

Raul Urrutia, M.D. 

Chair, Gastroenterology Research Unit 

Professor of Biophysics and Medicine 

Guggenheim Building 10-42C 

Mayo Clinic College of Medicine

Rochester, MN 55905 

June 18, 2015 

Dear Bill,

As you know, my habit is not to write support letters for NIH proposal unless I really believe that 

they have the potential to reach the outstanding category.

For this reason, it is with great enthusiasm that I put my pen to paper to express my excitement 

about this proposal. I have read it carefully and I believe that you focus on a remarkably exciting 

are of research, which is currently underrepresented though of great biomedical significance. 

This proposal logically addresses critical mechanistic questions of high implications, and the 

data provided indicate that it is highly feasible. Therefore, I congratulate you for your efforts.

Noteworthy, however, I would also like to complement this letter by expressing that it has been a 

pleasure for our laboratory to collaborate with you on the epigenetics, a subject on which my 

laboratory has made significant discoveries. We have also enjoyed our discussions and your 

presentations and open sharing of the data. Therefore, I assure you that you can count with all 

the conceptual framework, expertise, reagents, personnel and equipment from my laboratory to 

successfully help to achieve the goals outlined by you in the grant. Specifically, our work on 

EZH2 is in line with your proposed investigations. As our previous collaborative work suggests, 

I think your investigation into the regulation of this 3MeK27 writer HMT and immune cell 

function is mechanistically insightful and biomedically significant. As always, you have full 

access to our extensive collection of constructs, inhibitors, animal models, and other reagents 

you will find useful in your work. In particular, our previous work on AKT, STAT3, and EZH2 

led to a large inventory of reagents (inhibitors, gain of function mutants, etc) relevant to your 

work. Additionally, I am happy to provide expertise in the use of our protein chromatography 

system as you look into the EZH2, FOXP3 protein complex.

Lastly, the most important aspect of your proposal, I believe is that it will generate fundamental 

data, which can be applied to other systems in addition to your area of current focus. Therefore, 

this is what I call, a “proposal with a significant added value” which can benefit more areas than 

the one studied in the grant. 
Contact PD/PI: Faubion, William A

Letters Of Support Page 72

For all these reasons, I am looking forward to our collaborations, discussions, and planning of 

additional experiments. In simple words, my commitment to this proposal is the strongest that I 

can express.

Good Luck with your thoughtful and exciting experiments and I look forward to continuing with 

our already ongoing fruitful interactions. 

Best Regards,

Raul Urrutia, MD 

Professor of Biophysics and Medicine 

Director, Epigenetics and Chromatin Dynamics Laboratory 

Director, Epigenomics Education, Mayo Clinic 
Contact PD/PI: Faubion, William A

ResourceSharingPlans Page73

RESOURCE SHARING PLAN:

Sharing of Data and Model Organisms with the Broader Research Community: 

-We will comply with all NIH Public Access Policies including depositing peer-reviewed publications resulting 

from this research into National Library of Medicine PubMed Central within 12 months after the official date of 

publication.

-Additionally, all cell and mouse lines that are generated will be made available as requested by other 

investigators pursuant to Mayo Clinic and NIH guidelines.