PI: Lomvardas, Stavros Title: Principles of zonal olfactory receptor gene expression 

Received: 08/16/2019 FOA: PA19-056 

Clinical Trial:Not Allowed 

Council: 01/2020 

Competition ID: FORMS-E FOA Title: Research Project Grant (Parent R01 Clinical Trial Not Allowed) 

1 R01 DC018745-01 Dual: Accession Number: 4339093 

IPF: 1833205 Organization: COLUMBIA UNIVERSITY HEALTH SCIENCES 

Former Number: Department: Biochemistry and Mol. Biology 

IRG/SRG: ZRG1 IFCN-U (02)M AIDS: N Expedited: N 

Subtotal Direct Costs 

(excludes consortium F&A) 

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Animals: Y 

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Clinical Trial: N 

Current HS Code: 10 

HESC: N 

New Investigator: N 

Early Stage Investigator: N 

Senior/Key Personnel: Organization: Role Category: 

Stavros Lomvardas The Trustees of Columbia University in 

the City of New York 

PD/PI 
0MB Number 4040.0001 

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Principles of zonal olfactory receptor gene expression 

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Funding Opportun ty Number: PA 19 056 . Received Date: Tracking Number: GRANT12921503 

2019 08 16T10 05 36.000 04:00 Page 1 
Contact PD/Pl: Lomvardas, Stvros 

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Prefix: First Name•: Stavros Middle Name: Last Name•: Lomvardas Suffix: 

Position/Title: Professor

Organization Name·: The Trustees of Columbia University in the City of New York

Deparment: Biochemistry and Mol. Biology

Division: Zuckerman Institute

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Contact PD/PI: Lomvardas, Stavros Table of Contents 

424 R&R and PHS-398 Specific 

Table Of Contents 

SF 424 R&R Cover Page............................................................................................................1 

Table of Contents...................................................................................................................3 

Performance Sites.....................................................................................................................4 

Research & Related Other Project Information......................................................................5 

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

Project Narrative....................................................................................................................7 

Facilities & Other Resources................................................................................................8 

Equipment.............................................................................................................................11 

Research & Related Senior/Key Person................................................................................12 

Research & Related Budget Year - 1......................................................................................18 

Research & Related Budget Year - 2......................................................................................21 

Research & Related Budget Year - 3......................................................................................24 

Research & Related Budget Year - 4......................................................................................27 

Research & Related Budget Year - 5......................................................................................30 

Budget Justification................................................................................................................33 

Research & Related Cumulative Budget...............................................................................35 

PHS398 Cover Page Supplement...........................................................................................36 

PHS 398 Research Plan...........................................................................................................38 

Specific Aims........................................................................................................................39 

Research Strategy................................................................................................................40 

PHS Human Subjects and Clinical Trials Information......................................................52 

Vertebrate Animals..............................................................................................................53 

Bibliography & References Cited.......................................................................................55 

Resource Sharing Plan(s)...................................................................................................60 

Authentication of Key Biological and/or Chemical Resources.......................................61 

 Page 3
Contact PD/Pl: Lomvardas, Stavros 0MB Number: 4040-0010 

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Tracking Number: GRANT12921503 Funding Opportunity Number: PA 19 056. Received Date: 

2019 08 16T10:05:36.000 04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056. Received Date: 

OMB Number: 4040-0001

Expiration Date: 10/31/2019

RESEARCH & RELATED Other Project Information

Are Human Subjects Involved?* ❍ Yes ● No 

1.a. If YES to Human Subjects 

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If YES, check appropriate exemption number: 1 2 3 4 5 6 7 8 

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IACUC Approval Date: 

Animal Welfare Assurance Number A3007-01

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 ● No 

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 

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

❍ Yes ❍ No 

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5.a. If yes, please explain: 

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collaborators?* 

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Filename 

Project Summary/Abstract* Abstractzones.pdf

Project Narrative* Project_Narrativezones.pdf

Bibliography & References Cited ZonesBibliography.pdf

10.Facilities & Other Resources Facilities_and_Other_Resources.pdf

11.Equipment Major_Equipment.pdf

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Abstract 

The monogenic, monoallelic, and seemingly stochastic transcriptional choice of one out of > 1000 olfactory 

receptor (OR) genes remained elusive for decades after the discovery of the largest mammalian gene family. 

However, in the past few years we obtained significant understanding on the molecular underpinnings of this 

enigmatic gene regulatory process. Specifically, we showed that OR gene clusters become heterochromatic at 

the early stages of olfactory sensory neuron (OSN) differentiation and then they aggregate in distinct nuclear 

compartments that assure their stable repression. As a result of this interchromosomal convergence, intergenic 

OR enhancers (known as Greek Islands) that are found in most OR gene clusters come in close nuclear 

proximity and form a multi chromosomal super enhancer that in each OSN associates with the transcriptionally 

active OR allele. The formation of the Greek Island hub is dependent upon the recruitment of the adaptor 

protein Ldb1, which is essential for the stable interchromosomal interactions between Greek Islands and for 

OR transcription. This intricate network of activating and repressive interchromosomal interactions, together 

with a feedback signal elicited by the expression of the chosen OR, likely generate the regulatory framework 

transcriptional singularity. However, what remains unknown how this seemingly stochastic process 

operates under deterministic restrictions related to the spatial location of the OSN along the dorso ventral and 

apico basal axes of the MOE. These restrictions, known as zonal pattern of OR expression, restrict the 

expression of each OR gene in one of five zones of expression. Here we identified putative mechanisms of 

zonal restriction, by uncovering the molecular mechanisms that enable only zone 5 ORs to be expressed in 

zone 5. We show that transcription factors of the NFI family enable the transcriptional activation of zone 5 ORs, 

mediating the recruitment of these ORs to the interchromosomal OR compartment. Moreover, we show that 

the repressive histone modification H3K79me3 prevents the expression of out of zone ORs, possibly under the 

control of NFI factors, as well. We propose experiments that will decipher which NFI factors are required and 

sufficient for specification of zone 5 transcription programs, and experiment that will determine how NFI 

proteins accomplish these zonal restrictions. Our experiments will reveal novel mechanisms of regulation of 

nuclear architecture, and will uncover generally applicable principles for the regulation of developmental 

patterning. 

Contact PD/PI: Lomvardas, Stavros Project Summary/Abstract Page 6

 

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for 

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by 
Project Narrative 

Developmental patterning is a crucial biological mechanism perturbations of which are related to a plethora of 

developmental disorders. Developmental patterning in the olfactory epithelium, as manifested by the zonal 

expression of olfactory receptor genes remains mysterious for decades. Here, we describe genetic and 

epigenetic factors regulating zonal olfactory receptor expression, and we seek to uncover regulatory principles 

that are applicable to other systems undergoing developmental patterning. 

Contact PD/PI: Lomvardas, Stavros Project Narrative Page 7
FACILITIES AND OTHER RESOURCES 

Lomvardas Laboratory 

Laboratory: The laboratory is housed in ~2500 sq. feet in the 4th floor of the Jerome L. Green Science 

building that houses the Zuckerman mind brain and behavior institute (ZMBBI) at the new Manhattanville 

campus of Columbia University. Included in this area are the PI’s office, administrator’s office, 12 benches 12 

computer stations, one room for microscopy and histology, one room for cell culture and one room for 

electrophysiology and surgery. Shared facilities include dark room, cold room, bacterial incubator and shakers. 

There are 3 rooms for seminars and social gathering in the floor. My lab neighbors the laboratory of Dr. Tom 

Maniatis’s lab (open space configuration). 

Animal Facilities: We have access to the state of the art barrier mouse facility in the basement of the Jerome 

L. Green Science building with space for up to 1000 cages. Moreover, we have a satellite mouse facility next 

our laboratory with space for up to 250 cages. This facility is ideal for time-sensitive experiments that require 

continuous access to the mice. Next to the satellite mouse facility we also have a behavior room that is shared 

between the Maniatis and Lomvardas lab. All mice will be under the constant supervision of veterinary staff as 

well as the IACUC at Columbia. Moreover, we have access to the transgenic core facility of the Cancer Center 

at CUMC, which we used for the genetically modified mice described in this proposal. 

Computational resources: For analysis of deep sequencing data, every student and postdoc in the lab has their 

own personal computer and access to a Dual Xeon E5-2600 V3 Workstation, which has 24 cores, 4 x 32 GB RAM, 

and 24TB of local storage. An additional 60 TB of storage is available on backup workstations and digitally in an 

Amazon Cloud account. Additional large-scale computation resources are available through the Zuckerman Institute 

Research Computing Core. Finally, to keep up with our ever expanding needs for “scratch memory” in the analysis 

of deep sequencing databases by Next-seq we are renting 40TB of memory from Habanero, the high performance 

computing cluster at Columbia University. 

Office: In addition to the PI’s office, there is office space for one administrator and one lab manager and desks 

every student and postdoc in the lab (1desk per bench). We also have two color printers and one scanner 

that are used by members of the Lomvardas laboratory. 

Intellectual Environment: As a member of the Zuckerman Mind, Brain, and Behavior Institute I have the 

privilege to interact with some of the most creative and influential neuroscientists of our generation. First, Dr. 

Richard Axel, my postdoctoral mentor is located at the 8th floor of our building and we have weekly meetings to 

discuss common interests and ideas regarding olfaction (including the idea of using the olfactory system as a 

model for neurodegeneration). Furthermore, Tom Maniatis, with whom I share lab space, seeks to investigate 

the molecular mechanisms of monoallelic Protocadherin gene choice. As in OR gene choice, long range 

genomic interactions between intergenic enhancers and stochastically chosen promoters determine which 

Protocadherin allele will be expressed from each genomic cluster. The two laboratories have joined lab 

meetings and collaborate in various projects, including the development of powerful single cell HiC 

approaches. Moreover, we have monthly joint meetings with Dr. Richard Mann, Dr. Minoree Kohwi and their 

labs, which work on different aspects on genetic, epigenetic and nuclear architecture-based control of gene 

regulation. Finally, I meet regularly with Dr. Laura Landwerber and Dr. Sam Sternberg my colleague at the 

biochemistry department who are experts in regulated chromosomal DSBs (Landwerber) and CRISPR/Cas9 

technologies (Sternberg). As of recently, I am communicating with Dr. Gautier, who is an expert of DSBinduced chromatin mobility, discussing possible applications of his findings in our system. 

ZMBBI Jerome L. Greene Science Center 

The Cellular Imaging Core (Darcy Peterka, PhD, Director of Cellular Imaging) will provide access to the stateof-the-art imaging methods, broadly categorized into two critical imaging areas in neuroscience, high-speed 

functional imaging, and high resolution structural imaging, both across multiple scales, from sub-cellular 

structures, to circuits and entire brains. Second, the core will provide expert staff trained in sample preparation, 

microcopy, and image processing to assist investigators in complex experiments. Lastly, the core will be the 

central repository of imaging and image analysis expertise, within ZMBBI, and the greater Columbia 

Community, for both the hardware and the algorithms and software necessary for success. The Core will also 

provide strategic consulting services on current imaging modalities, feasibility and experimental design, and 

data processing and archiving. The Cellular Imaging Core has ~900 sq. ft. of dedicated imaging space on the 

lower level of the Greene Science Center, which currently houses a laser scanning confocal microscope, and a 

robotic slide scanner. Two-photon microscopes capable of in vivo imaging in awake animals, and a laser 

Contact PD/PI: Lomvardas, Stavros Facilities & Other Resources 

 

 

 

 

 

 

 

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scanning confocal microscope will be added to the facility before the commencement of this grant. A full animal 

monitoring suite will be maintained in this room to support in vivo imaging. The Core equipment on the 3rd floor 

includes the Zeiss Sigma HV electron microscope, and is the planned location of the lightsheet microscope 

budgeted for a Fall 2017 acquisition. 

The Cellular Imaging Core maintains local computer resources and workstations for image processing and 

analysis. There is also a suite of diagnostic instruments and electronics for support of advanced imaging 

including spectrophotometers and optical detectors. A large pool of optomechanics and optics are available to 

modify and enhance existing imaging platforms. 

The Cellular Imaging Core maintains a listing of all existing lasers, microscopes, and imaging related 

equipment for ZMBBI, with close relationships to the investigators. This allows Cellular Imaging to leverage a 

large pool of resources to bear on imaging intensive projects. 

The Zuckerman Institute Virology Core (ZIVIROLOGY) (Julia Sable, Director) is a full-service viral vector 

production organization that provides ZMMBI investigators access to state-of-the-art vector technology for 

basic animal model research applications. The core provides internal pricing structures for Z-MBBI faculty and 

Columbia University investigators which provides reduced costs compared to those currently incurred from 

purchasing via external viral vector cores. The core provides on-site same day pick up for in-stock catalog 

items. The Director, Julia Sable, has 18 years of experience in viral production and advanced imaging design. 

The Director and staff provide expert consultation services for advanced study design, safe use of viral vector 

technologies and viral construction services for multiple viral vector types. 

The ZIVIROLOGY Core is located in JLG in rooms L3.017. L3.010 and L3.014 and provides lenti and retro 

virus, rAAV and N2C G-deleted rabies customized for individual investigators needs; production and 

distribution of catalog/stock rAAV and N2C G-deleted rabies vectors expressing reporter and functional 

transgenes (e.g.) XFPs, GECIs, GEVIs and optogenetic tools. It provides consultation services on 

experimental design, development and use of custom viral vectors as well as novel client specific viral transfer 

plasmids. An example is the development of a fish Rhabdovirus genetic system to study cerebellar circuits in 

Mormyrid electric fish. The Center will soon provide local distribution of cloning vectors and cell lines with 

relevant MTA information to investigators prior to vector design and production (e.g. complete AAV plasmid 

collections housed on site from Deisseroth, Boyden, Roth, Uchida and Wickersham Labs). 

Zuckerman Institute Research Computing Core (Rajendra Bose, Director) is a five-person team that 

provides research computing and IT services, support and infrastructure to faculty, their research labs and 

other scientific service groups in the Greene Science Center. The Institute has launched a dedicated, shared, 

multi-petabyte storage system to house images and other research data and allow collaboration by Zuckerman 

researchers. This storage platform has an initial capacity of roughly two usable petabytes (PB, equal to 2000 

terabytes (TB)) and is designed to accommodate growth up to tens of PB over the next years. All three initial 

tiers of storage include remote backup for disaster recovery purposes. The Research Computing group also 

provides access to the university’s newest state-of-the-art high performance computing (HPC) cluster, 

launched in November 2016. This cluster has over 200 computer systems providing 5500 central processing 

unit (CPU) cores and 140,000 graphics processing unit (GPU) hardware accelerators for highly parallelized 

applications. In addition, a scalable virtual machine infrastructure (VMI) is in place to provide computational 

resources for data analysis approaches for which a shared HPC system is unsuited. The storage, VMI, HPC 

system and other local equipment is is housed in the dedicated, climate-controlled 1400 square foot 

Zuckerman Institute Data Center in the Greene Science Center with the ability to power and cool up to 30 

cabinets of equipment. The computational resources in the server room are accessed through a data network 

that has up to 10 Gigabit per second (Gbps) data transfer speeds within the building, and that connects to the 

40 Gbps campus backbone network and beyond to regional, national and international research networks. In 

addition, Columbia’s multiple commodity Internet connections provide an aggregate bandwidth of over 9 Gbps 

to the public Internet. 

The Advanced Instrumentation Core (Tanya Tabachnik, MEng, director) is available to help design, build 

and test instruments, tools, and software that are not available commercially. The group has complementary 

skills spanning mechanical, electrical, and optical engineering including manufacturing. The Advanced 

Instrumentation core supports an in-house machine and fabrication shop operated by and for the researchers 

themselves after completing a short training course. The Facility will continuously improve its design 

engineering and manufacturing capability to allow rapid and cost effective delivery of functional solutions. 

Contact PD/PI: Lomvardas, Stavros Facilities & Other Resources 

 

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all cases, the AIC’s philosophy is to collaborate closely with the research community to understand the 

neuroengineering problem to be solved. We believe that the synergy between life science and engineering will 

bring next-generation design solutions. We will support shared use of equipment and software by all interested 

Z-MBBI researchers. The AI group will have complementary skills spanning mechanical, electrical, and optical 

engineering including manufacturing. The Facility's goal is to provide in-house solutions and training to assist 

researchers through a complete life cycle of system development. 

The Advanced Instrumentation core is located with the Jerome L Greene Center encompassing 2,200 Sq. feet 

the basement level and 2 rows of the wet benches at the laboratory 3 level. Facilities include a fully 

equipped modern machine shop as well as electronic shop and engineering area. The shop includes a storage 

room, and two manufacturing and assembly spaces. The Manufacturing Shop is equipped with CNC and a 

manual milling machine, manual lathe, table and vertical saws, drill press, laser cutter and 3D printers. The 

Shop will also offer accessible hardware storage. Electrical equipment includes a Digital Storage Oscilloscope 

1Gs/s, with 4 channels capable of 100MHz and FFT Mathematical analysis, Logic Analyzer of 16 Channels at 

100MHz, decoding of I2C, SPI, RS232 interfaces, 25MHz Math Function and Arbitrary Signal Generator, Triple 

Channel intelligent programmable DC Power Supply 30V 1.5A, 5V 6A, with max load of 120W, 300 ft. 

Electrical and communications TDR Hand held meter, hot Air rework soldering station. The Facility will also 

have a metrology and optics lab, shared with the Cellular Imaging Core, suitable for optical design, integration, 

and assembly. 

Contact PD/PI: Lomvardas, Stavros Facilities & Other Resources 

 

In 

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Major Equipment: 

General Equipment: Our laboratory is equipped with the standard biochemistry, molecular biology and 

imaging equipment: 1 ultrancentrifuge (Sorval), 3 minicentrifuges, eppendorf thermomixer, hybridization 

ovens, cryostat (Leica), 4 PCR machines, two real time PCR machines, one epifluorescence microscope, 

one confocal microscope (Zeiss 710) with 4 lasers and 10x, 20x and 100x (oil immersion) lenses, tissue 

culture room (with 4 incubators, 2 hoods etch), an automatic sonicator (Covaris M220 fosused 

ultrasonicator), bioanalyzer, DNA library preparation hood (Air clean PCR workstation), gel 

documentation system, gel dryer, blue pipen DNA extraction instrument. We also have free access to a 

well head Biomek FX and 384 head Biomek FXp, purposed for single cell library preparation, which are 

owned by the Maniatis lab but are freely shared with us. 

FACS: We have access to he MoFlo XDP from Beckman Coulter, which is a high speed, high accuracy cell 

sorter with customizable lasers. It has a 32-bit high-resolution 5-decade multi-channel digital system to enable 

accurate sorting of rare events. It also has robust and stable microfluidics that enable both long sorts as well as 

high precision, for example, when sorting into 96, 384, and 1536-well plates. We also have direct access to a 

Sony SH800 flow cytometer, enabling direct sorting 24 hours a day, 7 days a week. Both instruments are ideal 

for our sorting needs achieving high viability, yield and purity combined with very high sensitivity. 

Deep Sequencing: We perform our sequencing reactions at ZMBBI using two NextSeq 550 instruments 

that are shared by a total of 4 labs in the institute. We also have access to the Columbia Cancer center 

core, which offers two more NextSeq 550 and two 10xGenomics single cell RNA-seq platforms. Thus, we 

have enough instrumentation for the completion of the proposed experiments and for the first time in my 

career we will not need to wait for sequencing. In the case that we need significantly deeper sequencing 

depths we will use the facilities of the NYC genome center, which is affiliated with Columbia University 

and directed by Dr. Maniatis. This facility has 80 Illumina Hi-seq stations,7 Nova-seq stations on two 

PAC-bio stations in a 30,000 square feet sequencing facility in NYC. 

Contact PD/PI: Lomvardas, Stavros Equipment 

 

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Contact PD/Pl: Lomvardas, Stavros 0MB Number. 4040-0001 

Exp ration Date: 10/31/2019 

RESEARCH & RELATED Senior/Key Person Profile (Expanded) 

PROFILE - Project Director/Principal Investgator

Prefix: Firt Name*: Stavros Middle Name Last Name*: Lomvardas Suffx: 

Position/Title*: Professor 

Organization Name*: The Trustees of Columbia University in the City of New York 

Department: Biochemistry and Mol. Biology 

Division: Zuckerman Institute 

Street1*: 

Street2: 

City*: 

County: 

State*: 

Province: 

Country*: USA: UNITED STATES 

Phone 

Zip/ Postal 

Number

Code*: 

*:­

-

Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

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

Degree Type: PHO Degree Year: 2002 

Attach Biographical Sketch*: File Name: Biosketch_Lomvardas_0S.12.19.pdf 

Attach Current & Pending Support: File Name: 

Page 12 

Track Funding Opportunity Number: PA 19 0560. Received Date: ing Number: GRANT12921503 

2019 08 16T10:05:36.000 04:00 
OMB No. 0925 0001 and 0925 0002 (Rev. 09/17 Approved Through 03/31/2020) 

BIOGRAPHICAL SKETCH 

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

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

NAME: Lomvardas, Stavros 

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

POSITION TITLE: Professor of Biochemistry and Molecular Biophysics and Neuroscience 

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 Crete, Heraklion, Greece B.S 06/1998 Molecular Biology 

Columbia University, New York, NY M.A. 09/1999 Genetics and 

Development 

Columbia University, New York, NY Ph.D. 09/2002 Biochemistry and 

Molecular Biophysics 

Columbia University, New York, NY Postdoctoral 12/2006 Biochemistry and 

Molecular Biophysics 

. Personal Statement 

My research program aims to understand the molecular mechanisms that generate the astounding diveristy of 

cell types that characterise the mammalian nervous system. As a model system for our studies we use the 

olfactory system and the study of the epigenetic mechanisms regulating olfactory receptor (OR) gene choice. 

OR genes compose the largest mammalian gene family consisted of >1000 members. These genes are 

expressed in a a mutually exclusive fashion, in such a way, that only one OR allele is expressed in each 

olfactory sensory neuron. In our effort to understand this extreme mode of stochastic gene expression we 

uncovered novel genetic and epigenetic principles that are likely deployed by other neuronal populations. For 

example, we previously showed that OR translation activates the unfolded protein response pathway (UPR) 

toward the generation of a feedback signal that prevents the expression of other OR alleles by silencing Lsd1. 

Moreover, we showed that the singularity of OR gene choice is orchestrated by an intricate network of 

interchromosomal interactions that culminates in the formation of a singular, multienhancer complex over the 

transcriptionally active OR. Our published work clearly indicates our expertise in genetics, epigenetics, nuclear 

architecture, gene regulation and developmental biology to lead a project seeking to decipher the zonal gene 

expression of olfactory receptor genes and the molecular mechanisms that recruit a single OR allele to the 

mult enhancer hub. My move from UCSF to Columbia in the end of 2014 posed significant delays to our 

research progress due to issue related to the mouse house of our interim space at the main campus at 

Columbia University. Due to technical problems at the vivarium we had limited mouse space that drastically 

delayed our genetic experiments. Importantly, due to delays in the completion of the Jerome L. Green Science 

building we spent 3 years instead of 6 months at this interim space. Since our move to the new state of the art 

science building we have expanded our colony and accelarated our productivity (one manuscript accepted in 

Nature, one in Cell and one in eLife in 2019 and three other manuscripts in preparatoin) 

Clowney E.J., LeGross M.A., Mosley C.M., Clowney F.G., Markenskoff Papadimitriou E.C., Myllys M., 

Barnea G., Larabell C.A. and Lomvardas, S. (2012). Nuclear aggregation of olfactory receptor genes 

regulates their monogenic expression. Cell, 151(4), 724 737. PMCID: PMC3659163. 

Lyons, B.D., Allen, W.E., Goh, T., Tsai, L., Barnea, G. & Lomvardas, S. (2013). An epigenetic trap 

stabilizes singular olfactory receptor expression. Cell, 154(2), 325 336. PMCID: PMC3929589. 

Contact PD/PI: Lomvardas, Stavros Biosketches Page 13

 

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Markenscoff Papadimitriou, E.C., Allen, W.E., Colquitt, B.M., Goh, T., Monahan, K., Murphy, K.K., Mosley, 

C.P., Ahituv, N., & Lomvardas S. (2014). Enhancer interaction networks as a means of singular olfactory 

receptor expression. Cell, 159(3), 543 557. PMCID: PMC4243057. 

Monahan, K., Horta A., and Lomvardas S (2019). Lhx2/Ldb1 mediated trans interactions regulate olfactory 

receptor choice. Nature, 565(7740): 448 453. PMCID: PMC6436840. 

. Positions and Honors 

Positions and Employment 

2007 2013 Assistant Professor, Department of Anatomy, University of California, San Francisco, CA 

2013 2014 Associate Professor, Department of Anatomy, University of California, San Francisco, CA 

2014 Professor, Biochemistry and Molecular Biophysics and Neuroscience, Columbia University, NY 

2014 Principal Investigator, Zuckerman Mind Brain Behavior Institute, Columbia University, NY 

Other Experience and Professional Memberships 

2010 2014 Scientific Board of the International Rett Syndrome Foundation 

2011 2014 European Research Council: Referee in Peer Review Evaluations 

2013 NIDA CEBRA review roster member (NIH) 

2012 2015 Somatosensory and Chemosensory Study Section, Ad Hoc Roster member (NIH) 

2015 2018 NIDA AVENIR phase II review roster member (NIH) 

2015 Simons Foundation Autism Research Initiative (SFARI) review roster member 

2015 4D Nucleome Program, review roster member for the Nucleomics Tools RFA (NIH) 

2015 2021 Chemosensory Study Section (NIH), regular member (chairperson from 2019) 

Honors 

2002 Harold M. Weintraub Award for outstand achievement during graduate studies in Biological 

Sciences, Fred Hutch Cancer Center, Seattle, WA 

2002 Ph.D. with highest distinction from Department of Biochemistry and Molecular Biophysics, 

Columbia University, New York, NY 

2002 Samuel W. Rover and Lewis Rover Awards for outstanding achievement in Biochemistry and 

Molecular Biophysics, Columbia University, New York, NY 

2003 Helen Hay Whitney Foundation Fellowship, Helen Hay Whitney Foundation, New City, NY 

2007 Innovations in Basic Sciences Award, Program for Breakthrough Biomedical Research, San 

Francisco, CA 

2008 Rett Syndrome Trust New Investigator Award, Rett Syndrome Research Trust, Trumbull, CT 

2009 NIH Director’s New Innovator Award, National Institutes of Health, Bethesda, MD 

2010 EUREKA Award, National Institutes of Health, Bethesda, MD 

2010 McKnight Scholar Award, McKnight Endowment for Neuroscience, Minneapolis, MN 

2014 Vilcek Prize for Creative Promise in Biomedical Promise, Vilcek Foundation, New York, NY 

2014 Young Investigator Award for Research in Olfaction, Association for Chemoreception Sciences, 

Glenview, IL 

2016 HHMI Faculty Scholar, Howard Hughes Medical Institute 

2017 Blavatnik Foundation National Award Finalist, Blavatnik Foundation 

Contribution to Science 

My scientific career is focused on deciphering regulatory mechanisms of gene expression, with major 

contributions in the understanding of mechanisms of inducible and stochastic gene activation. 

As a graduate student I examined the role of chromatin structure in the transcriptional activation of the 

IFN gene. My experiments revealed the order of events that lead to the activation of IFN transcription in 

vivo, established the role of histone acetylation in recruitment of nucleosome remodeling complexes, and 

demonstrated that TBP binding induces nucleosome sliding away from the core promoter, providing a 

novel mechanism by which general transcription factors facilitate transcription. Moreover, I showed that 

chromatin imposes specificity in gene activation; the position of a nucleosome determines the range of 

stimuli that lead to activation of IFN expression and defines the order of molecular events required for 

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initiation of transcription. These experiments provided the first insight into the role of chromatin in regulation 

of inducible gene transcription and constitute the textbook view of transcriptional activation in the context of 

chromatin. I had major conceptual and technical contribution to most of these experiments. 

a. Agalioti, T*., Lomvardas, S*., Parekh, B., Yie, J., Maniatis, T., & Thanos, D. (2000). Ordered 

recruitment of chromatin modifying and general transcription factors to the IFN beta promoter. Cell, 

103(4), 667 678. *Equally contributing authors 

b. Lomvardas, S. & Thanos, D. (2001). Nucleosome sliding via TBP DNA binding in vivo. Cell, 106(6), 

685 696. 

c. Lomvardas, S. & Thanos, D. (2002). Modifying gene expression programs by altering core 

promoter chromatin architecture. Cell, 110(2), 261 271. 

As an independent investigator I study the regulatory mechanisms of the monogenic and monoallelic 

olfactory receptor (OR) choice. Our experiments revealed an elegant molecular pathway that starts with the 

heterochromatic silencing of OR genes, continues with enzymatic de silencing of one OR allele by lysine 

demethylase Lsd1, and culminates with the OR dependent downregulation of Lsd1 expression, which 

stabilizes the singular OR choice. We also discovered that OR elicit this feedback signal via components of 

the unfolded protein response pathway, which detect OR protein expression in the endoplasmic reticulum. 

This regulatory loop, combined with our analysis of the unusual organization of OR loci in nuclei of olfactory 

sensory neurons provide molecular insight to a process that remained elusive since the cloning of olfactory 

receptor genes. These experiments, which I supervised and designed, provided significant insight to the 

elusive process of olfactory receptor choice, which remained mysterious since the discovery of olfactory 

receptor genes by Buck and Axel. 

a. Magklara, A., Yen, A., Colquitt, B.M., Clowney, E.J., Allen, W., Markenscoff Papadimitriou, E., 

Evans, Z.A., Kheradpour, P., Mountoufaris, G., Carey, C., Barnea, G., Kellis, M., & Lomvardas, S. 

(2011). An epigenetic signature for monoallelic olfactory receptor expression. Cell, 145(4), 555

570. PMCID: PMC3094500. 

b. Lyons, B.D., Allen, W.E., Goh, T., Tsai, L., Barnea, G. & Lomvardas, S. (2013). An epigenetic trap 

stabilizes singular olfactory receptor expression. Cell, 154(2), 325 336. PMCID: PMC3929589. 

c. Dalton, P.R., Lyons, B.D & Lomvardas S. (2013). Co opting the unfolded protein response to elicit 

olfactory receptor feedback. Cell, 155(2), 321 32 PMCID: PMC3843319. 

d. Lyons, B.D., Magklara, A., Goh, T., Sampath, S., Schaefer, A., Schotta, G. & Lomvardas, S. 

(2014). Heterochromatic silencing facilitates the diversification of olfactory neurons. Cell Rep, 9(3), 

884 892. PMCID: PMC4251488. 

In addition to our epigenetic studies focused on OR gene regulation we have performed comprehensive 

analyses on the epigenetic transitions occurring during the differentiation of olfactory neurons in vivo. By 

performing ChIP seq and DIP seq on FACsorted cells we provided the first detailed description of the 

changes in DNA methylation, DNA hydroxymethylation, chromatin accessibility occurring during 

differentiation. With genetic manipulation (loss of function and overexpression experiments) we 

demonstrated the role of hydroxylmethylcytosine in neuronal transcription as a facilitator of transcriptional 

elongation than a mere intermediate of cytosine de methylation. Finally, more recently by performing ChIP

seq for transcription factors we showed that transcription factors Lhx2 and Ebf bind cooperatively to OR 

enhancers and enable their function. 

a. Colquitt, B.M., Allen, W.E., Barnea, G., Lomvardas, S. (2013). Alteration of genic 5

hydroxymethylcytosine patterning in olfactory neurons correlates with changes in gene expression 

and cell identity. Proc Natl Acad Sci U S A, 110(36), 14682 14687 PMCID: PMC3767503. 

b. Colquitt, B.M., Markenscoff Papadimitriou, E., Duffié, R., Lomvardas, S. (2014). Dnmt3a regulates 

global gene expression in olfactory sensory neurons and enables odorant induced transcription. 

Neuron, 83(4), 823 838. PMCID: PMC4153871. 

c. Monahan K.,Schieren I., Cheung J., Mumbey Wafula A., Monuki E.S., and Lomvardas S. (2017). 

Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons. 

Elife PMCID: PMC5608512. 

d. Canzio D., Nwakeze C.,L., Horta A., Rajkumar S., M., Coffey E., L., Duffy E., E., Duffie R., 

Monahan K., O’Keeffe S., Simon M., D., Lomvardas S., and Maniatis T., (2019) Antisense lncRNA 

transcription mediates DNA demethylation to drive stochastic Protocadherin a promoter choice. 

Cell. 2019 Apr 18;177(3):639 653.e15. PubMed PMID: 30955885. 

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Importantly, my research program has revealed significant insight to the understanding of the role of 

nuclear architecture in epigenetic processes and gene regulation during neuronal differentiation. Using 

sequence capture technologies to generate a complex DNA FISH probe that detects the thousands of 

olfactory receptor alleles simultaneously we showed that during neuronal differentiation olfactory receptor 

loci converge to unique heterochromatic nuclear foci that contribute to the complete transcriptional 

silencing of olfactory receptor genes. Our experiments revealed that essential for this unusual nuclear 

reorganization is the downregulation of lamin b receptor from the differentiating neurons, which normally 

tethers olfactory receptor genes to the nuclear envelope. In each olfactory neuron, the transcriptionally 

active olfactory receptor allele resides outside of these repressive foci and is surrounded by a large number 

of intergenic enhancers that converge over the active allele from many different chromosomes, a discovery 

that constitutes the continuation of work that I started as post doc in Richard Axel’s lab. These findings

demonstrate the decisive role of nuclear architecture in gene regulation in vivo. I designed and supervised 

the experiments that led to these discoveries from my own lab and designed and executed the experiments 

that I published as post doc. 

a. Clowney E.J., LeGross M.A., Mosley C.M., Clowney F.G., Markenskoff Papadimitriou E.C., Myllys 

M., Barnea G., Larabell C.A. and Lomvardas, S. (2012). Nuclear aggregation of olfactory receptor 

genes regulates their monogenic expression. Cell, 151(4), 724 737. PMCID: PMC3659163. 

b. Markenscoff Papadimitriou, E.C., Allen, W.E., Colquitt, B.M., Goh, T., Monahan, K., Murphy, K.K., 

Mosley, C.P., Ahituv, N., & Lomvardas S. (2014). Enhancer interaction networks as a means of 

singular olfactory receptor expression. Cell, 159(3), 543 557. PMCID: PMC4243057. 

c. Le Gros, M.A., Clowney, E.J., Magklara, A., Yen, A., Markenscoff Papadimitriou, E., Colquitt, B., 

Myllys, M., Kellis, M., Lomvardas, S., & Larabell, C.A. (2016). Soft X Ray Tomography reveals 

gradual chromatin compaction and reorganization during neurogenesis in vivo. Cell Rep, 17(8), 

2125 2136. PMCID: PMC5135017. 

d. Monahan, K., Horta A., and Lomvardas S (2019). Lhx2/Ldb1 mediated trans interactions regulate 

olfactory receptor choice. Nature 565(7740): 448 453. PMCID: PMC6436840. 

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/stavros.lomvardas.1/bibliography/40755668/public/?sort=dat 

e&direction=descending 

Additional Information: Research Support and/or Scholastic Performance 

Ongoing Research Support 

R01 DC015451 Lomvardas (PI) 07/01/2016 06/30/2021 

Deciphering the Molecular Principles of Olfactory Receptor Gene Choice 

This grant seeks to decipher the molecular mechanisms by which transcription factors Lhx2 and Ebf activate 

intergenic transcriptional enhancers towards the singular olfactory receptor transcription. 

Role: PI 

U01 DA040582 Lomvardas, Brown, Larabell (MPI) 09/01/2015 08/30/2020 

Deciphering nuclear bodies and compartments that govern singular olfactory receptor expression 

This grant seeks to develop new technologies for the imaging, genomic, and biochemical analysis of the 

heterochromatic nuclear bodies formed by silent olfactory receptor genes. 

Role: MPI 

NSF 1921500 Narlikar (PI), Larabell, Lomvardas 08/01/2019 07/31/2024 

Collaborative Research: URoL: Epigenetics 2: Phase separated genome compartments as drivers of 

epigenetic phenotypes 

This grant seeks to decipher how droplet mediated DNA organization is regulated by cellular signals and how it 

impacts epigenetic phenotypes. 

Role: Co PI 

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R56 AG062454 Lomvardas (PI) 08/15/2019 07/31/2020 

Anosmia as a predictor of preclinical Alzheimer's disease 

This grant seeks to we explore the possibility that olfactory neurons could be used as early molecular sensors 

for neurodegenerative disorders. 

Role: PI 

HHMI Faculty ScholarLomvardas (PI) 11/01/2016 10/31/2021 

Molecular Mechanisms of Stochastic Gene Expression 

Role: PI 

Completed Research Support 

R01 DC014144 Lomvardas (PI) 08/01/2014 07/31/2019 

The Unfolded Protein Response as an Organizer of Chemosensory Response 

This grant seeks to characterize a newly discovered molecular pathway that uses components of the unfolded 

protein response for the stabilization of olfactory receptor gene choice. 

Role: PI 

R01 DC013560 Lomvardas (PI) 08/01/2014 07/31/2019 

Understanding the Nuclear Architecture in Olfactory Receptor Choice. 

This grant seeks to characterize a network of interchromosomal interactions formed between the 

transcriptionally active OR allele and distant regulatory enhancers in olfactory neurons. 

Role: PI 

R01 DA036894 Lomvardas, Barnea (MPI) 09/15/2013 9/14/2018 

Controling epigenetic states and nuclear architecture in the brain 

This grant seeks to develop technologies that will allow the cell type and sequence specific manipulation of 

gene loci, either at the level of epigenetic regulation and at the level of relative nuclear positioning. 

Role: MPI 

Contact PD/PI: Lomvardas, Stavros Biosketches Page 17

 

 

 

 

 

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OMB Number: 4040-0001 

Expiration Date: 10/31/2019 

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 2019-08-16T10:05:36.000-04:00 

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium 

Enter name of Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 1 

A. Senior/Key Person 

Prefix First Name* Middle 

Name 

Last Name* Suffix Project Role* Base 

Salary ($) 

Calendar 

Months 

Academic 

Months 

Summer 

Months 

Requested 

Salary ($)* 

Fringe 

Benefits ($)* 

Funds Requested ($)* 

Stavros Lomvardas PD/PI 2.0 

Total Funds Requested for all Senior Key Persons in the attached file 

Additional Senior Key Persons: File Name: Total Senior/Key Person 

B. Other Personnel 

Number of 

Personnel* 

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)* 

1 Post Doctoral Associates 6.0 

1 Graduate Students 12.0 

Undergraduate Students 

Secretarial/Clerical 

1 Staff Associate 6.0 

3 Total Number Other Personnel Total Other Personnel 

Total Salary, Wages and Fringe Benefits (A+B) 

RESEARCH & RELATED Budget {A-B} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 18

1 . 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 1 

C. Equipment Description 

List items and dollar amount for each item exceeding $5,000 

Equipment Item Funds Requested ($)* 

Total funds requested for all equipment listed in the attached file 

Total Equipment 

Additional Equipment: File Name: 

D. Travel Funds Requested ($)* 

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions) 

2. Foreign Travel Costs 

Total Travel Cost 

E. Participant/Trainee Support Costs Funds Requested ($)* 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 

RESEARCH & RELATED Budget {C-E} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 19

 

2019-08-16T10:05:36.000-04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 1 

F. Other Direct Costs Funds Requested ($)* 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual Costs 

6. Equipment or Facility Rental/User Fees 

7. Alterations and Renovations 

Animal costs 

Illumina sequencing, FACS Core Costs, Equipment Maintenance contracts 

GRA tuition plus GRA fees 

Total Other Direct Costs 

G. Direct Costs Funds Requested ($)* 

Total Direct Costs (A thru F) 

H. Indirect Costs 

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)* 

MTDC 

Total Indirect Costs 

Cognizant Federal Agency 

(Agency Name, POC Name, and POC Phone Number) 

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)* 

Total Direct and Indirect Institutional Costs (G + H) 

J. Fee Funds Requested ($)* 

K. Total Costs and Fee Funds Requested ($)* 

L. Budget Justification* File Name: 

Lomvardas.Zonal.R01.Budget.Justification.pdf 

(Only attach one file.) 

RESEARCH & RELATED Budget {F-K} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 20

 

8 . 

9 . 

10 . 

1 . 

2019-08-16T10:05:36.000-04:00 
OMB Number: 4040-0001 

Expiration Date: 10/31/2019 

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 2019-08-16T10:05:36.000-04:00 

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  2

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium 

Enter name of Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 2 

A. Senior/Key Person 

Prefix First Name* Middle 

Name 

Last Name* Suffix Project Role* Base 

Salary ($) 

Calendar 

Months 

Academic 

Months 

Summer 

Months 

Requested 

Salary ($)* 

Fringe 

Benefits ($)* 

Funds Requested ($)* 

Stavros Lomvardas PD/PI 2.0 

Total Funds Requested for all Senior Key Persons in the attached file 

Additional Senior Key Persons: File Name: Total Senior/Key Person 

B. Other Personnel 

Number of 

Personnel* 

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)* 

1 Post Doctoral Associates 6.0 

1 Graduate Students 12.0 

Undergraduate Students 

Secretarial/Clerical 

1 Staff Associate 6.0 

3 Total Number Other Personnel Total Other Personnel 

Total Salary, Wages and Fringe Benefits (A+B) 

RESEARCH & RELATED Budget {A-B} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 21

1 . 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 2 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 2 

C. Equipment Description 

List items and dollar amount for each item exceeding $5,000 

Equipment Item Funds Requested ($)* 

Total funds requested for all equipment listed in the attached file 

Total Equipment 

Additional Equipment: File Name: 

D. Travel Funds Requested ($)* 

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions) 

2. Foreign Travel Costs 

Total Travel Cost 

E. Participant/Trainee Support Costs Funds Requested ($)* 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 

RESEARCH & RELATED Budget {C-E} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 22

 

2019-08-16T10:05:36.000-04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 2 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 2 

F. Other Direct Costs Funds Requested ($)* 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual Costs 

6. Equipment or Facility Rental/User Fees 

7. Alterations and Renovations 

Animal costs 

Illumina sequencing, FACS Core Costs, Equipment Maintenance contracts 

GRA tuition plus GRA fees 

Total Other Direct Costs 

G. Direct Costs Funds Requested ($)* 

Total Direct Costs (A thru F) 

H. Indirect Costs 

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)* 

MTDC 

Total Indirect Costs 

Cognizant Federal Agency 

(Agency Name, POC Name, and POC Phone Number) 

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)* 

Total Direct and Indirect Institutional Costs (G + H) 

J. Fee Funds Requested ($)* 

K. Total Costs and Fee Funds Requested ($)* 

L. Budget Justification* File Name: 

Lomvardas.Zonal.R01.Budget.Justification.pdf 

(Only attach one file.) 

RESEARCH & RELATED Budget {F-K} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 23

 

 

8 . 

9 . 

10 . 

1 . 

2019-08-16T10:05:36.000-04:00 
OMB Number: 4040-0001 

Expiration Date: 10/31/2019 

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 2019-08-16T10:05:36.000-04:00 

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  3

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium 

Enter name of Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2022 End Date*: 03-31-2023 Budget Period: 3 

A. Senior/Key Person 

Prefix First Name* Middle 

Name 

Last Name* Suffix Project Role* Base 

Salary ($) 

Calendar 

Months 

Academic 

Months 

Summer 

Months 

Requested 

Salary ($)* 

Fringe 

Benefits ($)* 

Funds Requested ($)* 

Stavros Lomvardas PD/PI 2.0 

Total Funds Requested for all Senior Key Persons in the attached file 

Additional Senior Key Persons: File Name: Total Senior/Key Person 

B. Other Personnel 

Number of 

Personnel* 

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)* 

1 Post Doctoral Associates 6.0 

1 Graduate Students 12.0 

Undergraduate Students 

Secretarial/Clerical 

1 Staff Associate 6.0 

3 Total Number Other Personnel Total Other Personnel 

Total Salary, Wages and Fringe Benefits (A+B) 

RESEARCH & RELATED Budget {A-B} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 24

1 . 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 3 

ORGANIZATIONAL DUNS* 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2022 End Date*: 03-31-2023 Budget Period: 3 

C. Equipment Description 

List items and dollar amount for each item exceeding $5,000 

Equipment Item Funds Requested ($)* 

Total funds requested for all equipment listed in the attached file 

Total Equipment 

Additional Equipment: File Name: 

D. Travel Funds Requested ($)* 

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions) 

2. Foreign Travel Costs 

Total Travel Cost 

E. Participant/Trainee Support Costs Funds Requested ($)* 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 

RESEARCH & RELATED Budget {C-E} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 25

 

2019-08-16T10:05:36.000-04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 3 

ORGANIZATIONAL DUNS* 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2022 End Date*: 03-31-2023 Budget Period: 3 

F. Other Direct Costs Funds Requested ($)* 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual Costs 

6. Equipment or Facility Rental/User Fees 

7. Alterations and Renovations 

Animal costs 

Illumina sequencing, FACS Core Costs, Equipment Maintenance contracts 

GRA tuition plus GRA fees 

Total Other Direct Costs 

G. Direct Costs Funds Requested ($)* 

Total Direct Costs (A thru F) 

H. Indirect Costs 

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)* 

MTDC 

Total Indirect Costs 

Cognizant Federal Agency 

(Agency Name, POC Name, and POC Phone Number) 

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)* 

Total Direct and Indirect Institutional Costs (G + H) 

J. Fee Funds Requested ($)* 

K. Total Costs and Fee Funds Requested ($)* 

L. Budget Justification* File Name: 

Lomvardas.Zonal.R01.Budget.Justification.pdf 

(Only attach one file.) 

RESEARCH & RELATED Budget {F-K} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 26

 

 

8 . 

9 . 

10 . 

1 . 

2019-08-16T10:05:36.000-04:00 
OMB Number: 4040-0001 

Expiration Date: 10/31/2019 

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 2019-08-16T10:05:36.000-04:00 

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  4

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium 

Enter name of Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2023 End Date*: 03-31-2024 Budget Period: 4 

A. Senior/Key Person 

Prefix First Name* Middle 

Name 

Last Name* Suffix Project Role* Base 

Salary ($) 

Calendar 

Months 

Academic 

Months 

Summer 

Months 

Requested 

Salary ($)* 

Fringe 

Benefits ($)* 

Funds Requested ($)* 

Stavros Lomvardas PD/PI 2.0 

Total Funds Requested for all Senior Key Persons in the attached file 

Additional Senior Key Persons: File Name: Total Senior/Key Person 

B. Other Personnel 

Number of 

Personnel* 

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)* 

1 Post Doctoral Associates 6.0 

1 Graduate Students 12.0 

Undergraduate Students 

Secretarial/Clerical 

1 Staff Associate 6.0 

3 Total Number Other Personnel Total Other Personnel 

Total Salary, Wages and Fringe Benefits (A+B) 

RESEARCH & RELATED Budget {A-B} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 27

1 . 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 4 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2023 End Date*: 03-31-2024 Budget Period: 4 

C. Equipment Description 

List items and dollar amount for each item exceeding $5,000 

Equipment Item Funds Requested ($)* 

Total funds requested for all equipment listed in the attached file 

Total Equipment 

Additional Equipment: File Name: 

D. Travel Funds Requested ($)* 

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions) 

2. Foreign Travel Costs 

Total Travel Cost 

E. Participant/Trainee Support Costs Funds Requested ($)* 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 

RESEARCH & RELATED Budget {C-E} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 28

 

2019-08-16T10:05:36.000-04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 4 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2023 End Date*: 03-31-2024 Budget Period: 4 

F. Other Direct Costs Funds Requested ($)* 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual Costs 

6. Equipment or Facility Rental/User Fees 

7. Alterations and Renovations 

Animal costs 

Illumina sequencing, FACS Core Costs, Equipment Maintenance contracts 

GRA tuition plus GRA fees 

Total Other Direct Costs 

G. Direct Costs Funds Requested ($)* 

Total Direct Costs (A thru F) 

H. Indirect Costs 

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)* 

MTDC 

Total Indirect Costs 

Cognizant Federal Agency 

(Agency Name, POC Name, and POC Phone Number) 

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)* 

Total Direct and Indirect Institutional Costs (G + H) 

J. Fee Funds Requested ($)* 

K. Total Costs and Fee Funds Requested ($)* 

L. Budget Justification* File Name: 

Lomvardas.Zonal.R01.Budget.Justification.pdf 

(Only attach one file.) 

RESEARCH & RELATED Budget {F-K} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 29

 

 

8 . 

9 . 

10 . 

1 . 

2019-08-16T10:05:36.000-04:00 
OMB Number: 4040-0001 

Expiration Date: 10/31/2019 

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 2019-08-16T10:05:36.000-04:00 

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  5

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium 

Enter name of Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2024 End Date*: 03-31-2025 Budget Period: 5 

A. Senior/Key Person 

Prefix First Name* Middle 

Name 

Last Name* Suffix Project Role* Base 

Salary ($) 

Calendar 

Months 

Academic 

Months 

Summer 

Months 

Requested 

Salary ($)* 

Fringe 

Benefits ($)* 

Funds Requested ($)* 

Stavros Lomvardas PD/PI 2.0 

Total Funds Requested for all Senior Key Persons in the attached file 

Additional Senior Key Persons: File Name: Total Senior/Key Person 

B. Other Personnel 

Number of 

Personnel* 

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)* 

1 Post Doctoral Associates 6.0 

1 Graduate Students 12.0 

Undergraduate Students 

Secretarial/Clerical 

1 Staff Associate 6.0 

3 Total Number Other Personnel Total Other Personnel 

Total Salary, Wages and Fringe Benefits (A+B) 

RESEARCH & RELATED Budget {A-B} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 30

1 . 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 5 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2024 End Date*: 03-31-2025 Budget Period: 5 

C. Equipment Description 

List items and dollar amount for each item exceeding $5,000 

Equipment Item Funds Requested ($)* 

Total funds requested for all equipment listed in the attached file 

Total Equipment 

Additional Equipment: File Name: 

D. Travel Funds Requested ($)* 

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions) 

2. Foreign Travel Costs 

Total Travel Cost 

E. Participant/Trainee Support Costs Funds Requested ($)* 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 

RESEARCH & RELATED Budget {C-E} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 31

 

2019-08-16T10:05:36.000-04:00 
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 5 

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium 

Organization: The Trustees of Columbia University in the City of New York 

Start Date*: 04-01-2024 End Date*: 03-31-2025 Budget Period: 5 

F. Other Direct Costs Funds Requested ($)* 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual Costs 

6. Equipment or Facility Rental/User Fees 

7. Alterations and Renovations 

Animal costs 

Illumina sequencing, FACS Core Costs, Equipment Maintenance contracts 

GRA tuition plus GRA fees 

Total Other Direct Costs 

G. Direct Costs Funds Requested ($)* 

Total Direct Costs (A thru F) 

H. Indirect Costs 

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)* 

MTDC 62.0 

Total Indirect Costs 

Cognizant Federal Agency 

(Agency Name, POC Name, and POC Phone Number) 

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)* 

Total Direct and Indirect Institutional Costs (G + H) 

J. Fee Funds Requested ($)* 

K. Total Costs and Fee Funds Requested ($)* 

L. Budget Justification* File Name: 

Lomvardas.Zonal.R01.Budget.Justification.pdf 

(Only attach one file.) 

RESEARCH & RELATED Budget {F-K} (Funds Requested) 

Contact PD/PI: Lomvardas, Stavros Page 32

 

8 . 

9 . 

10 . 

1 . 

2019-08-16T10:05:36.000-04:00 
Budget Justification 

Key Personnel 

Stavros Lomvardas, P.I. (2.0 calendar months). Dr. Lomvardas has extensive experience in genetic and 

epigenetic analysis of the olfactory system and has an established track record in the study of 

interchromosomal genomic interactions in gene regulation. Dr. Lomvardas will supervise the proposed 

experiments, will provide technical assistance and troubleshooting, and will help with the computational 

analysis and interpretation of the data. 

Other Personnel 

Lisa Bashkirova, Graduate Research Assistant (12 calendar months). Ms. Bashkirova is a senior graduate 

student that performed all the zonal experiments presented in this grant and she has the skills to perform the 

proposed experiments and to analyze them computationally. 

Marianna Zazhytska, Postdoctoral Research Scientist (6 calendar months). Dr. Zazhytska is a new postdoc 

my lab that has experience with HiC experiments and computational analysis from her graduate work that 

included an extended collaboration with Erez Lieberman Aiden. She will assist Ms. Bashklrova with her HiC 

experiments. 

Alice Mumbey Wafula, M.S., Research Staff Associate. (6 calendar months). Ms. Mumbey Wafula has a 

Master’s degree in Molecular Biology and Biochemistry and has been working as a research staff associate in 

my lab for several years she will be responsible for managing the vast mouse colony related to this project, by 

assisting with genotyping, setting crosses, performing methimazol ablation experiments and preparing MOE 

sections for IF and RNA ISH experiments. 

Other Direct Costs 

General Lab Supplies. The proposed experiments require mouse genotyping via PCR (Taq 

polymerase+primer costs), use of primary and secondary antibodies, papain dissociation kits for FACS, 

generation of RNA probes for ISH experiments, primary and secondary antibodies, cDNA synthesis and 

cloning (for making riboprobes), mRNA purification kits and other standard lab supplies such as slides, 

coverslips, pipet tips, tubes, glassware and plasticware, tissue culture plates, serum and media. We calculated 

the sum of all these expenses as $20,000/year. 

Illumina Sequencing: The proposed experiments include multiple in situ HiC, ChIP seq, Cut and Run and 

RNA seq experiments for various mutants. Every experiment will be performed in biological triplicates. 

Moreover for our proposed HiC experiments, we seek the genomic resolution afforded by 1 billion HiC reads, 

that we will be using at least 4 Next seq flow cells per condition. Importantly, for each mutant strain we will be 

micro dissecting and comparing zones 1 and 5, and in certain experiments we will be characterizing the 

various developmental stages of each zone. 

Total sequencing costs are estimated at: 

FACS Core Costs. 

The proposed experiments require extensive FAC sorted populations of GFP or iRFP labeled olfactory sensory 

neurons. We calculate needing 100 hours of FAC sorting per year with hourly cost of $100/hour 

Total FACS core costs are estimated at 

Animal Costs. The current per diem cost for mouse cages at Columbia is 

We have proposed analysis of the following genotypes: 

NFIAfl/fl, Krt5CreER, TdTomato 

NFIB fl/fl; Krt5CreER, TdTomato 

NFIX fl/fl; Krt5CreER, TdTomato 

NFIA fl/fl; NFIX fl/fl; Krt5CreER, TdTomato 

5. NFIB fl/fl; NFIX fl/fl; Krt5CreER, TdTomato 

Gg8tTA; tetO NFIAT2ANFIBiresmCherry 

Contact PD/PI: Lomvardas, Stavros Budget Justification Attachment Page 33

 

 

 

 

 

 

 

 

 

- -

in 

-

-

-

-

-

-

-

-

1. 

2. 

3. 

4. 

6. -
Krt5CreER; Cre inducible tTA; tetO NFIAT2ANFIBiresmCherry 

Krt5CreER; NFIA,B fl/fl; Cre inducible tTA; tetO NFIAT2ANFIBiresmCherry 

Gg8 Cre; Dot1lfl/fl; TdTomato 

. Mash1 CreER; Dot1lfl/fl; TdTomato 

. Olfr1507iresCre; Dot1lfl/fl; TdTomato 

total we have 11 different genotypes that contain 3 or 4 different alleles. To obtain sufficient number of 

animals from each genotype we anticipate having 8 breeding cages for each genotype (88 cages total). 

Moreover, we will have 3 cages for maintenance of each one of the different alleles (11 different alleles 

described here, 33 cages). Finally, in addition to the breeding cages, male and female mice will be housed 

separately until the reach the desired age, thus we anticipate another 8 cages per genotype (88 cages). Total 

number of cages anticipated for this project in the coming year is (88+33+88 209). 

Total mouse costs: in year 1 with anticipated increases of per year. 

Publications. We anticipate publishing one manuscript per year describing these findings with an estimated 

publication cost of per year. 

ADP/Computer costs. An amount of per year is requested to access Columbia’s Habanero high 

performance computing server for the computational processing power required for data analysis. 

Equipment Maintenance Contracts. 

Confocal Service Contract (Zeiss). Our yearly service contract is and we have calculated that 1/3 of 

our microscope use will be dedicated to this project, thus we calculate a yearly cost of 

Cryostar Service Contract (Leica). Our yearly service contract for the cryostat is and we anticipate that 

/3 of its use will be dedicated to this project thus the yearly cost will be 

Conference travel. We are requesting per year to attend and present results at Neuroscience and 

Nuclear Architecture conferences. We envision a total of up to 2 conferences per year spread between 

the PI, Graduate Research Assistant and Postdoctoral Research Scientist. ( /conference on 

average). 

Graduate student tuition and fees. Funds are requested to cover the Graduate Research Assistant’s tuition 

and fees according to the schedule required by the Columbia University Graduate School of Biomedical 

Sciences. ( tuition and fees, in year 1 with annual increases.) 

Indirect Costs: 

Sponsored Projects Administration certifies that the Primary Place of Performance for the proposed research 

will occur at Jerome L. Green Science Center, 3227 Broadway New York NY, 10027, 4th floor L4.005, an on

campus location, and that the appropriate F&A rate is 

Contact PD/PI: Lomvardas, Stavros Budget Justification Attachment Page 34

 

 

 

11

1

7. - -

8. - -

9. -

10 -

In 

= 

-
Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

RESEARCH & RELATED BUDGET - Cumulative Budget 

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 15

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment

Section D, Travel

1. Domestic 

2. Foreign

Section E, Participant/Trainee Support

Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

6. Number of Participants/Trainees

Section F, Other Direct Costs

1. Materials and Supplies

 Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

7. Alterations and Renovations

8. Other 1

9. Other 2

10. Other 3

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

Section K, Total Costs and Fee (I + J)

Contact PD/PI: Lomvardas, Stavros 

Fees

 Page 35 2019-08-16T10:05:36.000-04:00 
PHS 398 Cover Page Supplement OMB Number: 0925-0001

Expiration Date: 03/31/2020

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

1. Vertebrate Animals Section

Are vertebrate animals euthanized? ● Yes ❍ No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

● Yes ❍ No

If "No" to AVMA guidelines, describe method and provide scientific justification

2. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ 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 *Anticipated Amount ($) *Source(s)

Contact PD/PI: Lomvardas, Stavros 

   

 Page 36 2019-08-16T10:05:36.000-04:00 
PHS 398 Cover Page Supplement

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 . Received Date: 

3. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

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

check the box indicating that one from the registry will be used:

❏ Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

4. Inventions and Patents Section (Renewal applications)

*Inventions and Patents: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

5. Change of Investigator/Change of Institution Section

❏ Change of Project Director/Principal Investigator

Name of former Project Director/Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Lomvardas, Stavros Page 37 2019-08-16T10:05:36.000-04:00 
PHS 398 Research Plan OMB Number: 0925-0001

Expiration Date: 03/31/2020

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056. Received Date: 

Introduction

1. Introduction to Application

(for Resubmission and Revision applications)

Research Plan Section

2. Specific Aims ZonesSpecificAims.pdf

3. Research Strategy* ZonesResearchStrategy.pdf

4. Progress Report Publication List

Other Research Plan Section

5. Vertebrate Animals Vertebrate_animals_zones.pdf

6. Select Agent Research

7. Multiple PD/PI Leadership Plan

8. Consortium/Contractual Arrangements

9. Letters of Support

10. Resource Sharing Plan(s) Resource_Sharing_Planzones.pdf

11. Authentication of Key Biological and/or

Chemical Resources Authenticationzones.pdf

Appendix

12. Appendix

Contact PD/PI: Lomvardas, Stavros 

 

 

 Page 38 2019-08-16T10:05:36.000-04:00 
Contact PD/Pl: Lomvardas, Stavros 

Specific Aims 

Olfactory receptor (OR) genes are expressed in a monogenic, monoallelic, and seemingly stochastic fashion in 

olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE)1

. This singular expression pattern is 

orchestrated by the formation of a multi-chromosomal enhancer hub over the transcriptionally chosen OR2

, 

followed by a feedback signal that prevents the transcriptional activation of additional OR alleles3. Although 

great progress was achieved towards the understanding of these two regulatory mechanisms, the process that 

restricts the transcription of specific OR gene repertoires to continuous "zones" along the dorso-ventral and 

apico-basal axes of the MOE4 remains elusive. The zonal pattern of OR gene expression was realized more 

than two decades ago5

·

6

, yet very little is known about cis regulatory elements and trans factors imposing these 

spatial restrictions. 

To obtain insight into this enigmatic process we perormed a comprehensive comparison of zone 1 and 

zone 5 OSNs and progenitor cells, which revealed striking differences in epigenetic marks and nuclear 

architecture of zone 1 and 5 OR genes at their respective zones. To identify sequence-specific factors that 

orchestrate these diferences we searched for zonally enriched motifs on OR promoters that correspond to 

zonally expressed transcription factors. We identified NFI motifs as being enriched on zone 5 OR promoters 

and paralogous transcription factors NFIA, B, and X as being enriched in zone 5 OSN progenitors (NFIA,B) 

and OSNs (NFIX). Conditional triple deletion of NFIA, B, and X in the developing MOE resulted in strong 

downregulation of zone 4-5 OR transcription and ectopic expression of zone 2-3 OR genes in zone 5, providing 

a striking example of homeotic transformation. In situ HiC of NFI KO OSNs revealed a similar transformation in 

nuclear architecture, supporting a role of NFI proteins as master regulators of zone 5 OR identity. We propose 

experiments that will determine the role of each NFI gene in establishing zonal OR identities and will define the 

developmental timing of their action, as well as experiments that will explore the molecular mechanism by 

which NFI proteins regulate zonal OR expression. 

Aim1: Determine which NFI genes are required and sufficient for establishment of zone 5 identity. 

Triple deletion of NFIA, B, and X in the stem cells of the MOE perurbs the expression of zone 4 and 5 OR 

genes and allows ectopic expression of zone 2-3 ORs. Deletion of only NFIA, B also results in downregulation 

of zone 4-5 ORs, but not in ectopic expression of zone 2 ORs, suggesting a role of NFIX in repressing these 

ORs in zone 5. Finally, triple deletion of NFI in mature OSNs does not alter zonal OR transcription, suggesting 

a transient role of these factors in specifying zone 5 OR identity. We seek to identify which of the 3 NFI genes 

are required and suficient for zone 5 OR expression and for repression of ectopic OR expression in zone 5. 

A. We will generate individual NFIA, B and X KO mice and ask which NFI genes are required for activation 

of zone 4-5 OR genes and for repression of ectopic ORs. 

B. We will ectopically express NFIA and NFIB proteins in progenitors and OSNs of zones 1-4 to determine 

the sufficiency of these factors in specifying zone 5 identity. 

Aim2: Determine how NFI proteins regulate zonal OR expression. 

Given that continuous NFI activity is not required for stable zone 5 OR expression in mature OSNs, NFI 

proteins do not act as traditional transcriptional activators but as factors that establish an early epigenetic 

"imprint" that enables transcription of zone 5 ORs in mature OSNs. Our data suggest that NFl-dependent 

recruitment to nuclear OR comparments contributes to this epigenetic memory. Furhermore, preliminary 

analysis suggests that zonal deposition of repressing histone modification H3K79me3 prevents "out of zone" 

ORs from being expressed in this zone. Thus, we seek to identify the mechanisms by which NFI proteins 

enable zone 4-5 ORs and prevent zone 2-3 ORs from expression in zone 5. We have three major goals: 

A. First, using Cut&Run, we seek to identify the direct genomic targets of NFI proteins on FAC-sorted 

progenitors and OSNs from zone 5 as well as in OSNs expressing the zone 5 OR Olfr1507. 

B. We wish to determine how NFI proteins affect the nuclear architecture of zone 4-5 and zone 2-3 ORs 

by performing zonal in situ HiC in NFI KO and transgenic mice expressing NFI in every zone. 

C. We will explore whether the repressive mark H3K79me3 keeps "out of zone" ORs silent in zone 5, and 

whether NFI proteins are mediating H3K79me3 deposition. We will also seek additional repressive 

histone and DNA modifications deposited on ORs in a zonal fashion. 

Our experiments will provide mechanistic insight to a fundamental process of developmental patterning that 

remained intractable since 1993 and will reveal general principles for the regulation of genomic interactions 

and epigenetic changes that govern transcriptional choices and cellular identities during differentiation. 

Page 39 

Specifc Aims 
Contact PD/Pl: Lomvardas, Stavros 

Significance 

Pattern formation is a fascinating phenomenon that governs fundamental developmental processes in plants 

and animals, vertebrates and invertebrates7 9. From the classic theoretical predictions on the chemical basis of 

morphogenesis1

0 to the seminal demonstration of the genetic control of segmentation in drosophila1 1•12; and 

from the elucidation of motor neuron pool identity along the spinal cord axes13 to the explanation of 

photoreceptor patterning in invertebrate omatidia14, developmental biologists have made strides in 

understanding various forms of patterning. However, patterning in the main olfactory epithelium (MOE), as 

6

manifested by the zonal expression of olfactory receptor (OR) genes5

• , remains intractable. This constitutes a 

glaring gap of knowledge for sensory biology for two main reasons: First, we cannot fully comprehend how an 

OR is chosen if we do not decipher why this choice is restricted to a zonal OR repertoire. The realization that 

OR gene choice is influenced by the position of an OSN begs the fundamental question: what mechanisms 

impose deterministic restrictions to an otherwise stochastic process? This question becomes even more 

pressing upon our recent discovery that OR gene choice is mediated by a multi-chromosomal enhancer hub 

16 that interacts in trans with the active OR allele, regardless of the zonal identity of the chosen OR15· . 

Therefore, determining how this zonally invariant enhancer hub exhibits differential afinity for distinct OR 

combinations along the 5 zones, may provide valuable insight into the mechanism of OR choice itself. 

Second, without 

knowing the regulatory 

mechanism that

orchestrate zonal OR 

expression, we cannot 

address the biological 

significance of this 

expression pattern. The 

conservation of zonal 

OR expression in most 

vertebrates suggests 

that spatial restrictions of 

OR gene choice is 

selected through 

evolution. The fact that 

Figuere!: The 5 major zones of OR gene expression. Coronal MOE section depicting the 5 zonal coordinates are 

zones (left) and the projections of zonal OSNs to the olfacto1y bulb. preserved in the olfactory 

bulb raises possible implications in olfactory perception 17. For example, there is evidence that dorsal glomeruli 

receiving input from zone I respond to odors associated with innate fear responses18. Thus, although most 

odorants lack strong innate valance19•20 and activate dispersed glomeruli21

, zonal boundaries may influence 

odor perception. Even if zonal OR expression is not contributing to odor perception, it may have diferent 

functional roles. For example, zonal restrictions may facilitate axon fasciculation and convergence of like axons 

to a common glomerulus; if all the OSNs expressing the same OR were evenly distributed across the MOE 

axes, then they may fail to converge to distinct glomeruli, because like axons will be rarely proximal to each 

other. Finally, we cannot exclude the possibility that zonal restrictions facilitate the singularity of OR gene 

choice; if ~1200 OR genes could be activated in each OSN, then the processes assuring singularity of choice 

may not be adequate. The OR-elicited feedback signal may not be fast enough if 2400 OR alleles are 

transcriptionally competent in each OSN, and more than one OR alleles may be able to associate 

simultaneously with the activating enhancer hub, violating the "one receptor per neuron rule". Therefore, our 

work is creating the foundation for a future experimental interrogation of the role of zones in olfactory coding, 

OSN targeting, and singular OR choice. 

In addition to its importance for olfaction, this project has general significance for developmental 

biology and for the emerging field of nuclear architecture. The role of long-range and trans genomic 

interactions in developmental patterning is starting to be appreciated in numerous fields. In developing 

cardiomyocytes, a multi-chromosomal gene hub coordinates the alternative splicing of cardiomyocyte-specific 

genes22

, promoting cardiogenesis. Further, photoreceptor gene choice in flies23

, Th1/Th2 lymphocyte 

26 differentiation in mice2

4, and X -chromosome inactivation in humans25

· are orchestrated by interchromosomal 

contacts, highlighting the diversity of cell specification processes and organisms utilizing this mechanism. 

Super-enhancers (SEs) tend to form interchromosomal compartments, likely promoting transcriptional 

Page 40 

Research Strategy 
44 

RNA speckles29, and similarly, the non-coding RNA Firre promotes interchromosomal interactions30 . In the 

during the process of antigenic

Contact PD/Pl: Lomvardas, Stavros 

robbustness27

·

28

. SPRITE, an alternative to HiC, revealed multi-chromosomal interactions organized by nuclear 

31

case of human antiviral responses, multi-chromosomal transcription factor "repositories" appear essential for 

the activation of INFβ

32·33

. Multi-chromosomal interactions are also used by trypanosome

3 4 and plasmodium35·

36 

y variation34 37. Therefore,

deciphering mechanisms that

regulate the formation of

interchromosomal interactions

across the axes of the MOE will

have general impact in

understanding 3D genome

organization, especially since

NFI proteins regulate a plethora

3 of developmental processesea

Finally, there is potential 

translational significance of our

proposal. Recent experiments

revealed the feasibility of cell

transplantation experiments in 

· Figure2: Transcriptionally Active ORs interact with multiple trans and cis Greek

model for congenital Islan ds rega r d less of zonael iden tity. a, Increased contcts between the active OR a 

restoring 

mouse 

olfactory sensitivity in

promoter and Greek Islads located in short range cis (grey), long range cis anosmia45. With anosmia and

(>5Mb, blue) and trans (red). Greek Island interactions are expressed as the hyposmia afecting the

faction of the totl HiC contcts mapped to each promoter. Olfl6 is a zone I emotional wellneSS46 and 

of affected 

such cell-based

OR, Olfrl 7 is zone 2, and Olfl507 is zone 5 b, Profile of the OR cluster longevity47

containing Olfl6 reveals increased contacts, expressed as faction of the total individuals, 

HiC contacts mapped to each position (5 Kb resolution), in Olfr16+ or Olfr17+

cells. approaches may soon reach

Olfr16+ c, Manhattan plot of Greek Island c ontacts withOR genes revea ls that in cells the the clinic. However, without

Olfrl 6 locus is the most significantly enriched for Greek Island contacts. being able to restore the full 

spectrum of odorant perception, 

such approaches will fall short. Deciphering the molecular regulators of zonal OR expression will eventually 

allow us to generate "designer" OSN progenitors that will produce zonal OSNs of our choice, allowing us to 

"correct" specific odor detection deficiencies or to restore damaged MOEs with the proper zonal consistency. 

Innovation 

Our proposal is conceptually and technically innovative. On the conceptual front we tackle a problem that has 

not been solved in any model organism, thus, by default, we break new ground. The correlation between 

formation of zone-dependent interchromosomal interactions in OSN progenitors and zonal transcriptional 

restrictions in mature OSNs (mOSNs) suggests that we uncovered a completely novel mechanism for 

establishing epigenetic memory during developmental patterning. In this note, this is the first demonstration 

that interchromosomal interactions can be subject to deterministic spatial restrictions that change along the 

dorso-ventral axis of an organ in a reproducible and stereotypic fashion. Until now such super long range 

genomic interactions were considered highly variable and appropriate only for stochastic transcriptional 

outcomes48

. Moreover, this is the first ever demonstration of a role of NFI proteins in the regulation of nuclear 

architecture and in establishing epigenetic memory, opening new routes for the study of these protein at the 

context of cell type specification. Finally, gradients of H3K79me3 along developmental axes represent a 

completely novel finding, adding a new potential function of this histone modification in patterning. 

From the technical standpoint we utilize cutting edge genomic approaches, such as in situ HiC and 

Cut&Run49

, which are applied in zonally dissected, FAC-sorted populations from mice carrying numerous 

genetically modified alleles. To our knowledge, we are the first to combine these state-of-the-art genomic 

approaches with sophisticated and complex genetics in any model organism. Thus, we approach an elusive 

and fundamental biological problem with a multi-disciplinary approach that deploys state of the art techniques 

and is poised to produce novel regulatory principles with general impact in developmental biology. 

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Research Strategy 
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Approach 

Soon after the identification of the mammalian OR superfamily5°, it became apparent that the expression of a 

given OR gene is spatially restricted to a specific segment, or zone, along the dorso-ventral and apico-basal 

axes of the MOE5

·

6. In mice there are 5 broad zones of OR expression51 (Figure 1) plus the septal organ52·

53 

·

55 and the Gruenemberg ganglion54 . Each zone represents an area that expresses only 50-200 ORs, in a highly 

reproducible and stereotypic fashion. Zones do not have sharp borders, but continuous boundaries in which 

ORs from neighboring zones are expressed in an intermingled fashion56

. Zonal OR expression is conserved 

59 across evolution and repored in flies57

, zebrafish58· , frogs60, salamanders61 , and various mammalian species, 

from rodents to goats62. This expression pattern is not an evolutionar remnant inherited from aquatic 

vertebrates; although about 100 of the mammalian ORs are class 1

63, or fish-like ORs, the majority of the 

6 mammalian chemoreceptors are class II ORs that emerged in amphibians60· . Thus, as the OR repertoire 

expanded dramatically during terrestrial adaptation65, the "logic" of zonal expression was preserved. Yet, 

nothing is known about the mechanisms that generate zonal boundaries. In fact, we only understand the 

spatial separations among the major chemoreceptor subfamilies, olfactory and vomeronasal receptors (VRs)66. 

Figu r e3: An OSN has th e pot enetial to ex press any OR fr om its zone. a, li neage tracing exp eri ment from a pseu dogenized 

zone 5OR (Olfrl507) and RNA-s eq a n aiysis of the OSNs that initi a lly c ho o se Olfrl507, revea ls preferenti al switc hi ng to z o ne 5

and 4.5 (border between zones 4-5), but also i n frequent but sign ificea nt switch to zo ne 4 ORs. b, In c ontrast a c lass I (fish-lieke 

OR) switcehes oniy to class ORs. I 

Briefly, ORs and VRs become restricted to the MOE and vomeronasal organ (VNO) respectively, under 

the control of transcription factors Fezf1 and Fezf267•

68, during embryonic development. Within these 

chemosensory organs there are two additional major divisions. In the VNO, the two main classes of VRs (V1 Rs 

and V2Rs) become spatially segregated in basally and apical sensor neurons (VSNs). This spatial distinction 

is initiated by transcription factors Bcl11 b69 and maintained by transcription factor AP2-E70. Bcl11 b appears to 

also act in the MOE, where it regulates the distinction between class I and II ORs71 . There are additional 

fundamental differences in the regulation of these ORs. Class II OR gene activation is mediated by multiple 

trans-interacting enhancers, the Greek Islands, which form a multi-chromosomal enhancer hub over the active 

OR in an Lhx2- and Ldb1-dependent fashion. In contrast a single enhancer, the J element that does not 

interact with Greek Islands in cis or trans, appears sufficient for the regulation of class I ORs72•

73, which all 

reside in a single genomic cluster. Consistent with a distinct regulator logic between the two major OR 

classes, straight Lhx2 KO mice express class I but not class II ORs74, and the same is true for Ldb1 KO 

Page 42 

Research Strategy 
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OSNs16. Finally, unclear are the mechanisms that determine which sensory neurons express other major types 

of chemoreceptors, such as MS4A7s and TAAR76 genes in the MOE, and FPRs77 in the VNO. However these 

gene families are also organized in a single cluster each, and have different nuclear organization than class II 

ORs16·78

, suggesting that class I-like mechanisms may by involved in their regulation. In contrast, the >1000 

class II ORs are organized in 60 genomic clusters distributed across 18 chromosomes making it difficult to 

regulate them as a group by targeting a single cis regulatory element. 

A challenge in deciphering zonal OR expression stems from the observation that the multichromosomal enhancer hub, which mediates class II OR choice, has similar constitution across zones79 and 

interacts at similar frequencies with the active OR irrespective of zonal identity1s

,,e6 (Figure 2). Moreover, the 

intergenic class II enhancers (known as Greek Islands) are homogeneously bound by Lhx2, Ebf and Ldb1, and 

have comparable accessibility in these 3 zones79

. Instead, what may be subject to zonal restrictions is not the 

activity of the Greek Islands, but their affinity for different OR genes. Thus, class II OR choice may be 

accomplished by a combination of deterministic and stochastic mechanisms: a deterministic mechanism that 

uses spatial cues to "mark" the ORs that can be chosen in each zone, and a stochastic mechanism that 

deploys the same multi-enhancer hub, across zones for a random selection among enabled ORs. Alternatively, 

the Greek Island hub could interact with any OR in any OSN, but transcription factor combinations would 

determine the consequences of hub-OR interactions via specific binding to OR promoters. 

Figu re4: NFI motifs enr iched on pr omo teres of zone 5 ORs. Usi ng HOMER for de novo moti f analysi s we i denti fied NFI moti fs 

as beieng enriched on zoene 5 promoters over zo ne 1 promoters, which are enriched for Ebf and Lhx2. We used zone 1 and 5OR 

definition s from Tan and Xi e 2018. 

Our data suggest that a combination of both models is in place: In zone I, only zone I ORs have the 

proper genomic positioning that would allow association with the Greek Island hub, whereas in zone 5 every 

OR is privy to these interactions and, therefore, eligible for activation. However, only zone 5 OR genes are 

chosen because zone 1-4 OR genes are repressed via H3K79me3. Our data suggest that different NFI family 

members are responsible for the recruitment of zone 5 ORs to the OR compartments and, possibly, for the 

zone 5-specific repression of "out of zone" ORs. Thus, an elegant mechanism for zonal OR expression deploys 

two competing processes: gradual "enabling" of an increasing number of OR genes via recruitment to the OR 

compartment and selective repression of the enabled "out of zone" ORs). 

Aim1: Determine which NFI genes are required and sufficient for establishment of zone 5 identity. 

Preliminar data: Before attempting to identify common features between ORs from the same zone we clarified 

whether an OSN has the potential to transcribe every OR from its respective zone. To answer this, we 

perormed RNA seq on FAC-sorted OSNs that had originally chosen a pseudogenized Olfr1507 allele 

(Olfr1507deliresCre from zone 5), using a lineage tracing strategy80• RNA seq of these FAC-sorted OSNs 

revealed that any zone 5 ORs1 can replace the mutant Olfr1507 allele (Figure 3a). Interestingly, zone 4 ORs 

are also detected in these cells, albeit at lower levels, consistent with the continuous nature of zones. The 

same analysis with a pseudogenized class I OR81

, revealed expression of other class I ORs only, without 

"leakage" to class II ORs, consistent with a sharp cell fate distinction between class I and II OSNs (Figure 3b). 

We searched for unique features that distinguish zone 5 ORs from ORs that cannot be expressed in 

Olfr150r OSNs. We compared the promoters of ORs expressed in the most distant zones, zones 1 and 5, by 

perorming de novo motif analysis on 1 Kb fragments centered at the transcriptional start site (TSS) of these 

genes, since these sequences were previously shown to contain zonal information82·

83. This revealed 

enrichment of Ebf and Lhx2 motifs on zone 1 promoters, consistent with previous reports on zone 1 OR 

Olfr15183•

84 (Figure 4a). Zone 5 promoters, however, have low quality Ebf motifs and strong enrichment for 

nuclear factor I (NFI) motifs (Figure 4b). NFI motifs were previously repored on genes expressed in mOSNs8s 

but the functional significance of these elements in the MOE is unclear. 

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Research Strategy 
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The NFI family of transcription factors has 4 members NFIA, 8, C and X, which bind on DNA as homoor hetero-dimers and act as activators or repressors of transcription86·

87. Zonal RNA-seq comparing the levels 

of NFI genes in mature OSNs and in various OSN progenitors from zones 1 and 5 revealed increased 

expression of NFIA, 8, and X in zone 5, with distinct transcriptional dynamics: NFIA and 8 are more highly 

expressed in zone 5 progenitor cells, whereas NFIX is upregulated in zone 5 OSNs (Figure 5a, b). Zonespecific OMNI-ATAC-seq analysis88 in the same FAC-sorted populations revealed NFI as the motif that is most 

highly enriched in zone 5 specific peaks, supporing a functional role for these zonally expressed factors 

(Figure 5c). To achieve these zonal dissections we used Olfr1 GiresGFP (zone 1) and Olfr1507iresGFP (zone 

5) mice to identify these zones. The identity of OR genes in each micro-dissection, as determined by RNA-seq,

was used in every experiment to confirm the accuracy of our dissection. These micro-dissections had low level 

contamination from the neighboring zones, however, there was no zone 1-3 ORs in micro-dissected zone 5 

and not zone 3-5 ORs in micro-dissected zone 1. 

We embarked in 

a genetic analysis of 

the functional role of 

the three zonal NFI 

genes, using

conditional KO mice for

the three zonal NFI

genes, shared with us

from Dr. Gronostasjki.

We first perormed a 

homozygote triple NFI 

deletion in the stem

cells (H8Cs) of the 

MOE, using the

Krt5CreER mice.

Deletion was induced 

by tamoxifen injection

of adult mice16. A Creinducible reporter

(TdTomato) was used 

Figu res: Zone 5ex pression of NFI pro teins. A, expr essi on of NFI genes in zo ne 1 (r ed) and 5 to mark the recombined 

(blue) duri ng differenti atieo n. Fr om leftto ri ght c ells ar e Mashl\ Mashl+/Ngnel

+

, Ngnel

+

Omp , + B, stem cells and of all 

NFIA immunof lu or escencee shows strong NFI A expressi on in apic ally located Sustentacu lar cells their progeny16. We 

ofboth zoenel andzone 5, a nd wea ker expr essieon in thebasal(progenitor) cell la yers o fzo ne 5. The induced differentiation 

area between the doted li n es conta i n sOSNs andOSN progenitors. C, omni -ATAC-seq fromzo ne 1 of the NFI KO stem cells and zo ne 5 s hows that in zo ne 5 specific peaks, the most c o mmo n motifis NFI , andthe s eco nd via methimazol ablation most commo n is a ha lfsite ofNFL of the MOE8992. We let 

the MOE to fully recover by waiting for 40 days, and then we FAC-sorted tomato+ cells from control and triple 

NFI KO mice. As previously described, we used tomato signal intensity to isolate OSNs, which were subjected 

to expression analysis16. 

RNA-seq comparing control and triple KO OSNs sorted from the whole MOE revealed a strong 

reduction in the levels of zone 4 and 5 ORs (Figure Ga), consistent with the hypothesis that NFI proteins 

regulate zone 4-5 OR expression. We also noticed a significant increase in zone 2 OR mRNA, suggesting 

ectopic expression in zones 4-5. Indeed, RNA-seq on OSNs sorted from micro-dissected zones 4-5 revealed 

strong expression of zone 2-3 ORs in the triple NFI KO OSNs, together with strong downregulation of zone 4-5 

ORs (Figure Gb). This ectopic OR expression explains why KO OSNs differentiate properly and express mOSN 

9 markers, since OR expression is required for OSN differentiation93· . Thus, triple NFI deletion in H8Cs results 

in a homeotic transformation of zone 4 and 5 OSNs to zone 2 and 3 OSNs, in regards of OR expression. 

Intriguingly, in NFIA, 8 double KO mice, this homeotic transformation shifted towards higher zone indexes, with 

zone 3 and 4 ORs replacing zone 5 ORs (Figure Ge). Thus, NFIX may be repressing zone 2 ORs in zone 5, 

while NFIA and 8 are responsible for zone 5 OR expression. Finally, it should be noted that among 200 other 

genes that are significantly enriched in zone 5 OSN and progenitors, only 30 are significantly downregulated in 

triple NFI KO (Figure Ge), with no transcriptional regulators being included in this list. Thus, most likely we are 

Page 44 

Research Strategy 
Contact PD/Pl: Lomvardas, Stavros 

not describing a drastic change in cellular identity but a specific alteration in zone 5 OR gene regulation that is 

likely mediated by the transiently expressed NFIA and B. 

To examine whether continuous NFI activity in mOSNs is required for stable zone 5 OR transcription 

we deleted NFIA, B, and X with OmpiresCre, which is only expressed in mOSNs. Although NFIA and B 

become downregulated with differentiation, they are still expressed in mOSNs, thus, they may still be required 

for OR expression. This is the case for Lhx2, which is downregulated during OSN differentiation yet, its 

79 continuous expression is necessary for OR transcription1 6• . However, triple NFI deletion with OMPiresCre, 

which is expressed only in mOSNs, revealed small downregulation of zone 4-5 ORs (Figure Gd) suggesting 

that NFI activity is predominantly required in OSN progenitors. Thus, our data confirm a role of NFI proteins in 

establishing zone 5 OR expression during differentiation and raise the following questions: 

Fig u re6: Trip le NFI dele tion abolis hes zone 4 aned 5 OR expr ession and allows ec topic ex pr ession of zone 2 and 3 ORs. A, 

RNA-seq in mOSNs so1ted from whole MOE s hows reduction of zone 4 a nd 5 OR mRNAs a nd i ncrease in zone 2 OR expression, 

in early (Ktt5CreER dri ver) NIF tri ple deleti on. B, RNA-seq in mOSNs s01ted from micro-di ssected zone 5 shows si gnific a nt 

downregulati on of zone 4 and 5 ORs and concomita nt upregulati on of zone 2 and 3 ORs. C, RNA-seq in mOSNs sorted from mic rodissected zone 5 shows strong downregu lation of zone 5 ORs, sustai ned expression of zone 4 ORs, ectopic expression of zone 3 but 

not zone 2 ORs in early NFIA, B double KO mi ce. D, RNA-seq in mOSNs s01ted from whole MOE shows small effects on zone 4 

an d 5 ORs in late (Ompi resCre) triple NFI KO mice. E, volca no plots showi ng the mOSN markers and zone I-enric hed genes are 

not affected by early triple NFI deleti on. Only a small fraction of zone 5-en riched genes is downregu lated in early triple NFI KO 

mice, a nd non of those downregu late d genes is a tra nscriptional regulator. Data represent a verages from biologiceal tripliceates 

1A. NFIA, NFIB, or both NFIA and NFIB are required for zone 5 OR transcription? Generation of individual 

NFIA and NFIB KO mice with Krt5CreER, will reveal which NFI gene regulates zone 4 and 5 OR transcription. 

Control and KO littermates will be characterized by RNA-seq performed on FAC-sored OSNs from the whole 

MOE or from micro-dissected zone 5. RNA-seq will be verified with IF and RNA ISH for zone 5 ORs. If the two 

factors were fully redundant, then individual NFI deletion would have a small effect in zone 4-5 OR 

transcription. If only one of the two factors is essential for zone 5 transcription, then its deletion will recapitulate 

the double deletion. The NFI gene that does not enable zone 5 ORs may be responsible for repression of zone 

1 or 3 OR genes, similarly with the role of NFIX in repressing zone 2 ORs. A third possibility is that NFIA and B 

Page 45 

Research Strategy 
Contact PD/Pl: Lomvardas, Stavros 

have differential affinity for zone 4 and 5 ORs, with each deletion affecting a fraction of the ORs that are 

downregulated in the double and triple KO. 

1 B. Are NFI proteins sufficient for specifying zone 5 OR expression? To explore if NFI proteins are 

sufficient for enabling the choice of zone 4-5 ORs we will induce ectopic NFI expression in OSN progenitors 

from other zones. We generated a tetO-NFIA2aNFIBiresmCherry transgenic line, which will allow conditional 

expression of the two key NFI genes in a non-zonal fashion. We have one transgenic line established (data not 

shown) and we are repeating injections for additional ones. We will cross the transgenic NFI mice with Gg8tTA 

mice, which will allow ectopic expression of the transgene in OSN progenitors throughout the MOE. If NFIA 

and B are sufficient for zone 5 OR transcription, then a transient pulse of these proteins should generate 

mOSNs that transcribe ectopically zone 4-5 ORs. If we fail to induce ectopic zone 5 OR expression, then either 

additional zone 5 specific factors are required, or repressors for "out of zone" ORs prevent ectopic induction of 

zone 5 ORs. Either result will be insightful and will not impact other aims. 

Caveats, pitfalls, alternative approaches: Given the effects of the triple and double NFI deletions, we do not 

anticipate technical 

issues 

explaining why NFIX 

alone cannot replace 

genes.

This is highly unlikely, 

since our data suppor

or unexpected

outcomes. An extreme 

scenario predicts that

at least 2 NFI genes

are needed for zone 5 

OR expression,

the other two

a different role for 

NFIX. However, if 

individual KO mice do 

not have strong 

phenotypes, we will 

generate NFIA, X and 

NFIB, X double KO to 

test this scenario. 

Finally, we will 

generate individual 

NFIX KO mice that will 

allow us to test if

zone2 ORs can be

ectopically expressed 

in zone 5 with zone 5

ORs, revealing 

Figu r e7: Cue t&Ruen on micr o-disse cted zone 5 re v ea ls NFI bin ding on G reek Islands. A, NFIA whether ectopic OR

Cut&Run peaks that co inci de with zone 5 specific omni-ATAC-seq pea ks B, NFI motifs are the expression can occur

most highly enriched motifs on NFIA Cut&Ruen peaeks. C, NFIA peaks are detected on Greek when native zonal ORs 

Islands. D, Heat map ofNFIA Cut&Run peaeks on the 63 Greek Islands. are transcribed. 

Induction of 

96 transgenic NFI proteins by Gg8tTA95· may not be induced early enough, failing to enable ectopic zone 5 OR

transcription. In this case we will induce NFI earier, using Krt5CreER crossed to a ere-inducible tTA. 

Repeated tamoxifen injections will induce permanent tTA expression in HBCs and their progeny, resulting in 

stable expression of the transgenic NFI proteins. Methimazol ablation of the MOE will be used to induce HBC 

differentiation into OSNs. If needed, doxycycline will turn off the expression of transgenic NFI, generating 

mOSNs that experienced a pulse of NFI at progenitor stages. To assure that transgenic NFI is expressed at 

physiological levels we will screen transgenic founders for ectopic NFI expression at the levels of the 

endogenous NFI proteins in zone 5, using IF. To exclude transgenic artifacts we will rescue NFIA, B double KO 

mice with transgenic NFIA, B expression. If the transgene restores expression of zone 4 and 5 ORs then the 

Page 46 

Research Strategy 
Contact PD/Pl: Lomvardas, Stavros 

timing and levels of transgenic NFI expression are appropriate. Alternatively, a CRISPR/dCAS9-AD strategy97 

will be deployed for the activation of the endogenous NFI proteins at physiological levels. We will use dCas9-

AD mice (Jackson Laboratories) and transgenic expression of sgRNAs under control of the tetO promoter 

induced by Gg8-tTA. The level of induction will be controlled by the number of sgRNAs used to recruit the 

activating dCAS9 to the NFI loci97. 

Figuer e8: Zonael HiC revea ls thaet 

NFI reguelaetes nuclear 

ar chitectu re of Zone 5 OSNs A, 

Circos p lots depi ctie ng the top

1000 trans i nter actions in zoene 1, 

2 and 5 OSNs, with OR-OR 

i nteractieones beieng dep icted in r ed. 

B, Same ciercos plot from triple 

NFI KO OSNs from zo ne 5, 

shows r eductieon of trans OR-OR 

c ontacts to zone 2 levels C, 

Plotti ng cumu lati ve tra ns contacts 

of a ll the ORs, separeated by theier 

zonael inedexes, acreoss a large OR 

c luster in chromo s ome 2 that 

c ontaiens OR genes from everey 

NFIABX KO zone 5OSNs zo ne 1

and 2 ORs have enric hed contacts 

zone ( depicted with our standard 

color code at the bottom of the 

p lot. In wi ld type Zo ne 5OSNs

the di stributieon among zones is 

homogeneous, but in early

with each other. D, A zoene 2-lieke 

tra n sformatieon is obvieous in a 

genomewiede anaelysies of OR-OR 

c ontacts accor di ng to zone i ndex. 

Data ar e a ver ages of bi o lo gic al 

dup licates 

Aim2: Determine how NFI proteins regulate zonal OR expression. 

Preliminar data: A question emerging from our studies is whether NFI proteins enable the expression of zone 

5 ORs by direct binding, or by activating secondar transcription factors. Due to the enrichment of NFI motifs 

on OR promoters of zone 5 ORs, we favor a model of direct NFI binding. In support of this, the 4 other 

transcription factors that are specifically expressed in zone 5 OSNs, Dlx4, Msx1, Meis1 and Meis2, do not 

change significantly in triple NFI KO. Obtaining evidence for direct binding of NFI proteins on zone 5 ORs is 

challenging since these factors are zonally expressed and dynamically regulated. Crosslinked ChlPs require 

hundreds of thousands of cells, which cannot be obtained for zonally isolated FAC-sorted cells. We therefore 

propose a novel approach for the detection of protein-DNA interactions named "Cut&Run", which was recently 

9s.100 applied on populations of a few hundred cells49• . 

We established conditions for NFIA Cut&Run on micro-dissected zone 5 cells. Despite using a mixed 

cell population in our proof of principle experiments we obtained NFIA peaks that, by and large, overlap with 

computationally predicted NFI motifs. In fact, de novo motif analysis of all the NFIA peaks returned NFI motifs 

as the most enriched in these peaks (Figure 7a, b). Interestingly, we detect NFIA peaks on Greek Islands, 

furher supporting a direct role of this protein on OR gene regulation (Figure 7c, d). In this mixed population we 

do not detect NFIA peaks on zone 5 OR promoters, which is expected since each promoter is active in at least 

~100 fold fewer cells than the Greek Islands. Alternatively, NFIB may be binding on OR promoters and NFIA 

on Greek Islands, mediating zone 5 OR-Greek Island contacts through NFIA-B interactions. Experiments on 

pure cell populations will resolve these models. 

2A. Perorm cut and run for NFI proteins in distinct cell types isolated from zone 5. With Cut&Run 

protocols in place we will use specific antibodies for NFIA, NFIB, and NFIX to determine their genomic targets 

in mash1, mash1/Ngn1, Ngn1, and OMP-expressing cells from zone 5. In addition, we will FACS Olfr150r 

cells, using an Olfr1507iresGFP knock-in mouse. We expect to detect transient binding of NFIA or NFIB (or 

both) on the majority of zone 5 ORs at the Mash1+

/Ngn1+ positive state that will be gradually eliminated from 

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Research Strategy 
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the non-chosen ORs as cells commit to a single OR. In this case we expect continuous NFI binding only on the 

transcriptionally active Olfr1507. Based on our genetic observations, we expect NFIX binding on the zone 2 

ORs, if NFIX directly represses these ORs in zone 5. 

28. Determine the role of NFI proteins in establishing zonal interchromosomal interactions.

Preliminar data: An important question emerging from our studies is how NFI proteins influence the nuclear

101 architecture of zone 5 ORs, which as we previously showed plays a critical role in OR gene choice 15•16· . We 

perormed HiC in mOSNs from zones 1, 2 and 5, which revealed a transient increases in trans OR-OR 

interactions as we transition from zone 1 to zone 5 (Figure 8a). Intriguingly, in triple NFI KO OSNs, trans OROR interactions are reduced to the levels of zone 2 OSNs (Figure 8b), consistent with the ectopic zone 2 OR 

transcription in these cells. Virtual 4 C analysis of the largest OR cluster in chr. 2, which contains ORs from 

ever zone, shows that in zone 2 OSNs, zone 1 and 2 ORs from the other chromosomes have increased trans 

contacts with the zone 1 and 2 ORs from this cluster, whereas in zone 5 OSNs the distribution is more uniform. 

However, in NFIABX KO OSNs from zone 5, this pattern reverts to the pattern observed in zone 2 OSNs 

(Figure 8c), suggesting that zone 1 and 2 ORs have more robust and selective contacts in the absence of NFI. 

This transformation in 30 nuclear architecture is confirmed by genomewide quantifications of HiC contacts, 

showing significant reduction of zone 5 trans OR-OR contacts in the NFI KO (Figure 8d). The dramatic 

differences in zonal genomic interactions is not an epiphenomenon stemming from diferences in 

transcriptional states: Even within their zone each zone 4 and 5 OR is transcribed, on average, in less than 1 % 

of the OSNs, making their interactions undetectable in the mixed population. Furthermore, the zonal 

differences in OR-OR interactions are first detected prior to OR gene activation by the Greek Island hub. Given 

the imporance of OR-OR interactions in OR transcription, these data suggest that NFl-dependent recruitment 

of zone 5 ORs to the OR compartment enables subsequent interactions with the Greek Island hub and, thus, 

transcriptional activation. Unpublished observations suggest the NFIA interacts with Lhx2 and H3K9me3 (data 

not shown), providing a possible mechanism for the role of this factor in mediating zone 5 OR interactions with 

the rest of the OR repertoire. 

Proposed experiments: To dissect the relationship between zonal OR transcription and NFl-dependent

genomic contacts we will perorm zonal in situ HiC in the various KO mice from Aim1. These experiments will 

uncover the contribution of every NFI factor in the establishment of interchromosomal OR-OR interactions. We 

anticipate to identify a strong correlation between transcriptional consequences and trans OR contacts in a 

zone-dependent fashion. Moreover, if ectopic expression of transgenic NFIA, B in progenitor cells induces 

ectopic expression of zone 4,5 ORs, we expect that we will also detect ectopic recruitment of these genes to 

the OR compartments, demonstrating sufficiency of NFI proteins in sculpting the OSN nucleome. 

Figuer e9: Zonael OR

markineg byH3K79me3 A,

H3K79me3 di stribution on 

OR genes from various zones 

by ChIP-seq on mOSNs from

the whole MOE. B, same

ChI P focused on a clu ster 

with zone 1 and 5ORs. C,

Heat m ap depicting 

H3K79me3 ChI P-seq s igna l

on OR genes from various

zonees in mOSNs i solated 

from micro-di sscted zones 1 

(r ed) an d 5 (blue). In zonee 1,

zon e 1 ORs have the highest 

H3K79me3 siegnal whereas i n

zonee 5 H3K79me3 is evenely 

di stri buted across zon a l

i ndexes D, Zone 5

H3K79me3 ChIP-seq at

diefferent OSN-progenitor 

populati ons reveaels delaeyed 

marking of zone 5ORs. Data 

ar e a verages of biologiceal 

dup licates 

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Research Strategy 
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2C. To explore the role of NFI proteins in H3K79me3-dependent repression of "out of zone" ORs. Our 

results may explain how zone 5 ORs become enabled for zone 5-specific transcription but they do not account 

for the lack of zone 1-3 OR expression in zone 5. The observation that NFIX prevents ectopic zone 2 OR 

expression in double KO mice, suggests that a repressive mechanism may keep "out of zone" ORs silent in 

zone 5. OR genes are marked by repressive H3K9me3, but in a non-zonal fashion. We, thus, searched for 

repressive histone modifications with zonal distribution. One such modification is H3K79me3, which is also 

found on pericentromeric and subtelomeric heterochromatin102 and was previously implicated in mono-allelic 

expression103

. H3K79me3, unlike H3K9me3 and H4K20me3, is irreversible, since no de-methylase for H3K79 

has been identified in yeast or higher animals. Thus, we hypothesized that H3K79me3 is used for irreversible 

repression of enabled ORs that should not be expressed. In this logic, in zone 1 OSNs, the zone 1 ORs that 

were not chosen for transcription should be the only ORs repressed via both H3K9me3/H3K79me3. In zone 2, 

all the zone 1 and 2 ORs should be H3K79me3-positive, but not zone 3-5 ORs, and so forth, predicting that 

zone 1 ORs will have the highest levels of H3K79me3 and zone 5 ORs the lowest. Indeed, native H3K79me3 

ChlP-seq in OSNs from the whole MOE revealed this exact distribution (Figure 9a, b). Furthermore, zonal 

dissections confirmed the zonal dependency on H3K79me3 distribution, with all the ORs having this mark in 

zone 5 (except class I ORs) and only zone1 ORs in zone 1 (Figure 9c). Importantly, in zone 5 H3K79me3 is 

deposited first on zone 1 ORs and last on zone 5 ORs during differentiation, consistent with the notion zone 5 

OR repression occurs after one is chose for transcription (Figure 9d). Indeed, the transcriptionally active 

Olfr1507 is devoid of H3K79me3, based on ChiP-seq experiments from sored Olfr1507+ OSNs (data not 

shown). 

Proposed Experiments: We seek to determine the role of NFI proteins in establishing zonal H3K79me3 

deposition and to test genetically the role of H3K79me3. For the first goal, if only ORs recruited to the OR 

compartment become marked by H3K79me3, then triple NFI deletion should result in loss of H3K79me3 from 

zone 4-5 ORs and delayed marking of the ectopically expressed zone 2-3 ORs. ChlP-qPCR experiments 

reveal the reduction of H3K79me3 from zone 5 ORs, without changes in H3K9me3, confirming that only the 

H3K79me3 is zonally regulated via NFI (Figure 10). Deep sequencing will determine the genomewide effects 

of NFI deletion in H3K79me3 distribution. We will repeat these ChlP-seq experiments in the double NFIA, B 

KO mice, determining if NFIX preserves high levels, and early deposition of H3K79me3 on zone 2 ORs. 

Finally, we will ask if ectopic expression of the NFIA, B transgene alters H3K79me3 deposition in other zones. 

Figur elO: NFI del etion de ple tes H3K79me3 from zone 

5 ORs. ChI P-qPCR from control and tri ple early NFI KO 

OSNs micro-di ssected from zone 5 shows strong reductieon 

of zone 5 ORs. Adcy3, whiech is not marked by 

H3K79me3, is used as negati ve coentrol. Data are a verages 

of two bi o lo gi cal replic ates. Re ductieon in zone 5 is 

siegnieficant (p<10·5, Student's T-test) 

For the functional interrogation of H3K79me3 we will 

perform conditional deletion of Dot1 L 102

, the only 

mammalian enzyme catalyzing H3K79 tri-methylation. 

Heterozygote Dot1 L deletion in HBCs, results in 

reduction of H3K79me3 on ORs, supporting the role of 

Dot1L in this process (data not shown). We anticipate 

that in homozygote KO OSNs we will detect complete 

loss of H3K79me3 and ectopic OR expression, the 

extent of which will be dependent on the timing of this 

deletion: Deletion in HBCs, may allow ORs from every 

zonal index to be expressed in zone 5; deletion in early 

GBCs may allow ORs from zones 2-3 to be expressed in 

zone 5; and deletion at the moment of OR choice may 

impair only zone 5 ORs, which may be co-expressed in 

zone 5 OSNs, violating the "one receptor per neuron" 

rule. Conditional deletions in GBCs will be performed 

with Mash1-CreER (early GBC) and Gg8-Cre (late GBC), 

whereas deletion at the stage of OR gene choice will be 

achieved with Olfr1507iresCre. Ectopic OR expression 

will be assessed by zonal RNA-seq, IF and RNA ISH, 

whereas OR co-expression will be determined by single 

cell RNA seq and two-color fluorescent RNA ISH. 

Caveats. petfalls. alternative approaches: Based on our

Cut&Run results we do not anticipate technical issues. There are commercially available antibodies that were 

successfully used for NFIA, B and X Ch IP-seq experiments 104 assuring that this approach will be successful for 

Page 49 

Research Strategy 
Contact PD/Pl: Lomvardas, Stavros 

each NFI factor. We have also established Ch IP-seq experiments on FAC-sored Olfr150r OSNs, thus, the 

more sensitive Cut&Run assay will provide cleaner results. If we discover that NFI binding on zone 5 ORs is so 

transient that it is not detectable even at pure populations, we will deploy alternative approaches. We could use 

the transgenic NFI mice to perform Cut&Run under conditions of stable NFI expression. A second approach 

will be to use CRISPR/Cas9 to generate fusions between NFIA or NFIB and a bacterial adenine 

methyltransferase105, allowing a OamlO-based106 approach for the identification of NFIA and NFIB targets. 

Also, we are currently experimenting with Cut&Tag, a newer version of Cut&Run applicable to single cell 

analysis, which would overcome issues of heterogeneity in progenitor populations. Finally, we can use 

CRISPR/Cas9 to delete predicted NFI motifs on zone 5 OR promoters and ask if this afects their expression. 

In the unlikely event that NFI proteins do not bind on zone 5 OR promoters, the other goals of the grant will not 

be afected. In fact, with the data presented on 2B and 2C we already have epigenetic mechanisms in place 

that likely explain these zonal restrictions. Since no other transcription factors are both highly and zonally 

expressed in an NFI dependent fashion we will explore other possibilities. For example, we will explore 

whether NFIA binding on Greek Islands alters the affinity of the hub towards zone 4-5 OR promoters. 

Alternatively, we will search for zonal differences on factors regulating gene expression at the posttranscriptional level. 

Regarding the proposed HiC experiments, based on the strong preliminary data, we do not anticipate 

technical difficulties in the execution and analysis. Similarly, the effects of the triple NFI deletion in 30 nuclear 

architecture render unexpected results very unlikely. 

H3K79me3 ChlP-qPCR from NFI KO mice show our ability to execute the proposed zonal experiments 

and support our hypothesis, thus, we do not expect challenges. We do not know if Oot1 L deletion will affect 

zonal OR expression, which constitutes the only goal of this proposal not guaranteed to generate positive 

results. If Oot1 L deletion does not affect OR expression, then we will seek additional repressive marks with 

zonal distribution. We will ask if Polycomb complexes are implicated in zonal OR silencing and whether DNA 

methylation plays a role in this process. Finally, we will enterain a different scenario: Our data show that zone 

1 ORs are recruited first in the OR comparment, followed by zone 2-3 ORs, and culminating in the recruitment 

of zone 4-5 ORs. Possibly, this gradual process results in a 'layered" assembly of the OR compartment, with 

zone 1 ORs in the center, surrounded by zone 2-3 ORs in the middle layer and zone 4-5 ORs in the external 

layer, which is proximal to the Greek Island hub15 101 . Ongoing super-resolution DNA FISH with zonal pan-OR ·

probes will test this model. 

Measures to assure rigor and 

Two-part model of zonal OR reproducibility: RNA-seq will 

expression be performed in 4 biological 

replicates, with each replicate 

being generated from a 

zones separate mouse. PCA will ORs I. Enabling 

recruitment into the OR compartment identify outliers among

zone4 enables ORs for future gene choice replicates, which will be

ORs removed and replaced with 

new replicates. In addition to 

zone3 zone3 zone3 including TdTomato in our 

ORs ORs ORs II . Repressing crosses, efficient deletion of the 

H3K79me3-md ated iriversible conditional KO genes will be silencing prevents "out of zone" 

expression of enabled ORs confirmed by measuring the

) reduction of reads on the flexed 

zonel zonel zonel zonel zonel exons, using Sashimi, as 

ORs ORs ORs ORs ORs previously described. Mice will 

be at the same exact age when 

Figur ell: A model for zona l OR reguelation. In eac h zoen eORs , from that zone and any induced by tamoxifen (P30)

previ ous zo ne are en abled by bei n g recruited to the OR compaiiment. ORs from the and after every methimazol 

previ ous zo n es are repressed by H3K79me3, a llowi ng only ORs from the proper zone to ablation we will wait 40 days be c ho s en for tra nscriptieo n. for the epithelium to be fully 

restored. Control and experimental mice will always represent littermates. Each HiC experiment will be 

perormed in biological duplicates, with 500 million HiC contacts per replicate. Replicates will be analyzed 

separately and tested for reproducibility (reproducibility of contact curves, fraction of HiC contacts mapping to 

OR genes, etch). Additional replicates will be performed needed. Replicates will then be combined to increase 

Page 50 

Research Strategy 
Aim Year 1 Year 2 Year 3 Year 4 Year 5 

1A 

Generate individual NF A 

NF B and NF X KO mice with 

Krt5CreER and d omato 

Analyze the 3 individual KO 

strains by RNA-seq on whole 

MOE and micro-dissected 

zone 5 

Con irm with F and RNA SH 

Generate double NF A X and 

NF B X KO mice with 

Krt5CreER and d omato 

Analyze the 2 double KO 

strainsby RNA-seq on whole 

MOE and micro-dissected zone 

5 

Con irm with F and RNA SH 

1B 

Generate multiple tetONF A2aNF BiresmCherry lines 

and screen them or 

expression levels similar 

endogenous NF A B in zone 5 

Analyse the e ects o ectopic 

NF expression in zonal OR 

expression 

Cross NF transgenic with NF 

KO mice 

Explore i transgenic NF 

expression can rescue NF 

deletion in zone 5 OR 

expression 

2A 

Per orm Cut&Run in FAC 

sorted cells rom microdissected zone 5 

Per orm Cut&Run in FAC 

sorted cells rom microdissected zone 5 rom triple 

NF KO mice 

Per orm Cut&Run in FAC 

sorted cells transgenic NF 

mice 

2B 

Per orm in situ HiC in late triple 

NF KO mice to con irm 

nuclear architecture is intact 

Per orm in situ HiC in individual 

NF KO mice and in double NF 

KO mice using zone 5 microdissections 

Per orm in situ HiC in 

itransgenic NF mice both in 

zone 5 and zones 1-3 

2C 

Per orm H3K79me3 Ch P-seq 

on triple NF KO mice using 

zone 5 microdissections 

Per orm H3K79me3 Ch P-seq 

on double and individual NF 

KO mice using zone 5 

microdissections (plus zone 1 

and 3 or the NF X KO) 

Generate Dot1L KO mice 

using Krt5CreER Gg8-Cre 

and Ol r1507iresCre drivers + 

d omato 

Analyze the various Dot1L 

mutants by RNA-seq and 

H3K79me3 using microdissected zone 5 

Analyze the various Dot1L 

mutants by HiC 

Contact PD/PI: Lomvardas, Stavros Research Strategy Page 51

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

genomic resolution. Our HiC analysis uses a stringent cutoff for removal of all multi-mappers. In situ HiC 

results will be independently confirmed by two- and three-color DNA FISH experiments as we have done in the 

past15,107. Control and experimental sections will be placed on the same slide and imaged under the same 

settings to minimize variability. Cut&run experiments will be performed in biological triplicates and NFI KO mice 

will be used as controls for antibody specificity, as well as, IgG isotype controls. 

Sex as biological variable: We have not observed sex dependent differences in the zonal expression of ORs. 

That said, in our 4 replicates we will have 2 males and 2 females, the data will be initially analyzed separately 

for each sex and only if sex differences are not detected they will be combined. In future publications we will 

have pre-prints with supplemental figures describing the data in a sex-segregated fashion. Also, we will deposit 

our data in GEO with each replicate separate, so other researchers will have the means to perform 

comparisons between sexes. We have not detected sexually dimorphic expression of NFI genes in the MOE, 

and none of the effects described in the double and triple KO mice are sexually dimorphic. Obviously, we 

would be very excited to detect sexual dimorphisms in OR expression, and if any of the experiments suggest 

that such differences exist, we will increase our analyses to properly powered numbers of males and females. 

Summary: Our data support a model of gradual increase in the number of ORs that participate in OR 

compartmentalization according to their zonal index and the zonal location of an OSN. Since OR 

compartmentalization is a pre-requisite for OR gene choice, we view this recruitment step as “enabling” an OR 

allele for future activation. Although this model explains how each OR becomes enabled for transcription in its 

cognate zone, it does not account for the fact that out of zone ORs that are enabled, they are never ectopically 

expressed. However, the observation that there is a concomitant increase in H3K79me3 along zonal indexes 

as we transition from zone 1 to zone 5, suggest that there is also a repressive process that prevents out of 

zone but enabled ORs to be ectopically activated. Our data show zone 5-enriched NFI proteins enable zone 5 

(and 4) OR genes and repress zone 2 and 3 ORs. To assist the reviewers with our working model we provide a 

visual depiction in Figure 11. Our data cannot explain how this process is regulated in zones 1-3, where NFI 

genes are lowly expressed, thus, future studies will explore if other NFI-like factors mediate a similar function. 

Our proposed genetic experiments will test this model and will reveal mechanisms by which each NFI protein 

regulates the epigenetic landscape and nuclear architecture of OR genes in zone 5 OSN. 

Timeline: The proposed experiments, although ambitious in regards of the extensive genetic crosses and 

genomic approaches, are within the expertise of our laboratory and feasible for the timeframe of an R01 

proposal. Importantly, all the genetically modified mice are in our colony and the proposed crosses are 

ongoing, thus no delays or technical challenges are expected for this application. 
PHS Human Subjects and Clinical Trials Information OMB Number: 0925-0001 and 0925-0002

Expiration Date: 03/31/2020

Tracking Number: GRANT12921503 Funding Opportunity Number: PA-19-056 Received Date: 

2019-08-16T10:05:36.000-04:00 

Are Human Subjects Involved ❍ Yes ● No 

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

Exemption Number ❏ 1 ❏ 2 ❏ 3 ❏ 4 ❏ 5 ❏ 6 ❏ 7 ❏ 8 

Does the proposed research involve human specimens 

and/or data ❍ Yes ● No 

Other Requested information 

Contact PD/PI: Lomvardas, Stavros Page 52
Vertebrate Animals 

Description of procedures 

3 aims will require the use of Mus musculus (the house mouse). We will study both male and female mice 

but we will analyze them separately in case there are unexpected sexual dimorphisms. Also in our future 

publications, we will provide supplemental figures with where data from males and female mice will be 

segregated. This is not something we have done yet, but we will start following this principle from now on. 

Approximately 935 young adult transgenic mice (mixed background) of both sexes will be used per year for the 

purposes of this proposal. This will amount to approximately 4675 mice over the 5 year period of support 

requested. This total number of animals includes mice that will be used for breeding, mice used for the 

retention of the different strains, as well mice that will be used for experimental analysis. Careful planning and 

periodic meetings between lab members and the PI ensure that animals are effectively shared between lab 

members, minimizing the numbers of animals that need to be bred. 

Justification of animal use 

Our proposal aims to study mechanism of olfactory receptor gene choice in mammalian olfactory neurons. 

Because there are no available olfactory neuron cell lines that recapitulate the in vivo gene regulation process 

our proposed experiments can only be performed in the mouse. We cannot use invertebrates as alternative 

because the mechanisms of olfactory receptor gene regulation and olfactory neuron differentiation in flies and 

worms are very different from the mechanisms we uncovered in the mouse. Thus, mice constitute the only 

model organism that is genetically accessible and utilizes long range genomic interactions for stochastic OR 

gene choice. Furthermore, mouse olfaction provides the closet model for human olfaction among genetically 

tractable model organisms. 

Minimization of Pain and Distress 

Our animals our housed in the state of the art barrier mouse facility in the basement of the Jerome L. Green 

Science building. Moreover, we have a satellite mouse facility next to our laboratory with space for up to 250 

cages. This facility is ideal for time sensitive experiments that require continuous access to the mice. There is 

24/7 access to the veterinary staff of the campus for any case of emergency. My lab members as well as the 

animal care staff monitor our mice for any visible infection, injury, visible guarding, sensorimotor abnormalities 

(as evidenced by abnormal gait or paralysis), seizures or loss of >15% body weight and inform us about it in 

order to euthanize the animal. Usually, when animal care stuff inform us about a mouse experiencing 

discomfort, we respond within 15 30minutes by either instructing them to euthanize the animal or by 

euthanizing the animal in the lab for collection of its olfactory epithelium. 

Minimizing Pain and Distress during Genotyping and Identification of animals 

We will use metal ear tags to mark our animals when they are 10 days old. All our mice are genotyped by 

PCR, which requires minimal amount of DNA for the analysis. Therefore we use tail snips, which are 

performed at the same time with the insertion of ear tags under isofluarane anesthesia to avoid discomfort and 

stress for the mice. We use sharp sterile scissors and the tail is swabbed with a dilute betadine solution. For all 

the experiments proposed, except for aims 1C and 2A mice are euthanized prior to experimentation, so there 

will be no additional source of pain and distress. 

Viral infections: We deliver Lenti and adeno associated viruses through intra nasal delivery of 20ul high tighter 

virus using a 1ml syringe. The syringe is placed in the nasal cavity and administered during inhalation on un

anesthetized pups (daily from P4 till P7). Mice are analyzed 23 days later. The procedure is approved by 

IACUC and abides to AVMA guidelines. 

Euthanasia 

Mice will be euthanized before the extraction of the olfactory epithelium. All methods are consistent with 

American Veterinary Medical Association (AVMA) guidelines. 

Contact PD/PI: Lomvardas, Stavros Vertebrate Animals Page 53

 

 

 

 

- -

All 

-

-

-

-

-
Strains and number of mice 

We have proposed analysis of the following genotypes: 

NFIAfl/fl, Krt5CreER, TdTomato 

NFIB fl/fl; Krt5CreER, TdTomato 

NFIX fl/fl; Krt5CreER, TdTomato 

NFIA fl/fl; NFIX fl/fl; Krt5CreER, TdTomato 

5. NFIB fl/fl; NFIX fl/fl; Krt5CreER, TdTomato 

Gg8tTA; tetO NFIAT2ANFIBiresmCherry 

Krt5CreER; Cre inducible tTA; tetO NFIAT2ANFIBiresmCherry 

Krt5CreER; NFIA,B fl/fl; Cre inducible tTA; tetO NFIAT2ANFIBiresmCherry 

Gg8 Cre; Dot1lfl/fl; TdTomato 

10. Mash1 CreER; Dot1lfl/fl; TdTomato 

11. Olfr1507iresCre; Dot1lfl/fl; TdTomato 

total we have 11 different genotypes that contain 3 or 4 different alleles. To obtain sufficient number of 

animals from each genotype we anticipate having 8 breeding cages for each genotype (88 cages total). 

Moreover, we will have 3 cages for maintenance of each one of the different alleles (11 different alleles 

described here, 33 cages). Finally, in addition to the breeding cages, male and female mice will be housed 

separately until the reach the desired age, thus we anticipate another 8 cages per genotype (88 cages). Total 

number of cages anticipated for this project in the coming year is (88+33+88 209). 

We house a maximum of 5 mice per cage thus we will use a total of 1045 mice per year. This includes mice 

that will not be used for experiments and represent genotypes that cannot serve as experimental or control 

subjects. As described in the proposal, male and female mice will be analyzed separately and if these analyses 

suggest existence of sexual dimorphisms we will increase the number of mice to statistically significant results. 

Contact PD/PI: Lomvardas, Stavros Vertebrate Animals Page 54

 

1. 

2. 

3. 

4. 

6. -

7. - -

8. - -

9. -

-

In 

= 
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Resource Sharing Plan 

My laboratory always deposits raw and normalized sequencing data to public databases (GEO for ChIP seq 

and RNA seq, 4D nucleome portal for HiC). Raw images and the quantifications that we performed on them 

will be deposited at Mendeley Data, a new practice that my lab has started to follow. Detailed protocols will 

also be deposited in Mendeley Data plus, as we always do, we will be happy to host scientists that require 

hands on training for new protocols. 

Mice will be deposited to Jackson laboratories and until the colonies are available for distribution we will be 

sharing them directly with investigators who request them. We are strong believers in sharing our published 

mice without requiring authorship or collaboration (for example: Ericksson and colleagues, 2015 and Brooks 

and colleagues, 2018). 

Timeline of Resource Sharing Plan: We are strong advocates of pre prints, which make discoveries and 

technologies available to the community prior to the completion of the peer review process. Thus, our 

manuscripts will be deposited at BioRxiv prior to submission to peer review journals and this will immediately 

initiate the resource sharing plan described above. Specifically, as we have done in the past with BioRxiv 

submissions, we will provide GEO accession numbers for the sequencing data and we will make our mice 

freely available (without requesting collaboration) to the community. 

Contact PD/PI: Lomvardas, Stavros Resource Sharing Plan(s) Page 60

 

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Authentication of Key Biological and Chemical Resources 

Mice: 

NFIA, B, and X conditional knockout mice were provided by Dr. Gronostasjki from University of Buffalo. We 

confirmed that crossing the conditional KO alleles to Cre drivers results in deletion of the floxed exons using 

RNA seq and Sashimi software for exon usage analysis. We further confirmed the loss of protein by western 

blots for the three NFI factors. 

The Gg8 tTA and Gg8 Cre drivers were donated to us by Dr. Nick Ryba. The expression pattern of Gg8 was 

described in the following publication: 

Ryba, N. J. & Tirindelli, R. A novel GTP binding protein gamma subunit, G gamma 8, is expressed 

during neurogenesis in the olfactory and vomeronasal neuroepithelia. The Journal of biological 

chemistry 270, 6757 6767 (1995). 

We confirmed that Gg8 is expressed only in GBCs/INPs by performing RNA seq in HBCs, GBCs, INPs and 

mOSNs, which detects no Gg8 expression in mOSNs. 

The activity of the tTA driver was described in the following publication: 

Nguyen, M. Q., Marks, C. A., Belluscio, L. & Ryba, N. J. Early expression of odorant receptors distorts 

the olfactory circuitry. J Neurosci 30, 9271 9279, doi:10.1523/JNEUROSCI.1502 10.2010 (2010). 

We confirmed that the Gg8 tTA driver is only active in the GBCs/INPs by visualizing the expression pattern of 

tetO GFP reporters, as well as the expression pattern of the tetO OlfrCDSdeliresGFP mice (Fig. 7b) which are 

both tTA dependent and their expression is completely restricted to basal layers of the MOE corresponding to 

these cell types. Similar results for the Cre driver. 

The OMPirestTA driver was gifted to us from the Axel lab, and has been used in numerous publications by 

 and others to induce expression of tetO dependent genes specifically in mOSNs. 

D) The Olfr1507iresCre and Olfr1507deliresCre lines were gifted by Dr. Axel and behaved exactly as described 

in Shykind et al 2004. 

Transgenic expression of NFIA and B will be confirmed by RNA seq, RNA ISH and IF experiments. 

Chemicals: 

Methimazol: We perform methimazol injections as previously described by performing peritoneal injections 

(0.1mg/g) of Methimazol. We have performed careful timecourse experiments that confirm the almost 

complete ablation of the MOE and the timely reformation after 30 days. RNA seq analysis of OMP, Ngn1, and 

Mash1 populations 30 days after methimazol ablation shows that these major MOE populations are extremely 

similar with the same populations from intact animals. 

Tamoxifen: We perform peritoneal tamoxifen injection of (400mg/Kg) and we confirm the activation of Cre 

drivers with the use of the cre dependent fluorescent driver TdTomato 

Doxycycline: We use a high doxycycline feeding diet (200mg/Kg) that turns off tetO GFP reporters less 

than a week after initiation of the feeding. We only keep the food pellets for 4 months to assure high Dox 

potency. Importantly, for every experiment we deploy an OmpirestTA;tetOOlfr17iresGFP control as well as a 

Gg8 tTA;tetO GFP control. These two lines do not express GFP under dox diet, so they provide assurance for 

the effectiveness of the dox diet. 

Antibodies: 

ntibodies used in ChIP seq have been evaluated in two ways: 

H3K9me3, H4K20me3 and H3K79me3 were evaluated with the use of the Modified histone peptide array 

from active motif, which contains histone tails with various modifications. Every time we purchase a new 

Contact PD/PI: Lomvardas, Stavros Authentication of Key Biological and/or Chemical Resources Page 61

 

 

 

 

 

 

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A) 

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-

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us -

E) -

2. 

A) 

of 

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A) -

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A -

A) 
antibody lot, we screen it with this array and we reject antibodies that cross react with other modifications than 

the ones they are supposed to interact with. 

NFIA, B and X antibodies were evaluated by doing IF in the triple NFI KO MOEs. We continued using only 

antibodies in which immunoreactivity was diminished from the triple KO mice. 

Contact PD/PI: Lomvardas, Stavros Authentication of Key Biological and/or Chemical Resources

 

B) 

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