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SUMMARY STATEMENT 

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 12/19/2019 

Revised Date: 

Application Number: 1 R01 DC018745-01 

Principal Investigator 

LOMVARDAS,STAVROS 

Applicant Organization: COLUMBIA UNIVERSITY HEALTH SCIENCES 

Review Group: ZRG1 IFCN-U (02) 

Center for Scientific Review Special Emphasis Panel 

Member Conflict: Sleep, Movement, Mood and Olfaction 

Meeting Date: 11/19/2019 RFAIPA: PA19-056 

Council: JAN 2020 PCC: CS04 

Requested Start: 04/01/2020 

Project Title: Principles of zonal olfactory receptor gene expression 

SRG Action: Impact Score:13 Percentile:1 # 

Next Steps: Visit https://grants.nih.gov/grants/next_steps.htm 

Human Subjects: 10-No human subjects involved 

Animal Subjects: 30-Vertebrate animals involved - no SRG concerns noted 

Project Direct Costs Estimated 

Year Requested Total Cost 

1 

2 

3 

4 

5 

TOTAL 

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been adjusted 

to reflect any recommendations made by reviewers. If an award is planned, the costs will be calculated by 

Institute grants management staff based on the recommendations outlined below in the COMMITTEE 

BUDGET RECOMMENDATIONS section. 
 

 

 

 

 

 

 

1 R01 DC018745-01 2 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

1R01DC018745-01 Lomvardas, Stavros 

RESUME AND SUMMARY OF DISCUSSION: This is an R01 application from an established 

investigator seeking to elucidate the mechanisms underlying the zonal expression patterns of specific 

olfactory receptor (OR) genes in the olfactory epithelium. The investigator will use an array of state-ofthe-art tools, including Cut and Run, to establish the genomic targets of different transcription factors, 

HiC, deep sequencing, and 11 different mouse lines, as well as CRISPR/dCas9 approaches, to parse 

the epigenetic role of NFI transcription factors and examine the influence of the zonal repressive mark 

H3K79me3 to explicate their collective critical contributions to zonal OR expression. During the 

discussion, there was considerable excitement for this application. The panelists remarked on the many 

strengths of the application, including the powerful, innovative techniques that will be applied to the 

research goals, the high value of the research goals for the field, the convincing, high quality 

preliminary data, and the “gorgeous” experiments proposed. Further, the panelists commented on the 

superb investigative team. Only two weaknesses were raised by the panelists: (i) whether the proposed 

full conditional knockout of three independently segregating alleles, using an independent inducible 

driver, would yield artifacts affecting interpretation, and (ii) a concern over whether the number of 

replicates were sufficient for the proposed analyses and interpretations, particularly with respect to sex 

as a biological variable. Overall, however, these weaknesses did not significantly affect the study 

section’s enthusiasm for this exceptional, high impact application that was considered likely to advance 

fundamentally our understanding of the mechanisms by which activating and repressive 

interchromosomal interactions incorporate feedback signals to regulate selective zonal transcription of 

a particular OR in the olfactory epithelium. 

DESCRIPTION (provided by applicant): 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 for 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 dorsoventral and apicobasal 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, by 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. 
 

 

 

1 R01 DC018745-01 3 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

PUBLIC HEALTH RELEVANCE: Developmental patterning is a crucial biological mechanism 

perturbation 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. 

CRITIQUE 1 

Significance: 1 

Investigator(s): 1 

Innovation: 1 

Approach: 1 

Environment: 1 

Overall Impact: The experimental program addressed by this application is focused on how olfactory 

sensory neurons express different odorant receptors in different regions of the olfactory epithelium. 

Recent studies, largely from the Principal Investigator's laboratory, have elucidated a complex multi 

enhancer complex that activates transcription from singular OR loci, followed by a feedback mechanism 

that helps to keep all other OR loci silent. In this application very promising preliminary studies suggest 

that the transcription factors NFIA, B, and X contribute to both the selective expression of olfactory 

epithelium Zone 5 ORs and the repression of Zone 2 and 3 ORs. The Aims of this application are 

designed to better understand NFIs' roles in zonal OR expression. A triple deletion of all three NFIs 

perturbs zonal OR expression. In Aim 1, each NFI will be tested individually for its ability to perturb 

expression through loss of function experiments, and its sufficiency for perturbation through gain of 

function experiments. This is worthwhile as different NFI factors may have specialized roles. Aim 2 

addresses the mechanisms through which NFIs regulate zonal OR expression. In one series of 

experiments their direct binding sites will be probed using a method named 'cut and run'. In a second 

series of experiments interchromosomal contacts will be assayed using HiC in normal and NSI mutant 

OSNs collected from specific zones of the epithelium. One potential outcome is that zone appropriate 

OR to OR enhancer contacts will be detected, and that these will be disrupted in some of the knockout 

animals produced in Aim1. In a third series of experiments a candidate suppressive epigenetic mark, 

H3K79me3, will be tested for its contribution to zonal OR expression with an OSN knockout of the 

enzyme that produces it, and by determining whether any of the NFIs contribute to its deposition in 

zones that would otherwise be available for OR expression. 

This is an outstanding application in every way. The biological problem it addresses is fundamental to 

understanding olfactory system function, it builds upon the seminal contributions the Principal 

Investigator has already made towards understanding OR gene expression, its preliminary findings are 

extensive, informative and provide a strong basis for the proposed experiments, and the experimental 

approach is innovative, powerful, and will almost certainly yield exciting results. 

1. Significance 

Strengths 

• Understanding the mechanisms of the zonal expression patterns of specific OR genes in the 

olfactory epithelium is fundamental to understanding the development and function of the 

system. This is largely unexplored territory and an advance would have the potential to be of 

more general developmental interest. 

Weaknesses 

• None noted. 

2. Investigator(s) 
 

 

1 R01 DC018745-01 4 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

Strengths 

• This Principal Investigator has been one of the primary leaders in efforts to understand how OR 

gene expression is controlled, especially through his laboratory's discovery of a complex multienhancer-based mechanism of gene choice and activation. 

• This laboratory, by building on its previous studies and using some of the leading-edge 

technologies it has mastered, is in a unique position to address the mechanism of zonal OR 

expression. 

Weaknesses 

• None noted. 

3. Innovation 

Strengths 

• There is a growing appreciation that epigenetic mechanisms control developmental pathways 

and cell identities. This research plan explores epigenetic contributions to an important regional 

pattern of gene expression whose origins are as yet unknown 

• The investigator's laboratory has pioneered the use of HiC, deep sequencing, and other 

advanced techniques to solve a fundamental and fascinating developmental and genetic 

problem. 

Weaknesses 

• None noted. 

4. Approach 

Strengths 

• The preliminary findings reported in the proposal are unusually strong and informative. They 

demonstrate the technical ability of the laboratory to perform the proposed experiments and 

point the research in what looks like a very promising direction. Not only are the NSI 

transcription factors identified as candidate contributors to zonal OR expression, but preliminary 

tests of necessity demonstrate their involvement. Target NSI motifs are identified in and 

enriched in zone 5 OR promoter sequences and NSIA binds general OR-activating enhancer 

sequences. The H3K79me3 suppressive mark is shown to be deposited in a zone dependent 

manner. 

• It is noteworthy that an attractive and ultimately testable model for regional OR choice is 

presented in which ORs appropriate for a particular zone are made available for expression and 

then silenced, starting with zone 1 and then sequentially through zone 5. 

• Each of the proposed experiments appears technically feasible for this laboratory and 

addresses a specific clearly formulated question. Reasonable alternative approaches are 

mentioned where appropriate. 

Weaknesses 

• From a purely technical perspective some of the proposed experiments may be challenging, but 

this lab has shown through past publications and the preliminary findings that they should be 

capable of accomplishing most of what they propose. 

5. Environment 

Strengths 

• The Principal Investigator is situated in an institution with first rate resources and renowned 

Neuroscience and Genetics faculty. 

• The high quality and significance of the publications coming from the Principal Investigator's 

laboratory over the past several years attest to the suitability of the research environment. 

Weaknesses 
 

 

 

1 R01 DC018745-01 5 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

• None noted. 

Protections for Human Subjects 

Not Applicable (No Human Subjects) 

Vertebrate Animals 

Acceptable 

Biohazards 

Acceptable (No Biohazards) 

Budget and Period of Support 

Recommend as Requested 

CRITIQUE 2 

Significance: 2 

Investigator(s): 1 

Innovation: 2 

Approach: 2 

Environment: 1 

Overall Impact: The intellectual formulation of this application places the details of transcriptional 

regulation of zonally restricted odorant receptor expression into a very clear and compelling biological 

framework. The identification of a family of transcription factors, the NFIs, that may be key determinants 

of expression of multiple genes within a single zone provides a starting point to discern several 

mechanistic dimensions of patterned gene expression. Some concern is raised by the multi-allelic/ 

conditional recombination approaches necessary to test some of the hypotheses and the potential for 

low yield from these experiments and also genetic artifacts that reflect the technique but nevertheless 

influence execution and interpretation of the data. One wishes that the Principal Investigator had dealt 

more with this technical issue. 

1. Significance 

Strengths 

• This application considers the mechanism and biological significance of zone restricted 

expression of odorant receptors based upon its transcriptional and chromosomal regulation and 

its expression as an example of patterned gene expression. 

• The application places the problem of odorant receptor gene expression, and by extension 

coordinated expression of multiple genes in a distinct domain, in the appropriate context—how 

chromatin confirmation/architecture may or may not regulate this critical feature of 

morphogenesis and tissue architecture. 

Weaknesses 

• The application focuses on the mechanism of transcriptional control for patterning, but does not 

discuss whether the experiments and potential results address issues of the functional/ 

behavioral consequences of this developmental/cellular regulation. 

2. Investigator(s) 

Strengths 
 

1 R01 DC018745-01 6 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

• The Principal Investigator has outstanding accomplishments in the field of transcriptional 

regulation and in the study of the olfactory epithelium as a model for highly regulated gene 

expression. 

Weaknesses 

• None noted. 

3. Innovation 

Strengths 

• The focus on chromatin architecture, its regulation by identified transcription factors and the 

integrated contribution to patterning of these mechanisms is truly innovative. 

Weaknesses 

• The ambitious use of multi-allelic conditional inactivation to fully eliminate a set of related factors 

is technically demanding, but one wonders about the potential for artifact in this approach that 

takes a “cutting edge” technique to near to its known limits (full conditional knock out of 3 

independently segregating alleles using an independent inducible driver). 

4. Approach 

Strengths 

• The experiments to evaluate positive and negative regulation (expression versus exclusion of 

zonal OR genes) are particularly well designed and will yield high resolution data both in terms 

of developmental output, and the underlying mechanism of interchromosomal interaction and 

chromatin architecture in determining patterns of gene expression. 

Weaknesses 

• The multi-allelic, cell class specific recombination experiments though theoretically compelling 

have the potential for artifact and also resource-intensive but still low or slow yield data because 

of low probability of obtaining the needed genotypes. It would have been good to address these 

issues more thoroughly. 

5. Environment 

Strengths 

• The environment at the Zuckerman Institute of Columbia University is outstanding in all ways for 

the completion of this work. 

Weaknesses 

• None noted. 

Protections for Human Subjects 

Not Applicable (No Human Subjects) 

Vertebrate Animals 

Acceptable 

Biohazards 

Acceptable (No Biohazards) 

Budget and Period of Support 

Recommend as Requested 

CRITIQUE 3 
 

1 R01 DC018745-01 7 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

Significance: 2 

Investigator(s): 1 

Innovation: 2 

Approach: 2 

Environment: 1 

Overall Impact: This application’s main goal is to elucidate the mechanisms underlying the specific 

zonal expression of odorant receptors in the mouse olfactory system. This mechanism has been very 

hard to determine, and thus the successful completion of this project will significantly advance the field. 

It is innovative in that it proposes a novel mechanism and uses cutting edge technology as well. The 

Principal Investigator is a leader in the field and has made important contributions to the mechanisms 

underlying singular expression of odorant receptors. His research environment is excellent, with 

numerous core facilities and there are exceptional opportunities to interact with prominent scientists. He 

presents convincing preliminary data identifying a zone 5 specific NFI motif on zone 5 OR promotors to 

support the research. A negative is that there are too few replicates to examine for sex differences if 

performed as proposed (4 replicates, 2 male and 2 female, which the Principal Investigator proposes to 

examine for sex differences prior to combining the data). 

Protections for Human Subjects 

Not Applicable (No Human Subjects) 

Vertebrate Animals 

YES, all criteria addressed 

Biohazards 

Acceptable 

Resource Sharing Plans 

Acceptable 

Authentication of Key Biological and/or Chemical Resources 

Acceptable 

Budget and Period of Support 

Recommend as Requested 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS’ 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 

VERTEBRATE ANIMALS: ACCEPTABLE 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

Footnotes for 1 R01 DC018745-01; PI Name: Lomvardas, Stavros 

# Ad hoc or special section application percentiled against "Total CSR" base. 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-
 

 

1 R01 DC018745-01 8 ZRG1 IFCN-U (02) 

LOMVARDAS, S 

14-074.html. The impact/priority score is calculated after discussion of an application by 

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring. 
MEETING ROSTER 

The roster for this review meeting is displayed as an aggregated roster that includes reviewers from 

multiple CSR Special Emi,hasis Panels of the SEP A regated Roster IFCN IRG