STEVEN C. MUNGER, Ph.D.
The Jackson Laboratory  Center for Genome Dynamics  600 Main Street  Bar Harbor, ME 04609
steven.munger@jax.org  w 207.288.6715  h 207.244.0011
PROFILE
I explore the genetic variation segregating within natural populations to discover how
common or rare alleles that assert subtle individual effects act in combination to
disrupt the normal network state and confer genetic susceptibility to developmental
anomalies, adult disorder, and disease. I have established the bipotential embryonic
mouse gonad at the critical stage of primary sex determination as an ideal
mammalian model of cell differentiation and organogenesis that is particularly
amenable to systems genetic approaches. My research strategy integrates
experimental and computational methods to infer and test predictive transcription
network models governing sex determination in a population of genetically diverse
mice. I have extensive interdisciplinary training in developmental biology, complex
trait genetics, genomics, and bioinformatics. I value and promote a collaborative
research environment. I have a strong passion for education, and extensive
experience teaching and mentoring high school through graduate-level students.
EDUCATION & TRAINING
2011-present
The Jackson Laboratory, Bar Harbor, ME
Postdoctoral Training, Center for Genome Dynamics
Advisor: Gary Churchill, Ph.D.
Led an interdisciplinary team of post-docs and software engineers to develop
advanced analytical methods and computational tools for analyzing RNA-seq data
derived from genetically diverse mice. Developed Seqnature software to impute and
construct individualized diploid genome sequences for RNA-seq read alignment.
Demonstrated that the individualized alignment strategy increases read mapping
accuracy, improves estimates of transcript abundance, yields direct estimates of
allele-specific expression, reduces spurious expression quantitative trait locus (eQTL)
associations, and unmasks thousands of real eQTL confounded by the common
reference alignment strategy. Implemented novel pipelines to integrate liver eQTL
and ENCODE datasets to systematically predict causative strain variants underlying
cis- and trans-eQTL.
Designed and conducted a comprehensive sensitized RNA-seq eQTL screen of the
gonad transcriptome during sex determination that employs these same analytical
methods.
2010-2011
Duke University, Durham, NC
Postdoctoral Training
Advisor: Blanche Capel, Ph.D.
Developed a medium-throughput gonad primary cell assay and optimized a lentiviralmediated RNAi knockdown method to experimentally validate genetic interactions
predicted from the temporal expression and eQTL data.
2010
Duke University, Durham, NC
Ph.D. Genetics
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
Advisor: Blanche Capel, Ph.D.
A systems-level view of mammalian sex determination.
Designed and conducted the first eQTL study in a developing vertebrate organ, the
embryonic mouse gonad at the critical time point of sex determination. Characterized
the fine temporal patterns of gene activation and repression in the critical 24-hour
window of sex determination, and showed that sensitivity to sex reversal in the
C57BL/6J strain background stems from delayed activation of the testis pathway and
delayed repression of the ovarian pathway.
1999
The University of Michigan, Ann Arbor, MI
B.S. Biology
ADDITIONAL TRAINING
2014
The Jackson Laboratory, Bar Harbor, ME
The Whole Scientist Course (one-week course)
2012-2014
The Jackson Laboratory, Bar Harbor, ME
Short Course on Medical and Experimental Mammalian Genetics (two-week course)
2011-2013
The Jackson Laboratory, Bar Harbor, ME
Short Course on Systems Genetics (one-week course)
2000-2003
Dow Chemical Company, Midland, MI
Research & Development Technologist
Advisor: Mark Bernius, Ph.D.
Developed flexible flat panel displays to test new light-emitting polymers.
POSITIONS HELD
2011-present
The Jackson Laboratory, Bar Harbor, ME
Postdoctoral Research Fellow
2010-2011
Duke University Medical Center, Durham, NC
Postdoctoral Associate
2003-2004
Dow Chemical Company, Midland, MI
Quality Control Technologist
2000-2003
Dow Chemical Company, Midland, MI
Research & Development Technologist
1999-2000
Waterford Kettering High School, Waterford, MI
Substitute 9-12 Grade Science Teacher
HONORS & AWARDS
2015-2017 term
Elected to Secretariat of the International Mammalian Genome Society
2013-2015
Ruth L. Kirschstein National Research Service Award
Individual postdoctoral fellowship
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
2013
Chicago Prize for Outstanding Oral Presentation
12th Annual Meeting of the Complex Trait Community
2012
Best Oral Presentation by a Post-Doctoral Fellow
11th Annual Meeting of the Complex Trait Community
2011
Verne Chapman Memorial Young Scientist Award
International Mammalian Genome Society
2008
Best Oral Presentation by a Graduate Student
7th Annual Meeting of the Complex Trait Consortium
2004-2006
NIH Predoctoral Training Grant
Institution-awarded fellowship
PUBLICATIONS
Google Scholar page: http://scholar.google.com/citations?user=j6C32yAAAAAJ&hl=en
Peer-reviewed Primary Research
8.
French JE, Gatti DM, Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard K, Price HC, King D,
Witt KL, Pedersen LC, Svenson KL, Munger SC, and Churchill GA. Benzene exposure to genetically
diverse outbred mice reveal inter-individual differences in susceptibility to chromosomal damage
and implications for risk assessment. In Press at Environmental Health Perspectives.
7.
Munger SC, Raghupathy N, Choi K, Simons AK, Gatti DM, Hinerfeld DA, Svenson KL, Keller MP, Attie
AD, Hibbs MA, Graber JH, Chesler EJ, and Churchill GA. RNA-seq alignment to individualized genomes
improves transcript abundance estimates in multiparent populations. Genetics 2014, 198(1): 59-73.
6.
Munger SC*, Natarajan A*, Looger LL, Ohler U, and Capel B. Fine timecourse expression analysis
reveals cascades of activation and repression and maps a regulator of mammalian sex determination.
PLoS Genetics 2013, 9(7): e1003630. * Equal contributors.
5.
Churchill GA, Gatti DM, Munger SC, and Svenson KL. The diversity outbred mouse population.
Mammalian Genome 2012, 23(9-10): 713-718.
4.
Jameson SA, Natarajan A, Matouk DM, DeFalco T, Cool J, Mork L, Munger SC, and Capel B. Temporal
transcriptional profiling of somatic and germ cells reveals lineage priming of sexual fate in the fetal
mouse gonad. PLoS Genetics 2012, 8 (3): e1002575.
3.
Cook MS, Munger SC, Nadeau JH, and Capel B. (2011) Regulation of male germ cell cycle arrest and
differentiation by DND1 is modulated by genetic background. Development 2011, 138 (1): 23-32.
2.
Munger SC, Aylor DL, Syed HA, Magwene PM, Threadgill DW, and Capel B. Elucidation of the
transcription network governing mammalian sex determination by exploiting strain-specific
susceptibility to sex reversal. Genes & Development 2009, 23: 2521-2536.
1.
Ross A, Munger SC, and Capel B. Bmp7 regulates germ cell proliferation in mouse fetal gonads. Sexual
Development 2007, 1 (2): 127-137.
Peer-reviewed Review Articles/ Meeting Reports
2.
Musser MA*, Munger SC*, and Gunn TM. Meeting report of the 26th International Mammalian
Genome Conference. Mammalian Genome 2013, 24(5-6): 179-89. Meeting Report.
* Equal contributors.
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
1.
Munger SC and Capel B. Sex and the circuitry: Progress toward a systems-level understanding of
vertebrate sex determination. Wiley Interdisciplinary Reviews – Systems Biology and Medicine 2012,
doi: 10.1002/wsbm. 1172. Review article.
MANUSCRIPTS IN REVIEW/ IN PREPARATION
1.
Morton NM, Beltram J, Carter R, Gorjanc G, Munger SC, Svenson KL, Rodriguez-Cuenca S, MorenoNavarrete JM, Gibbins M, McFadden C, Gastaldello A, Stott H, Naredo G, Zeyda M, Wang Z, Howie AF,
Saari A, Sipila P, Stulnig T, Gudnasson V, Kenyon CJ, Seckl JR, Walker BR, Webster SP, Dunbar DR,
Vidal-Puig A, Churchill GA, Fernandez-Real JM, Emilsson V, and Horvat S. Genetic selection for
extreme low adiposity identifies a healthy leanness gene. In review at Nature Medicine.
2.
Raghupathy N, Choi K, Munger SC, Korstanje R, Pardo-Manuel de Villena F, and Churchill GA. EMASE:
Accurate quantitation of allele-specific expression in diploid genomes. Manuscript in preparation.
3.
Munger SC, Choi K, Raghupathy N, Gatti DM, Simecek P, Svenson KL, Keller MP, Attie AD, and
Churchill GA. Systematic prediction and validation of causative genetic variants underlying cis- and
trans-eQTL in the mouse liver transcriptome. Manuscript in preparation.
4.
Munger SC, Choi K, Khalife M, Montagutelli X, Panthier JJ, Churchill GA, and Jaubert J. An
individualized RNA-seq alignment strategy resolves reads of bacterial origin in mouse spleen
samples infected with the plague Yersinia pestis. Manuscript in preparation.
5.
Liang ZS, Munger SC, Raghupathy N, Soria XI, and Logan DW. Allele-specific and imprinted gene
expression in the mouse olfactory system. Manuscript in preparation.
6.
Munger SC, Eicher EM, and Churchill GA. A predictive network model of the mammalian gonad
transcriptome during primary sex determination. Manuscript in preparation.
INVITED PRESENTATIONS
“Systems genetic approaches to understanding normal development, disorder, and disease.”
Genome Science Seminar Series, Host: Charles Farber
University of Virginia, Charlottesville, VA
Scheduled visit for November 19, 2014
“Exploring the genetics of transcript and protein abundance in the murine liver.”
Invited Seminar, Host: Steven Gygi, Harvard Medical School, Boston, MA
October 10, 2014
“RNA-seq alignment to individualized genomes.”
Environmental Genomics Short Course, Mount Desert Island Biological Laboratory, Salisbury Cove, ME
August 6, 2014
“RNA-seq alignment to individualized genomes.”
Invited Seminar, Host: Darren Logan, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK
May 23, 2014
“Finding sex in the circuitry: Toward a predictive network model of mammalian sex determination.”
Behavior, Ecology, Evolution, and Systematics Invited Seminar, Host: Thomas Kocher
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
University of Maryland, College Park, MD
March 24, 2014
“A systems genetic analysis of the dynamic transcription network governing primary sex determination.”
Invited Seminar, Host: April Binder, National Institute of Environmental Health Sciences, Triangle Park, NC
August 22, 2011
PRESENTATIONS & POSTERS
“Accurate global prediction of transcript and protein abundance in the Diversity Outcross and Collaborative
Cross mice.” (oral presentation selected from submitted abstract – Conference scheduled for October 2014)
28th International Mammalian Genome Conference 2014, Bar Harbor, Maine
“Allele specific expression and eQTL in diploid genomes.” (poster)
American Society of Human Genetics Meeting 2014, San Diego, CA
“Reconsidering the laboratory mouse.” (oral presentation)
Genomics of Common Diseases 2014 Meeting, Potomac, MD
“Dissection of expression quantitative trait loci in the Diversity Outbred mouse population.”
(oral presentation selected from submitted abstract)
13th Annual Meeting of the Complex Trait Community 2014, Berlin, Germany
“RNA-seq alignment to individualized transcriptomes.” (poster)
American Society of Human Genetics Meeting 2013, Boston, MA
“RNA-seq alignment to individualized diploid transcriptomes reveals extensive local genetic regulation and
differential allelic expression in outbred DO mice.” (oral presentation selected from submitted abstract)
27th International Mammalian Genome Conference 2013, Salamanca, Spain
“RNA-seq alignment to individualized genomes.”
(oral presentation selected from submitted abstract)
12th Annual Meeting of the Complex Trait Community 2013, Madison, WI
Chicago Prize for Outstanding Oral Presentation
“RNA-seq alignment to individualized genomes.”
(oral presentation selected from submitted abstract)
40th Maine Biological and Medical Sciences Symposium 2013, Salisbury Cove, ME
“Expression QTL mapping in the Diversity Outbred mouse population.”
(oral presentation selected from submitted abstract)
26th International Mammalian Genome Conference 2012, St. Pete Beach, FL
“Expression QTL mapping in the Diversity Outbred mouse population.” (poster)
Annual Meeting of the National Centers for Systems Biology 2012, Chicago, IL
“Expression QTL mapping in the Diversity Outbred mouse population.”
(oral presentation selected from submitted abstract)
11th Annual Meeting of the Complex Trait Community 2012, Paris, France
Best Oral Presentation by a Post-Doctoral Fellow
“Systems genetic analysis reveals a complex and highly dynamic transcription network governing sex
determination.” (oral presentation selected from submitted abstract)
Mouse Genetics 2011, Washington, DC
Verne Chapman Memorial Young Scientist Award
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
“Systems genetic analysis of gonadogenesis in mice reveals a dynamic transcription network governing sex
determination and testis organogenesis.” (poster)
Duke School of Medicine 1st Annual Basic Science Day 2010, Durham, NC
“Systems genetic analysis of gonadogenesis in mice reveals a dynamic transcription network governing sex
determination and testis organogenesis.” (poster)
Annual Meeting of the National Centers for Systems Biology 2009, Bethesda, MD
“Integrating genetics and genomics to elucidate the transcription networks governing sex determination and
testis organogenesis in mice.”
(oral presentation selected from submitted abstract)
Symposium on the Biology of Vertebrate Sex Determination 2009, Kona, HI
“Integrating genetics and genomics to elucidate the transcription networks governing sex determination and
testis organogenesis in mice.”
3rd Annual Duke Center for Systems Biology Symposium 2008, Durham, NC
“Integrating genetics and genomics to elucidate the transcription networks governing organogenesis.”
(oral presentation selected from submitted abstract)
7th Annual Meeting of the Complex Trait Consortium 2008, Montreal, Quebec
Best Oral Presentation by a Graduate Student
“Whole-genome expression profiling of embryonic male gonads at the time of sex determination reveals
significant differences between the C57BL/6J and 129S1/ImJ inbred mouse strains.” (poster)
Society for Developmental Biology Southeast Regional Conference 2007, Chapel Hill, NC
FUNDING
9/2013-9/2015
F32 HD074299-01
NIH/NICHD
Sensitized Screen in the Diversity Outcross Mouse Population
Individual Ruth L. Kirschstein National Research Service Award Fellowship
TEACHING EXPERIENCE & MENTORING
Fall 2014
Mentored local high school student on senior independent research project.
August 2014
23rd Annual JAX Short Course on Experimental Models of Human Cancer
Developed and led a 2-hour problem-driven training session that explored the power
of common genome browsers for guiding gene discovery in cancer biology.
Summer 2014 &
Summer 2013
Journal Club – The Jackson Laboratory Summer Student Program
Mentored two groups of talented high school and college students to present and
critique recent high-profile genome-wide association and next generation sequencing
studies.
Spring 2014 &
Spring 2013
Mammalian Genetics Course - Tufts University/University of Maine
Taught two 2-hour classes on genetic mapping and RNA-seq analysis. Designed datadriven exam questions to test students’ comprehension of concepts and applications.
October 2013 &
2012
Short Course on Systems Genetics – The Jackson Laboratory
Developed and led a 3-hour training session on all aspects of RNA-seq analysis.
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
Fall 2013 & 2014
Applied Bioinformatics Course – Mount Desert Island Biological Laboratory
Presented a lecture on methods for RNA-seq analysis in diverse populations.
Fall 2013 & 2014
Computational Methods in Biology/Genomics – University of Maine
Presented a one-hour lecture on methods for RNA-seq alignment and quantitation.
May 2013
Workshop on Modern Approaches to High Throughput Sequencing
Presented a one-hour lecture on methods for RNA-seq alignment and quantitation.
Spring 2013
Genetics Journal Club – Tufts University
Facilitated three journal club sessions focused on genome-wide association studies.
Mentored student presenters on experimental design and results.
Spring 2012
RNA-seq Interest Group – The Jackson Laboratory
Initiated a new interest group at the laboratory centered on RNA-seq, and led seven
monthly 1-hour sessions covering all aspects of RNA-seq analysis from RNA
extraction to expression quantitation and differential expression analysis.
Summer 2011
Summer Fellows Program – Duke IGSP Center for Systems Biology
Mentored a talented undergraduate student from North Carolina State University on
an independent systems biology research project.
Spring 2010 &
Spring 2009
Undergraduate Independent Study Program – Duke University
Mentored two Duke undergraduate students on independent research projects.
Spring 2007
Genetics and Molecular Biology – Biology 118, Duke University
Served as a graduate teaching assistant in a large undergraduate genetics course. Led
weekly study sections, held office hours, and graded midterm and final exams.
PROFESSIONAL MEMBERSHIPS & SERVICE
Reviewer for:
GENETICS
Circulation: Cardiovascular Genetics
Experimental Cell Research
Member of:
International Mammalian Genome Society (Served on the Secretariat 2011-2013,
Elected to the Secretariat for 2015-2017 term)
American Society of Human Genetics
Complex Trait Community
Session Chair:
13th Annual Meeting of the Complex Trait Community 2014, Berlin, Germany
27th International Mammalian Genome Conference 2013, Salamanca, Spain
26th International Mammalian Genome Conference 2012, St. Pete Beach, FL
11th Annual Meeting of the Complex Trait Community 2012, Paris, France
REFERENCES
Gary Churchill, Ph.D.
Professor and Director, Center for Genome Dynamics
The Jackson Laboratory
gary.churchill@jax.org
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
Blanche Capel, Ph.D. (dissertation advisor)
James B. Duke Professor, Department of Cell Biology
Duke University Medical Center
blanche.capel@duke.edu
David Threadgill, Ph.D.
Professor and Director, Whole Systems Genomic Initiative
Veterinary Medicine & Biomedical Sciences
Texas A&M University
dwthreadgill@neo.tamu.edu
Mary Ann Handel, Ph.D.
Senior Research Scientist
The Jackson Laboratory
maryann.handel@jax.org
Curriculum Vitae: Steven Munger, Ph.D.
Date prepared: October, 2014
The Systems Genetics of Development
Congenital abnormalities present in 2-3 percent of live births1, and result from insults to the normal processes
of development. As we refine our understanding of birth defects as well as disease and disorder in general, it is
clear that most anomalies have a genetic component, and regardless of the timing of their presentation (e.g.
congenital or adult onset), are rooted at least in part in embryonic life. The heritable component may be a single
gene of large effect (i.e. Mendelian) or it may be many variants that assert subtle individual effects but in
combination sensitize the genetic background. Mutations in critical developmental genes often confer
pleiotropic effects on multiple organs. Genes active during sex determination are illustrative. Mutations in the
transcription factors SOX9, FOXL2, and WT1 disrupt gonadogenesis but also cause skeletal defects, eyelid
malformations, renal disease, and cancer. The transcriptional architecture underlying organogenesis is not
distinct to each organ but rather builds upon a conserved framework to which unique elements are added that
differentiate function. Therefore, elucidating the network of one developing organ can inform us about the core
structure and vulnerabilities of all developmental networks.
Despite the significance of early development to adult organ function, most genetic mapping studies in
mammalian models have been limited to adult subjects. Genetic variation may be masked in the adult by
buffering mechanisms (e.g. hormones) that are largely absent in the fetus until after most organs have
developed. Studies that seek to understand the genetic etiology of congenital abnormalities should focus on a
developmental stage that is both amenable to genetic approaches and likely to coincide with the timing of the
causative genetic lesion(s). For this reason, the mouse is the premier genetic model for characterizing
organogenesis and congenital defects. Furthermore, previous studies have largely focused on “end phenotypes”
- distal measurements of adult organ structure or function such as blood pressure - rather than
“endophenotypes” - proximal responses to perturbation such as transcript abundance. Gross organ physiology
may be buffered from genetic variation, yet any significant perturbation to the system must elicit a proximal
response to maintain homeostatic balance. Consequently, I plan to measure primary cellular responses at the
onset of perturbation to fully characterize the combinatorial effects of genetic and epigenetic variation on a
system. By taking advantage of advances in high throughput sequencing and low-input amplification methods, I
will apply powerful “systems genetics” approaches to dissect these combinatorial effects with nucleotide
resolution in individual differentiating cells.
My research goals are to 1) characterize the transcription network architecture and dynamics underlying
normal gonad development in the mouse, 2) infer genes, interactions, and pathways with major roles in this
process, and 3) predict and validate genetic variants with individually small effects that in specific combinations
disrupt the buffering capacity of the transcription network and cause defect. I will combine advanced
computational methods with experimental validation techniques in high-resolution genetic mapping
populations, the mouse Diversity Outcross (DO) and Collaborative Cross (CC), to elucidate the transcription
network governing sex determination of the unique bipotential gonad. My long-term goal is to translate this
predictive model of sex determination to the human population to guide the discovery of novel mutations
underlying disorders of sex development (DSD) and infertility.
Doctoral and Postdoctoral Accomplishments
I have spent the past decade advancing this new paradigm for developmental genetics research. Evolution has
provided a powerful resource in the form of abundant genetic variation segregating within and between
species. For example, the eight founder strains of the mouse DO differ at 50 million SNPs, equivalent to the
variation among humans, chimpanzees, and gorillas. I have developed the computational and experimental
expertise to exploit this variation to dissect the transcription network governing sex determination of the
embryonic mouse gonad. I joined Dr. Blanche Capel’s lab in 2005, and soon recognized the potential of the
gonad for a systems genetics approach. The gonad arises as a bipotential primordium competent to differentiate
as a testis or ovary. Antagonistic signals confer this unique plasticity by establishing a transient, unstable
transcription network state balanced between the testis and ovarian differentiation pathways2,3. Sex
determination proceeds by disrupting this balance toward the testis or ovarian fate, a process that requires the
activation of one sexual pathway and concurrent repression of the alternative pathway (Fig. 1a). Failure to
establish or canalize one sex results in trans-differentiation to the alternative fate. My doctoral research built
upon the observation that the C57BL/6J (B6) mouse strain was particularly sensitive to XY sex reversal in
Research Statement: Steven Munger, Ph.D.
Date prepared: October, 2014
Figure 1. A dynamic transcription network underlies primary sex determination of the bipotential gonad.
response to genetic perturbations4. Linkage mapping later revealed that at least three loci were involved5. I
hypothesized that transcriptional differences in the testis at the critical timepoint of sex determination
conferred this sensitivity in B6. I profiled transcript abundance by microarray in sensitive B6 and resistant
129S1/SvImJ (129S1) E11.5 XY gonads. I found that hundreds of genes associated with ovarian differentiation
were upregulated in sensitive B6 XY gonad relative to 129S1, while many genes associated with testis
differentiation were downregulated in B6 XY gonads relative to 129S16. This shift toward the ovarian fate likely
accounted for the observed sensitivity of B6 to sex reversal. More recently I extended this analysis to profile B6
and 129S1 XY and XX gonads at six 4-hour intervals between E11.0-12.03. Using a hidden Markov model to
resolve the time of onset of sexually-dimorphic expression, I showed that the gonad transcriptome is highly
dynamic during this critical 24-hour window of development and involves five distinct cascades of gene
activation and repression (Fig. 1b). By comparing this temporal data to cell-type specific expression data, I
showed that sexual dimorphism during this critical period was largely confined to testis supporting cells7.
Moreover, I identified a consistent 5-hour delay in the activation of the testis pathway and repression of the
ovarian pathway in B6 XY gonads compared to 129S1. This delay likely accounts for the sensitivity of B6 to sex
reversal and highlights the importance of expression timing for canalizing sexual fate.
I hypothesized that linkage mapping could be used to identify the genetic loci underlying strain differences
in gonad gene expression. To explore the feasibility of this approach, I dissected 80 E11.5 XY F2 gonads from a
B6x129S1 intercross, profiled the gonad expression of 51 sex-associated genes by nanoliter-scale quantitative
PCR, and genotyped each F2 individual at 128 strain-informative SNPs across the genome. I mapped regions of
the genome where SNP genotype was correlated with differences in gene expression (expression quantitative
trait loci, eQTL) for 44/51 genes, including novel autosomal loci that controlled Sry and Sox9 expression, master
regulators of testis differentiation6. Further, I identified eQTL that influenced multiple genes, including a region
of distal Chromosome 3 that controlled the expression of 16/51 genes. These “trans-band” eQTL were quite
broad, however my temporal expression data proved informative for predicting candidate genes. On distal Chr 3
for example, Lmo4 is upregulated early in the testis but delayed in B6 compared to 129S1, a pattern predicted
for the causative gene underlying that eQTL. To test this hypothesis, I developed a gonad primary cell assay and
lentivirus-mediated RNAi method to knockdown Lmo4 expression. I showed by qPCR that multiple target genes
were specifically affected by perturbation of Lmo4, validating it as a novel early regulator in the differentiating
testis. This study proved the feasibility of an eQTL mapping approach for identifying genes and interactions
across the transcriptome, and represented the first eQTL analysis of a developing vertebrate organ.
My growing interest in systems genetics was tempered by my lack of computational training. To remedy
this, I moved to Dr. Gary Churchill’s group at The Jackson Laboratory to lead the analysis of a large DO liver
RNA-seq eQTL study. I found that existing methods for RNA-seq analysis were insufficient to account for the
high genetic diversity in the DO, where nearly half of all 100bp RNA-seq reads contain one or more transcribed
SNPs. Alignment of DO samples to the reference genome - the standard method employed in RNA-seq - caused
numerous mapping errors, inaccurate estimates of transcript abundance, and spurious eQTL associations. To
resolve this problem, I developed the Seqnature software package (https://github.dom/jaxcs/Seqnature).
Research Statement: Steven Munger, Ph.D.
Date prepared: October, 2014
Figure 2. Nucleotide resolution of liver expression QTL in the Diversity Outcross population.
Seqnature takes the 36-state founder diplotypes inferred for each DO animal from SNP genotypes and
incorporates strain-specific SNPs and indels from the founder strains to construct individualized diploid
genome sequences. I demonstrated that read alignment to individualized genomes increases read mapping
accuracy, improves transcript abundance estimates, yields direct estimates of allele-specific expression, reduces
spurious eQTL associations, and reveals thousands of real eQTL8. Figure 2 illustrates the power of this approach
for eQTL resolution. A local eQTL controls Slc22a2 gene-level abundance (blue line in Fig. 2a), however this
association is masked by alignment to the NCBIM37 reference (red line). The additive effects of each of the eight
DO founder strains can be inferred at the locus and show that DO animals that derive this locus from the 129S1,
NZO, or PWK strains exhibit lower expression of Slc22a2 (Fig. 2b). Alignment to individualized diploid genomes
yields direct estimates of allele-level expression that are tagged with their founder strain origin. By comparing
the allele abundance estimates for Slc22a2, it is clear that the 5-3 strain pattern observed in the additive genelevel eQTL model is conferred at the allele level, confirming that the causative variant acts in cis on one but not
both transcribed alleles (Fig. 2c). The genotypes of all 50k SNPs in the locus region can be imputed and tested
for association with the observed expression differences, yielding 22 SNPs that exhibit the expected 5-3 strain
genotype pattern (Fig. 2D). Indeed, the eight DO founder strain alleles can segregate in 127 different biallelic
patterns (compared to two biallelic patterns for a backcross or intercross), making this population particularly
powerful for fine-mapping. Further, I have integrated profiles of histone modifications (e.g. mouse ENCODE)
and transcription factor (TF) binding site databases to identify SNPs or indels that reside in enhancer elements
or consensus TF binding sites. This yields 8 SNPs near Slc22a2 that reside in active enhancers marked by
H3K27ac, one of which is found in a predicted binding site for NKX3-2. I am validating a subset of the highest
scoring predictions with quantitative ChIP-seq assays in F1 hybrids from crosses of founder and CC strains.
From this powerful DO eQTL dataset, I have developed novel computational methods to resolve cis and transeQTL down to one or a few variants, and furthermore I can now predict with high accuracy the liver transcript
abundance of nearly 10,000 genes in any animal derived from the eight DO founder strains (In preparation).
Plans for future research
I recognized the power of the gonad as a model for systems genetics research early on, however my
graduate research was largely exploratory in nature, focused on a small subset of genes, and frankly lacked
mapping power and statistical rigor. Further, because most known sex determination genes are not genetically
variable between B6 and 129S1, an intercross of these strains is insufficient to fully elucidate the genetic
interactions underlying sex determination. With Dr. Capel’s encouragement and Dr. Churchill’s support, I have
designed and nearly completed a large-scale sensitized screen of gonad eQTL in the Diversity Outcross (DO)
population that will serve as the foundation for my independent research. This screen integrates RNA-seq
Research Statement: Steven Munger, Ph.D.
Date prepared: October, 2014
transcriptional profiles with high density SNP genotyping in 200 XY gonads derived from crossing sensitized
B6.Dax1/Nr0b1 heterozygous females to 100 unrelated DO males. Mutation of Dax1 causes XY male-to-female
sex reversal in B69, and I have observed varying degrees of sex reversal in hemizygous DO progeny, indicating
that sex determination is being compromised. The resulting data will match the format of my liver data, and
therefore I will apply the same eQTL analysis strategies outlined above to systematically dissect the gonad
transcriptome and generate a predictive network model of sex determination (Aim 1).
Based on the results from my liver study, I expect to be able to identify hundreds of local eQTL that can be
resolved down to one or a few candidate SNPs/indels and a similar number of distant eQTL for which I can
predict the trans regulatory gene with high certainty. Importantly, I will integrate my earlier analyses of
temporal gonad gene expression and cell-type specific signatures to prioritize validation efforts and anchor novel
genes to known regulators of sex determination (Aim2a). First, I will characterize the initial steps of sexual fate
specification by focusing my validation efforts to genes upregulated specifically and early in the somatic
supporting cell population. Next, I will test predicted genetic interactions between Sertoli- and germ cellspecific genes to characterize novel intercellular interactions that reinforce the testis pathway. Alternatively, I
will anchor my search to specific annotated cellular functions or pathways, for example by focusing on genes
with known roles in cell migration or extracellular matrix deposition. I will interrogate this predictive model
using a set of standard methods and assays that I have optimized for validating putative gene-gene interactions or
polymorphisms that alter transcription factor binding in the gonad (Aim2b). These include immunocytochemistry
and confocal microscopy, quantitative ChIP-/ATAC-seq and RNA-seq in tissue samples from F1 hybrid mice, and
RNAi knockdown in gonad primary cell assays. The emergence of CRISPR/Cas technology makes it now possible
to edit multiple sites in the genome in a single step, and I will use this technology extensively in cell culture and in
vivo to test combinations of small-effect alleles that I predict together will compromise the buffering capacity of the
transcription network and cause sex reversal (Aim 3). Inferring and validating novel genes and genetic
interactions will require a collaboration of computational and experimental students/postdocs, and I expect to
cross-train my students in both dry and wet bench methods. I have designed a research strategy that can easily
be partitioned into modular projects suitable for undergraduate, graduate, or postdoctoral research, and
requires only a small standing colony of 12-15 mouse strains. Long term, I will seek to translate this predictive
network model to the human population to discover novel variants that cause DSD, and I will build
collaborations to extend this approach to reproductive biology at later stages, for example to elucidate the
genetic architecture underlying strain differences during spermatogenesis. Ultimately, I will apply my
understanding of critical/vulnerable genes and network interactions in the differentiating gonad transcriptome
to determine whether the same pathways affect decision processes in other organs.
References
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Martin, J.A. et al. 2008. Pediatrics 121:788-801.
Munger, S.C. and Capel, B. 2012. WiReS:SysBioMed 4:401-412.
Munger, S.C., Natarajan, A. et al. 2013. PLoS Genet: e1003630.
Eicher, E.M. et al. 1982. Science 217:535-537.
Eicher, E.M. et al. 1996. Nat Genet 14:206-209.
Research Statement: Steven Munger, Ph.D.
Date prepared: October, 2014
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Munger, S.C. et al. 2009. Genes & Dev 23(21):2521-2536.
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