Steven C. Munger

Genetic variation impacts biological response to chemical and environmental exposures, contributing to differences in resistance or susceptibility. Forward genetic screens of genetically diverse cell populations can be used to identify the precise genetic variants that drive response variation. Cell lines from laboratory mouse genetic reference populations like the Diversity Outbred (DO) population are well powered for genetic mapping. Diverse stem cell panels are especially beneficial for studying early developmental effects of chemical exposures like triphenyl phosphate (TPHP), an organophosphate flame retardant linked to adverse developmental effects, including altered cell cycle in stem cells. Self-renewal and pluripotency are defining properties of stem cells during development; therefore, it is important to understand how chemicals like TPHP may alter these critical characteristics. To better understand the influence of genetic variation on TPHP exposure response, we utilized a genetically diverse panel of DO induced pluripotent stem cells (DO iPSCs). Our analyses characterize interline variation in gene expression through differential gene expression analysis and expression quantitative trait locus (eQTL) mapping. We identified genomic loci that contribute to variation in gene expression following TPHP exposure, including a regulatory hotspot on chromosome 15. Gene set enrichment highlighted several pathways affected by TPHP including lipid metabolism, steroid hormone signaling, and cell cycle. Our study shows that genetic variation modulates the effects of TPHP on gene expression and cell cycle in stem cells. Additionally, our work demonstrates that genetically diverse cell panels like the DO iPSC resource offer a tractable and scalable approach for identifying the genetic determinants of chemical toxicity.

Phenotypic variation in pluripotent stem cells arises largely from genetic variants that impact stability of the pluripotent state, response to differentiation cues, and cell fate diversification. Panels derived from genetic reference populations leverage genetic diversity to map the loci influencing these molecular and cellular traits. Here we describe the first publicly available resource for in vitro systems genetics in Mus musculus: 299 genetically diverse, highly heterozygous embryonic stem cell lines derived from Diversity Outbred mice, with minimal kinship across the panel. We characterize genomic integrity and allelic variation across the panel and describe differences in pluripotency and differentiation networks in embryoid bodies. Mapping the genetic determinants of gene expression differences in response to cadmium exposure in embryoid bodies identified 13 regulatory hotspots, the largest acting on ribosome biogenesis genes. This panel enables mapping of loci and gene-by-environment interactions that influence molecular identity in pluripotent cells and their derived cell types.

Genetically diverse panels of human pluripotent stem cells enable genetic dissection of cellular phenotypes, but comparable induced pluripotent stem cell (iPSC) resources in model organisms remain limited. We generated a panel of iPSCs from the Diversity Outbred (DO) mouse population and established 288 genetically unique lines that retain the allele frequency distribution, heterozygosity, and low population structure of the source population. The lines exhibit consistent growth, pluripotent gene expression profiles, and capacity to form embryoid bodies. Transcriptomic profiling of the lines revealed significant variation in gene expression driven in part by genetic background. We used expression quantitative trait locus (eQTL) mapping to identify over 10,000 regulatory loci that influence gene expression variation, including multiple distal eQTL hotspots that are key gene regulatory hubs and are shared with DO embryonic stem cells (ESCs). The largest hotspot, mediated by Lifr , showed a shift in founder allele effects relative to ESCs, consistent with differences in cellular state and culture conditions. These results establish the DO iPSC panel as a genetically diverse, publicly accessible platform derived from a laboratory mouse genetic reference population, enabling integration of in vitro cellular phenotypes with in vivo traits within a closed, outbred population.

Motivation: Over the past two decades, integrating “-omics” scale molecular profiling with genetic mapping in diverse populations—termed “systems genetics”—has proven powerful for linking complex disease phenotypes to genetic variants and their regulatory effects. However, these combined genomic and genetic analyses remain computationally intensive and often inaccessible to researchers without a strong computational background, a problem compounded in studies with multiple tissues or thousands of molecular -omics phenotypes. Results: We developed QTLretrievR, an R package designed to simplify and streamline the identification and downstream analysis of molecular quantitative trait loci in genetically diverse populations. QTLretrievR builds on widely used R packages such as r/qtl2 for quantitative trait locus mapping and r/intermediate for mediation analysis, and incorporates substantial parallelization features to improve computational efficiency. Benchmarking demonstrates that QTLretrievR can reproduce results from previous quantitative trait locus studies while significantly reducing the time required for quantitative trait locus peak detection and downstream analyses. Additionally, QTL-retrievR includes plotting functions to visualize results of the included downstream analyses. Availability: Source code is available at https://github.com/deweyhannah/QTLretrievR . Contact: Steven Munger, steven.munger@jax.org

Genetic variation leads to phenotypic variability in pluripotent stem cells that presents challenges for regenerative medicine. Although recent studies have investigated the impact of genetic variation on pluripotency maintenance and differentiation capacity, less is known about how genetic variants affecting the pluripotent state influence gene regulation later in development. Here, we characterized expression of 12,000 genes in a large panel of donor-matched Diversity Outbred mouse embryonic stem cell and mouse neural progenitor cell lines. QTL mapping identified 4,060 expression QTLs in mouse neural progenitor cells, including 2,998 local and 1,062 distant expression QTLs. In a comparison of mouse neural progenitor cell and mouse embryonic stem cell expression QTLs, we found that local expression QTLs were more likely than distant expression QTL to be detected in both cell types. Distant expression QTLs were largely unique to 1 cell type, and we mapped 3 mouse neural progenitor cell–specific expression QTL hotspots on chromosomes 1, 10, and 11. Mediation analysis of the chromosome 1 hotspot identified Rnf152 as the best candidate mediator expressed in mouse neural progenitor cells, while cross-cell-type mediation using mouse embryonic stem cell gene expression along with partial correlation analysis strongly implicated genetic variant(s) affecting Pign expression in the mouse embryonic stem cell state as regulating the mouse neural progenitor cell chromosome 1 hotspot. These findings highlight that local mouse neural progenitor cell expression QTLs are more likely than distant expression QTLs to be shared with mouse embryonic stem cells; distant mouse neural progenitor cell expression QTLs are numerous but largely unique to that cell type, with many colocalizing to mouse neural progenitor cell–specific hotspots; and mediation analysis across cell types suggests that expression of Pign in mouse embryonic stem cells shapes the transcriptome of the more specialized mouse neural progenitor cell state.

Summary: It is currently thought that growing mammalian oocytes receive only small molecules via gap junctions from surrounding support cells, the granulosa cells. From the study of chimeric preantral oocyte and granulosa cell reaggregations, we provide evidence that growing mouse oocytes receive mRNAs from granulosa cells. Among the >1,000 granulosa-transcribed RNAs we identified in the oocyte, those that contribute to proper oocyte maturation and early embryo development were highly enriched. Predicted motifs for two RNA-binding proteins that function in RNA trafficking, FMRP and TDP43, were abundant in the UTRs of the granulosa-derived transcripts. Immunostaining demonstrated that both FMRP and TDP43 co-localize with the actin-rich granulosa cell protrusions that span the zone pellucida and connect to the oocyte, suggesting their role in importing mRNAs. Our results offer the possibility that oocyte failure may not always reflect an intrinsic oocyte deficiency but could arise from insufficient supply of maternal transcripts by granulosa cells during oocyte growth.

Genetic variation leads to phenotypic variability in pluripotent stem cells that presents challenges for regenerative medicine. Although recent studies have investigated the impact of genetic variation on pluripotency maintenance and differentiation capacity, less is known about how genetic variants affecting the pluripotent state influence gene regulation in later stages of development. Here, we characterized expression of more than 12,000 genes in 127 donor-matched Diversity Outbred (DO) mouse embryonic stem cell (mESC) and neural progenitor cell (mNPC) lines. Quantitative trait locus (QTL) mapping identified 2,947 expression QTL (eQTL) unique to DO mNPCs and 1,113 eQTL observed in both mNPCs and mESCs with highly concordant allele effects. We mapped three eQTL hotspots on Chromosomes (Chrs) 1, 10, and 11 that were unique to mNPCs. Target genes of the Chr 1 hotspot were overrepresented for those involved in mRNA processing, DNA repair, chromatin organization, protein degradation, and cell cycle. Mediation analysis of the Chr 1 hotspot identified Rnf152 as the best candidate mediator expressed in mNPCs, while cross-cell type mediation using mESC gene expression along with partial correlation analysis strongly implicated genetic variant(s) affecting Pign expression in the mESC state as regulating the mNPC Chr 1 eQTL hotspot. Together these findings highlight that many local eQTL confer similar effects on gene expression in multiple cell states; distant eQTL in DO mNPCs are numerous and largely unique to that cell state, with many co-localizing to mNPC-specific hotspots; and mediation analysis across cell types suggests that expression of Pign early in development (mESCs) shapes the transcriptome of the more specialized mNPC state.

The genetic integrity of pluripotent stem cells (PSC) is integral to their applications in research and therapy, but it is compromised by frequent development of copy number variations. Little is known about the basis of the variable genomic integrity among different PSC lines. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We identified 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression and that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. Possible mechanisms for this protective effect of X chromosome dosage include our discovery that the lines with two active X Chromosomes have a higher expression of 2-cell-like state genes, and differential expression of X-linked tumor suppressor genes associated with DNA damage response. Highlights First genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures Chromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities X-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells XX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response

Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions has previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic-epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic-epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of three- dimensional chromatin structure.

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A . Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of-function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate disease variants identified through human genetic studies.