Biology subjects

Czechanski, A.

Publications and source records attributed to Czechanski, A..

15 recordsLinked to original sources

bioRxiv2026

The Flame Retardant Triphenyl Phosphate Induces Varying Responses in Genetically Diverse Mouse Induced Pluripotent Stem Cells

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.

genomics↗

bioRxiv2026

Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

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.

genetics↗

bioRxiv2025

A shared genetic regulator of metabolism and addiction-related behavior in mice and humans

Substance use disorders and other mental health conditions often co-occur with metabolic disorders, suggesting shared biological underpinnings1. These heightened behavioral and physiological responses may have evolved to promote survival during resource scarcity but can become maladaptive in modern environments2. The genetic mechanisms linking these traits have remained elusive. Here, we show that a variable gene enhancer in mice jointly regulates genes encoding an epigenetic factor (Eed) and a mitochondrial enzyme (Me3) thereby influencing propensity to addiction-related behaviors and mitochondrial function. We further identify variation in a corresponding enhancer in humans regulating EED and ME3 associated with substance use, psychiatric and metabolic disorders. These findings reveal a convergent genetic regulatory network linking mitochondrial biology to behavioral and metabolic risk, offering insight into how genetic variation in beneficial regulatory pathways can predispose individuals to substance use disorders and related conditions.

genetics↗

bioRxiv2024

Anapc5 and Anapc7 as genetic modifiers of KIF18A function in fertility and mitotic progression

The kinesin family member 18A (KIF18A) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Functional dependency on KIF18A varies by cell type and genetic context but the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association at a Chr5 locus where anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We found that both genes were differentially expressed in a sensitive strain background when compared to resistant strain background at key timepoints in gonadal development. We also identified a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of ANAPC7 (APC7) and exacerbated by co-depletion of ANAPC5 (APC5). These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies.

cell biology↗

bioRxiv2024

The structural diversity of telomeres and centromeres across mouse subspecies revealed by complete assemblies

It is over twenty years since the publication of the C57BL/6J mouse reference genome, which has been a key catalyst for understanding mammalian disease biology. However, the mouse reference genome still lacks telomeres and centromeres, contains 281 chromosomal sequence gaps, and only partially represents many biomedically relevant loci. We present the first T2T mouse genomes for two key inbred strains, C57BL/6J and CAST/EiJ. These T2T genomes reveal significant variability in telomere and centromere sizes and structural organisation. We add an additional 213 Mbp of novel sequence to the reference genome containing 517 protein-coding genes. We examined two important but incomplete loci in the mouse genome - the pseudoautosomal region (PAR) on the sex chromosomes and KRAB zinc finger proteins (KZFPs) loci. We identified distant locations of the PAR boundary, different copy number and sizes of segmental duplications, and a multitude of amino acid substitution mutations in PAR genes.

genomics↗

bioRxiv2024

Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

The genetic integrity of pluripotent stem cells (PSC) is critical to their applications in research and therapy, but it is compromised by frequent development of structural chromosome variants associated with malignancy. Many cell lines exhibit remarkable genetic stability, but little is known about the basis of the known variation in genomic integrity amongst different PSC isolates. 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 found 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 a prospective study confirmed that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. We identify potential mechanisms for this protective effect of X chromosome dosage, including our findings that the lines with two active X Chromosomes have a higher proportion of 2-cell-like cells, a state associated with maintenance of genetic integrity of mESC, and that they show differential expression of X-linked tumor suppressor genes associated with the DNA damage response. HighlightsO_LIFirst genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures C_LIO_LIChromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities C_LIO_LIX-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells C_LIO_LIXX 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 C_LI

genetics↗

bioRxiv2024

A pluripotent stem cell platform for in vitro systems genetics studies of mouse development

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.

developmental biology↗

bioRxiv2022

Resolution of structural variation in diverse mouse genomes reveals chromatin remodeling due to transposable elements

Diverse inbred mouse strains are among the foremost models for biomedical research, yet genome characterization of many strains has been fundamentally lacking in comparison to human genomics research. In particular, the discovery and cataloging of structural variants is incomplete, limiting the discovery of potentially causative alleles for phenotypic variation across individuals. Here, we utilized long-read sequencing to resolve genome-wide structural variants (SVs, variants [≥] 50 bp) in 20 genetically distinct inbred mice. We report 413,758 site-specific SVs that affect 13% (356 Mbp) of the current mouse reference assembly, including 510 previously unannotated variants which alter coding sequences. We find that 39% of SVs are attributed to transposable element (TE) variation accounting for 75% of bases altered by SV. We then utilized this callset to investigate the impact of TE heterogeneity on mouse embryonic stem cells (mESCs), and find multiple TE classes that influence chromatin accessibility across loci. We also identify strain-specific transcription start sites originating in polymorphic TEs that modify gene expression. Our work provides the first long-read based analysis of mouse SVs and illustrates that previously unresolved TEs underlie epigenetic and transcriptome differences in mESCs.

genomics↗

bioRxiv2022

Imputation of 3D genome structure by genetic-epigenetic interaction modeling in mice

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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/479436v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14d4221org.highwire.dtl.DTLVardef@130a812org.highwire.dtl.DTLVardef@16a6069org.highwire.dtl.DTLVardef@df9d9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype

Neural progenitor cells (NPC) represent potential cell transplantation therapies for CNS injuries. To understand how lesion environments influence transplanted NPC fate in vivo, we derived NPC expressing a ribosomal protein-hemagglutinin tag (RiboTag) for transcriptional profiling of transplanted NPC. Here, we show that NPC grafted into uninjured CNS generate cells that are transcriptionally similar to healthy astrocytes and oligodendrocyte lineages. In striking contrast, NPC transplanted into serum-exposed CNS lesions after stroke or spinal cord injury generate cells that share transcriptional, morphological and functional features with newly proliferated host astroglia that restrict inflammation and fibrosis and thereby protect adjacent neural tissue. Our findings reveal overlapping differentiation potentials of grafted NPC and proliferating host astrocytes; and show that in the absence of other interventions, non-cell autonomous cues in CNS lesions direct the differentiation of grafted NPC predominantly towards a naturally occurring neuroprotective wound repair astroglial phenotype.

neuroscience↗

bioRxiv2021

Deficiency of SYCP3-related XLR3 Disrupts the Initiation of Meiotic Sex Chromosome Inactivation in Mouse Spermatogenesis

In mammals, the X and Y chromosomes share only small regions of homology called pseudo-autosomal regions (PAR) where pairing and recombination in spermatocytes can occur. Consequently, the sex chromosomes remain largely unsynapsed during meiosis I and are sequestered in a nuclear compartment known as the XY body where they are transcriptionally silenced in a process called meiotic sex chromosome inactivation (MSCI). MSCI mirrors meiotic silencing of unpaired chromatin (MSUC), the sequestration and transcriptional repression of unpaired DNA observed widely in eukaryotes. MSCI is initiated by the assembly of the axial elements of the synaptonemal complex (SC) comprising the structural proteins SYCP2 and SYCP3 followed by the ordered recruitment of DNA Damage Response (DDR) factors to effect gene silencing. However, the precise mechanism of how unsynapsed chromatin is detected in meiocytes is poorly understood. The sex chromosomes in eutherian mammals harbor multiple clusters of SYCP3-like amplicons comprising the Xlr gene family, only a handful of which have been functionally studied. We used a shRNA-transgenic mouse model to create a deficiency in the testis-expressed multicopy Xlr3 genes to investigate their role in spermatogenesis. Here we show that knockdown of Xlr3 in mice leads to spermatogenic defects and a skewed sex ratio that can be traced to MSCI breakdown. Spermatocytes deficient in XLR3 form the XY body and the SC axial elements therein, but are compromised in their ability to recruit DDR components to the XY body. Author SummaryA key event in the production of sperm is the pairing and synapsis of homologous chromosome pairs to facilitate genetic recombination during the first meiotic division. Chromosomal abnormalities that undermine pairing and synapsis in spermatocytes trigger a checkpoint that leads to removal of the abnormal cells via programmed cell death. In mammals, the sex chromosomes, X and Y, lack homology through most of their length and remain largely unpaired. To avoid triggering the checkpoint the X and Y are sequestered in a specialized nuclear compartment called the XY body. DNA damage response (DDR) proteins are recruited to the XY body in a highly ordered progression leading to repression of all gene transcription from the sex chromosomes. We show that knocking down expression of the X-linked SYCR3-like gene, Xlr3, disrupts sex chromosome gene silencing by interfering with recruitment of DDR factors, leading to compromised sperm production.

genetics↗

bioRxiv2021

Genetic Control of Pluripotency Epigenome Determines Differentiation Bias in Mouse Embryonic Stem Cells

Genetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naive networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naive pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

genomics↗

bioRxiv2019

Genetic variation influences pluripotent ground state stability in mouse embryonic stem cells through a hierarchy of molecular phenotypes

Mouse embryonic stem cells (mESCs) cultured under controlled conditions occupy a stable ground state where pluripotency-associated transcriptional and epigenetic circuitry are highly active. However, mESCs from some genetic backgrounds exhibit metastability, where ground state pluripotency is lost in the absence of ERK1/2 and GSK3 inhibition. We dissected the genetic basis of metastability by profiling gene expression and chromatin accessibility in 185 genetically heterogeneous mESCs. We mapped thousands of loci affecting chromatin accessibility and/or transcript abundance, including eleven instances where distant QTL co-localized in clusters. For one cluster we identified Lifr transcript abundance as the causal intermediate regulating 122 distant genes enriched for roles in maintenance of pluripotency. Joint mediation analysis implicated a single enhancer variant ~10kb upstream of Lifr that alters chromatin accessibility and precipitates a cascade of molecular events affecting maintenance of pluripotency. We validated this hypothesis using reciprocal allele swaps, revealing mechanistic details underlying variability in ground state metastability in mESCs.

genomics↗

bioRxiv2018

Mitotic chromosome alignment is required for proper nuclear envelope reassembly

Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell division, however, its importance to genomic stability and cellular fitness are not understood. Normal mammalian somatic cells lacking Kif18A function complete cell division without aligning chromosomes. These alignment-deficient cells display normal chromosome copy numbers in vitro and in vivo, suggesting that chromosome alignment is largely dispensable for maintenance of euploidy. However, we find that loss of chromosome alignment leads to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit, and the formation of micronuclei in vitro and in vivo. These defects slow cell proliferation and reduce postnatal growth and survival with variable penetrance in mice. Our studies support a model in which the alignment of mitotic chromosomes promotes proper nuclear envelope reassembly and continued proliferation by ensuring that chromosomes segregate as a compact mass during anaphase.

cell biology↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗