Biology subjects

Waxman, D. J.

Publications and source records attributed to Waxman, D. J..

At least 19 recordsLinked to original sources

bioRxiv2025

Disease-associated mutations in the STAT5B SH2 domain reprogram hepatic cholesterol and lipid metabolism

Growth hormone (GH) signaling through STAT5B is a central regulator of hepatic metabolism, yet the functional consequences of disease-associated STAT5B variants remain poorly understood. Here, we analyzed mice carrying STAT5BY665F (gain-of-function) and STAT5BY665H (loss-of-function) variants and dissect their impact on metabolic regulation. STAT5BY665F mice developed hepatic lipid accumulation, hypercholesterolemia, and enhanced insulin sensitivity, whereas STAT5BY665H mice displayed reduced body weight and impaired insulin responsiveness. Transcriptomic analyses revealed that STAT5BY665F activated lipid, cholesterol, and immune transcriptional programs, while STAT5BY665H failed to induce these pathways. Notably, STAT5BY665F substantially feminized male liver gene expression, inducing 77% of female-biased genes while repressing 51% of male-biased genes, thereby mimicking the persistent STAT5B activation characteristic of female livers. ChIP-seq demonstrated extensive STAT5BY665F enhancer occupancy at metabolic and immune loci, contrasting with the minimal chromatin engagement of STAT5BY665H. Beyond the liver, STAT5BY665F broadly reprogrammed adipose tissue gene expression, activating lipid metabolism and immune regulatory networks, whereas STAT5BY665H exerted more restricted effects. Together, these findings illustrate how alterations in STAT5B activity affect enhancer activation and can lead to changes in metabolic function and hepatic sexual dimorphism.

genomics↗

bioRxiv2025

Single Nucleus MultiOmics and In Vivo Massively Parallel Reporter Assays Identify Functional Sex-Biased Enhancers Linked to Liver Metabolism and Disease

Growth hormone (GH) controls sexual dimorphism in hepatocyte gene expression programs governing lipid metabolism, bile acid synthesis and xenobiotic processing, which contribute to sex differences in metabolic dysfunction-associated steatotic liver disease (MASLD) risk. Despite extensive study of GH-regulated sex differences in gene transcription, the functional cis-regulatory hepatocyte enhancers that orchestrate these sex-dependent metabolic programs remain largely unknown. Here, we integrated single-nucleus multiomic profiling of hepatocyte chromatin accessibility with in vivo functional enhancer assays to identify and validate GH-responsive, sex-biased hepatocyte enhancers in intact mouse liver. We constructed a tiled HDI-STARR-seq library of 23,912 reporters spanning 1,839 liver ATAC regions and delivered it to liver by hydrodynamic injection, enabling functional assessment of enhancer activity in vivo across distinct biological conditions. Reporters representing 840 ATAC regions showed sex-biased and/or GH-regulated enhancer activity, in many cases mirroring the regulation of their chromatin accessibility in hepatocytes, validating these sites as functional, physiologically regulated enhancers. The regulated enhancers were enriched for activating histone marks (H3K4me1, H3K27ac), for binding sites for the GH-activated transcriptional regulator STAT5, and for the STAT5-dependent, sex-specific repressors BCL6 and CUX2. Further, de novo motif analysis identified binding sites for HNF4A and for several novel factors specifically enriched at the regulated enhancers. Sex-biased and GH-regulated functional enhancers were linked to both MASLD-enabling and MASLD-protective genes, suggesting that GH-dependent chromatin remodeling at these loci contributes to sex-differential metabolic disease susceptibility. This integrated in vivo approach defines a validated set of GH-regulated hepatocyte enhancers through which chromatin accessibility and transcription factor binding drive sexual dimorphism in hepatic metabolism and sex-specific MASLD risk.

genomics↗

bioRxiv2025

Single Nucleus MultiOmics Links Novel Transcription Factor Motifs to Murine Hepatic Sex Differences in Chromatin Accessibility and Metabolic Dysfunction-Associated Steatotic Liver Disease

The liver exhibits striking sexual dimorphism in gene expression that impacts drug and lipid metabolism and disease susceptibility, with males showing substantially higher predisposition to metabolic dysfunction-associated steatotic liver disease (MASLD) and its complications including hepatocellular carcinoma. These sex differences are primarily controlled by sexually dimorphic pituitary growth hormone (GH) secretion patterns; however, the underlying transcriptional and epigenetic regulatory networks remain only partially understood. Here, we generated paired single-nucleus chromatin accessibility (snATAC-seq) and gene expression (snRNA-seq) profiles from 46,188 liver nuclei isolated from male, female and continuous GH-infused male mice to comprehensively map the epigenetic basis of hepatic sexual dimorphism. We identified 127,957 accessible chromatin regions genome-wide, including thousands of novel regions enriched specifically in non-parenchymal cells. Sex-biased differentially accessible chromatin regions (DARs) were almost exclusively hepatocyte-localized, and continuous GH infusion feminized their accessibility, demonstrating that plasma GH patterns alone are sufficient to reprogram sex-biased hepatocyte chromatin landscapes. Correlation-based peak-to-gene linkage analysis mapped these DARs to sex-biased gene targets and revealed that regulatory interactions are constrained by topologically associated domain boundaries. Motif enrichment analysis identified both established regulators (STAT5, CUX2, BCL6) and novel transcription factors (TFs) at sex-biased DARs. ATAC-seq footprinting revealed novel TF motifs predicted to be occupied at DARs linked to sex-biased genes implicated in MASLD, providing mechanistic insights into the male bias in fatty liver disease. Further, motif co-occurrence analysis revealed TF clusters likely cooperating to regulate sex-dependent gene expression programs. We also identified stringently cell type-specific regulatory regions with cell type-specific TF motifs that define the regulatory architecture underlying hepatocyte and non-parenchymal cell identities. This comprehensive multiOmic atlas elucidates TF networks controlling sex-dependent liver gene expression and serves as a foundational resource for understanding molecular mechanisms underlying sex disparities in MASLD and other liver diseases.

genomics↗

bioRxiv2024

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of [~]50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

genomics↗

bioRxiv2024

Steatotic liver disease induced by TCPOBOP-activated hepatic constitutive androstane receptor: Primary and secondary gene responses with links to disease progression

Constitutive Androstane Receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to subsequent disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP, a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase levels, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared to males. Early (1-day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipid and xenobiotic metabolism and oxidative stress protection pathways; late (2-wk) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for immune response, macrophage activation, and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to pro-inflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle activated carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Thus, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary expression changes in liver non-parenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP gene responses include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease.

pharmacology and toxicology↗

bioRxiv2023

Plasma Growth Hormone Pulsatility Directly Regulates Male-biased Chromatin Accessibility in Adult Mouse Liver

Sex-differences in plasma growth hormone (GH) profiles, pulsatile in males and persistent in females, regulate sex differences in hepatic STAT5 activation linked to sex differences in gene expression and liver disease susceptibility, but little is understood about the fundamental underlying, GH pattern-dependent regulatory mechanisms. Here, DNase hypersensitivity site (DHS) analysis of liver chromatin accessibility in a cohort of 18 individual male mice established that the endogenous male rhythm of plasma GH pulse-stimulated liver STAT5 activation induces dynamic, repeated cycles of chromatin opening and closing at several thousand liver DHS and comprises a novel mechanism conferring male bias to liver chromatin accessibility. Strikingly, a single physiological replacement dose of GH given to hypophysectomized male mice restored, within 30 min, liver STAT5 activity and chromatin accessibility at 83% of the pituitary hormone-dependent dynamic male-biased DHS. Sex-dependent transcription factor binding patterns and chromatin state analysis identified key genomic and epigenetic features distinguishing this dynamic, STAT5-driven mechanism of male-biased chromatin opening from a second GH-dependent mechanism operative at static male-biased DHS, which are constitutively open in male liver. Dynamic but not static male-biased DHS adopt a bivalent-like epigenetic state in female liver, as do static female-biased DHS in male liver, albeit using distinct repressive histone marks in each sex, namely, H3K27me3 at female-biased DHS in male liver, and H3K9me3 at male-biased DHS in female liver. Moreover, sex-biased H3K36me3 marks are uniquely enriched at static sex-biased DHS, which may serve to keep these sex-dependent hepatocyte enhancers free of H3K27me3 repressive marks and thus constitutively open. Pulsatile chromatin opening stimulated by endogenous, physiological hormone pulses is thus one of two distinct GH-determined mechanisms for establishing widespread sex differences in hepatic chromatin accessibility and epigenetic regulation, both closely linked to sex-biased gene transcription and the sexual dimorphism of liver function.

genomics↗

bioRxiv2023

Growth Hormone Accelerates Recovery From Acetaminophen-Induced Murine Liver Injury

Background and AimsAcetaminophen (APAP) overdose is the leading cause of acute liver failure, with one available treatment, N-acetyl cysteine (NAC). Yet, NAC effectiveness diminishes about ten hours after APAP overdose, urging for therapeutic alternatives. This study addresses this need by deciphering a mechanism of sexual dimorphism in APAP-induced liver injury, and leveraging it to accelerate liver recovery via growth hormone (GH) treatment. GH secretory patterns, pulsatile in males and near-continuous in females, determine the sex bias in many liver metabolic functions. Here, we aim to establish GH as a novel therapy to treat APAP hepatotoxicity. Approach and ResultsOur results demonstrate sex-dependent APAP toxicity, with females showing reduced liver cell death and faster recovery than males. Single-cell RNA sequencing analyses reveal that female hepatocytes have significantly greater levels of GH receptor expression and GH pathway activation compared to males. In harnessing this female-specific advantage, we demonstrate that a single injection of recombinant human GH protein accelerates liver recovery, promotes survival in males following sub-lethal dose of APAP, and is superior to standard-of-care NAC. Alternatively, slow-release delivery of human GH via the safe nonintegrative lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP), a technology validated by widely used COVID-19 vaccines, rescues males from APAP-induced death that otherwise occurred in control mRNA-LNP-treated mice. ConclusionsOur study demonstrates a sexually dimorphic liver repair advantage in females following APAP overdose, leveraged by establishing GH as an alternative treatment, delivered either as recombinant protein or mRNA-LNP, to potentially prevent liver failure and liver transplant in APAP-overdosed patients.

pathology↗

bioRxiv2023

TCDD dysregulation of lncRNA expression, liver zonation and intercellular communication across the liver lobule

The persistent environmental aryl hydrocarbon receptor agonist and hepatotoxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces hepatic lipid accumulation (steatosis), inflammation (steatohepatitis) and fibrosis. Thousands of liver-expressed, nuclear-localized lncRNAs with regulatory potential have been identified; however, their roles in TCDD-induced hepatoxicity and liver disease are unknown. We analyzed single nucleus (sn)RNA-seq data from control and chronic TCDD-exposed mouse liver to determine liver cell-type specificity, zonation and differential expression profiles for thousands of IncRNAs. TCDD dysregulated >4,000 of these lncRNAs in one or more liver cell types, including 684 lncRNAs specifically dysregulated in liver non-parenchymal cells. Trajectory inference analysis revealed major disruption by TCDD of hepatocyte zonation, affecting >800 genes, including 121 IncRNAs, with strong enrichment for lipid metabolism genes. TCDD also dysregulated expression of >200 transcription factors, including 19 Nuclear Receptors, most notably in hepatocytes and Kupffer cells. TCDD-induced changes in cell-cell communication patterns included marked decreases in EGF signaling from hepatocytes to non-parenchymal cells and increases in extracellular matrix-receptor interactions central to liver fibrosis. Gene regulatory networks constructed from the snRNA-seq data identified TCDD-exposed liver network-essential lncRNA regulators linked to functions such as fatty acid metabolic process, peroxisome and xenobiotic metabolic. Networks were validated by the striking enrichments that predicted regulatory IncRNAs showed for specific biological pathways. These findings highlight the power of snRNA-seq to discover functional roles for many xenobiotic-responsive lncRNAs in both hepatocytes and liver non-parenchymal cells and to elucidate novel aspects of foreign chemical-induced hepatotoxicity and liver disease, including dysregulation of intercellular communication within the liver lobule.

pharmacology and toxicology↗

bioRxiv2022

Dysregulation of murine long non-coding single cell transcriptome in non-alcoholic steatohepatitis and liver fibrosis

LncRNAs are typified by low expression, nuclear enrichment, high tissue-specificity and functional diversity, but the vast majority are uncharacterized. Here, we develop a catalog of 48,000 mouse liver-expressed lncRNAs and use to elucidate lncRNA dysregulation in high fat diet-induced non-alcoholic steatohepatitis and carbon tetrachloride-induced liver fibrosis by single cell RNA-seq. LncRNA zonation patterns across the liver lobule were discovered in five cell populations. Perturbations in lncRNA expression were common in disease-associated cell types, including non-alcoholic steatohepatitis-associated macrophages, a hallmark of fatty liver disease progression, and collagen-producing myofibroblasts, key to liver fibrosis. Gene regulatory network analysis linked individual lncRNAs to biological pathways and network centrality metrics identified network-essential, disease-associated regulatory lncRNAs. Regulatory lncRNAs with network-predicted target gene promoters enriched for triplex-based lncRNA binding were also identified. These findings elucidate hepatic lncRNA cell-type specificities, spatial zonation patterns, linked biological networks and dysregulation in disease progression. A subset of the disease-associated lncRNAs have human orthologs and are promising candidates for biomarkers and therapeutic targets.

systems biology↗

bioRxiv2022

Long non-coding RNA G23Rik attenuates fasting-induced lipid accumulation in the liver

Peroxisome proliferator-activated receptor (PPAR) is a key mediator of lipid metabolism and metabolic stress in the liver. A recent study revealed that PPAR-dependent long non-coding RNAs (lncRNAs) play an important role in modulating metabolic stress and inflammation in the livers of fasted mice. Here hepatic lncRNA 3930402G23Rik (G23Rik) was found to have active peroxisome proliferator response elements (PPREs) within its promoter and is directly regulated by PPAR. Although G23Rik RNA was expressed to varying degrees in several tissues, the PPAR-dependent regulation of this lncRNA was only observed in the liver. Pharmacological activation of PPAR induced PPAR recruitment at the G23Rik promoter and a pronounced increase in hepatic G23Rik lncRNA expression. A G23Rik-null mouse line was developed to further characterize the function of this lncRNA in the liver. G23Rik-null mice were more susceptible to hepatic lipid accumulation in response to acute fasting. Histological analysis further revealed a pronounced buildup of lipid droplets and a significant increase in neutral triglycerides and lipids as indicated by enhance oil red O staining of liver sections. Hepatic cholesterol, non-esterified fatty acid, and triglyceride levels were significantly elevated in G23Rik-null mice and associated with induction of the lipid-metabolism related gene Cd36. These findings provide evidence for a lncRNA dependent mechanism by which PPAR attenuates hepatic lipid accumulation in response to metabolic stress through lncRNA G23Rik induction. Key pointsO_LI3930402G23Rik (G23Rik) is a liver-enriched long non-RNA. C_LIO_LIG23Rik is a PPARA target gene. C_LIO_LILoss of G23Rik increases hepatic lipid accumulation during fasting C_LI

physiology↗

bioRxiv2022

Constitutively active STAT5b feminizes mouse liver gene expression

STAT5 is an essential transcriptional regulator of the sex-biased actions of growth hormone (GH) in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the gene expression changes observed were associated with STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, overexpression of STAT5CA resulted in some histopathology not seen in continuous GH-infused male liver, including hepatocyte hyperplasia and distorted liver architecture. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the female-biased expression of a majority of female-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering such STAT5 derivatives for therapeutic use in treating liver disease.

genomics↗

bioRxiv2022

Impact of neonatal activation of nuclear receptor CAR (Nr1i3) on Cyp2 gene expression in adult mouse liver

Perinatal exposure to environmental chemicals is proposed to reprogram development and alter disease susceptibility later in life. Supporting this, neonatal activation of the nuclear receptor CAR (Nr1i3) by TCPOBOP induces persistent expression of mouse hepatic Cyp2 genes into adulthood, attributed to long-term epigenetic memory of the early life exposure [Hepatology (2012) 56:1499-1509]. Here, we confirm that the same high-dose (15x ED50) neonatal TCPOBOP exposure used in that work induces prolonged (12 weeks) increases in hepatic Cyp2 expression; however, we show that the persistence of expression can be fully explained by the persistence of residual TCPOBOP in liver tissue. When the long-term presence of TCPOBOP in tissue was eliminated by decreasing the neonatal TCPOBOP dose 22-fold (0.67x ED50), strong neonatal increases in hepatic Cyp2 expression were still obtained but did not persist into adulthood. Furthermore, the neonatal ED50-range TCPOBOP exposure did not sensitize mice to a subsequent, low-dose TCPOBOP treatment. In contrast, neonatal treatment with phenobarbital, a short half-life (t1/2=8 h) agonist of CAR and of PXR (Nr1i2), induced high-level neonatal activation of Cyp2 genes and also altered their responsiveness to low-dose phenobarbital exposure at adulthood by either increasing (Cyp2b10) or decreasing (Cyp2c55) expression. Thus, neonatal xenobiotic exposure can reprogram hepatic Cyp2 genes and alter their responsiveness to exposures later in life. These findings highlight the need to carefully consider xenobiotic dose, half-life and persistence in tissue when evaluating the long-term effects of early life environmental chemical exposures.

pharmacology and toxicology↗

bioRxiv2022

Interplay between murine sex-biased gene expression and hepatic zonation revealed by single nucleus RNA sequencing

The zonation of liver metabolic processes is well-characterized; however, little is known about the cell type-specificity and zonation of sexually dimorphic gene expression or its growth hormone (GH)-dependent transcriptional regulators. We address these issues using single nucleus RNA sequencing of 32,000 nuclei representing nine major liver cell types. Nuclei were extracted from livers from young adult male and female mice, from male mice infused with GH continuously to mimic the female plasma GH pattern, and from mice treated with TCPOBOP, a xenobiotic agonist ligand of the liver nuclear receptor CAR (Nr1i3). Analysis of these rich transcriptomic datasets revealed: 1) expression of sex-biased genes and their key GH-dependent transcriptional regulators is primarily restricted to hepatocytes and is not a feature of liver non-parenchymal cells; 2) many sex-biased transcripts show sex-dependent zonation within the liver lobule; 3) gene expression is substantially feminized in both periportal and pericentral hepatocytes when male mice are infused with GH continuously; 4) sequencing nuclei increases the sensitivity for detecting thousands of nuclear-enriched lncRNAs and enables determination of their liver cell type-specificity, sex bias and hepatocyte zonation profiles; 5) the periportal to pericentral hepatocyte cell ratio is significantly higher in male than female liver; and 6) TCPOBOP exposure disrupts sex-specific gene expression and hepatocyte zonation within the liver lobule. These findings highlight the complex interconnections between hepatic sexual dimorphism and zonation at the single cell level and reveal how endogenous hormones and foreign chemical exposure can alter these interactions across the liver lobule with large effects on both protein-coding genes and lncRNAs. SynopsisSingle nucleus RNA-seq analysis elucidated the cell type-specificity and zonation of the sex-biased murine liver transcriptome, including thousands of long non-coding RNAs. Xenobiotic exposure induced widespread dysregulation, including both gain and loss of sex-biased gene expression and changes in zonation.

systems biology↗

bioRxiv2021

Type-I interferon signaling is essential for robust metronomic chemo-immunogenic tumor regression in murine triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by poor prognosis and aggressive growth, with limited therapeutic options for many patients. Here, we use two syngeneic mouse TNBC models, 4T1 and E0771, to investigate the chemo-immunogenic potential of cyclophosphamide and the mechanistic contributions of cyclophosphamide-activated type-I interferon (IFN) signaling to therapeutic activity. Chemically-activated cyclophosphamide induced robust IFN/{beta} receptor-1-dependent signaling linked to hundreds of IFN-stimulated gene responses in both TNBC lines. Further, in 4T1 tumors, cyclophosphamide given on a medium-dose, 6-day intermittent metronomic schedule induced strong IFN signaling but comparatively weak immune cell infiltration associated with long-term tumor growth stasis. Induction of IFN signaling was somewhat weaker in E0771 tumors but was followed by extensive downstream gene responses, robust immune cell infiltration and prolonged tumor regression. The immune dependence of these effective anti-tumor responses was established by CD8 T-cell immunodepletion, which blocked cyclophosphamide-induced E0771 tumor regression and led to tumor stasis followed by regrowth. Strikingly, IFN/{beta} receptor-1 antibody blockade was even more effective in preventing E0771 immune cell infiltration and blocked the major tumor regression induced by cyclophosphamide treatment. Type-I IFN signaling is thus essential for the robust chemo-immunogenic response of these TNBC tumors to cyclophosphamide administered on a metronomic schedule. SignificanceTNBC has poor prognosis and few therapeutic options. We show that cyclophosphamide treatment can induces extensive tumor regression in syngeneic mouse models of TNBC via a chemo-immunogenic mechanism linked to type-I IFN production. Our findings establish that IFN signaling is essential for the robust anti-tumor actions of cyclophosphamide and suggest that treatment resistance may stem from silencing the IFN pathway. This suggests a new avenue for improving TNBC treatment efficacy.

cancer biology↗

bioRxiv2021

Harnessing natural variation to identify cis regulators of sex-biased gene expression in a multi-strain mouse liver model

Sex differences in gene expression are widespread in the liver, where a large number of autosomal factors act in tandem with growth hormone signaling to regulate individual variability of sex differences in liver metabolism and disease. Here, we compare hepatic transcriptomic and epigenetic profiles of mouse strains C57Bl/6J and CAST/EiJ, representing two subspecies separated by 0.5-1 million years of evolution, to elucidate the actions of genetic factors regulating liver sex differences. We identify 144 protein coding genes and 78 lncRNAs showing strain-conserved sex bias; many have gene ontologies relevant to liver function, are more highly liver-specific and show greater sex bias, and are more proximally regulated than genes whose sex bias is strain-dependent. The strain-conserved genes include key growth hormone-dependent transcriptional regulators of liver sex bias; however, three other transcription factors, Trim24, Tox, and Zfp809, lose sex-biased expression in CAST/EiJ mouse liver. To elucidate these strain specificities in expression, we characterized the strain-dependence of sex-biased chromatin opening and enhancer marks at cis regulatory elements (CREs) within expression quantitative trait loci (eQTL) regulating liver sex-biased genes. Strikingly, 208 of 286 eQTLs with strain-specific, sex-differential effects on expression were associated with a complete gain, loss, or reversal of expression sex differences between strains. Moreover, 166 of the 286 eQTLs were linked to the strain-specific gain or loss of localized sex-biased CREs. Remarkably, a subset of these CREs lacked strain-specific genetic variants yet showed coordinated, strain-dependent sex-biased epigenetic regulation. Thus, we directly link hundreds of strain-specific genetic variants to the high variability in CRE activity and expression of sex-biased genes, and uncover underlying genetically-determined epigenetic states controlling liver sex bias in genetically diverse mouse populations. Author summaryMale-female differences in liver gene expression confer sex differences in diverse biological processes relevant to human health and disease, but are difficult to model in inbred mice given their identical genetic backgrounds. Outbred mice provide some variability, but cross-strain studies of sex bias in rodents have not been well studied. Here we elucidate the actions of genetic factors regulating liver sex differences in two Diversity Outbred mouse founder mouse strains, C57Bl/6 and CAST/EiJ. We find that many of the strain differences in sex-biased gene expression can be linked to the gain or loss of a cis regulatory element associated with one or more strain-specific sequence variants. Strikingly, in many cases, the associated cis regulatory element lacked strain-specific variants, yet was subject to coordinated, strain-dependent epigenetic regulation. Thus, harnessing the power of naturally occurring genetic diversity of Diversity Outbred mice, we integrated biological data at the genetic, epigenetic, and transcriptomic levels across evolutionary divergent mouse strains to discover hundreds of localized genomic regions that control phenotypic sex differences in the liver. These findings may serve as a model for studies of human genetic variation and the effect of population-wide variation on sex differences in health and disease.

genetics↗

bioRxiv2021

Global analysis of expression, maturation and subcellular localization of mouse liver transcriptome identifies novel sex-biased and TCPOBOP-responsive long non-coding RNAs

While nuclear transcription and RNA processing and localization are well established for protein coding genes (PCGs), these processes are poorly understood for lncRNAs. Here, we characterize global patterns of transcript expression, maturation and localization for mouse liver RNA, including more than 15,000 lncRNAs. PolyA-selected liver RNA was isolated and sequenced from four subcellular fractions (chromatin, nucleoplasm, total nucleus, and cytoplasm), and from the chromatin-bound fraction without polyA selection. Transcript processing, determined from normalized intronic to exonic sequence read density ratios, progressively increased for PCG transcripts in going from the chromatin-bound fraction to the nucleoplasm and then on to the cytoplasm. Transcript maturation was similar for lncRNAs in the chromatin fraction, but was significantly lower in the nucleoplasm and cytoplasm. LncRNAs were 11-fold more likely to be significantly enriched in the nucleus than cytoplasm, and 100-fold more likely to be significantly chromatin-bound than nucleoplasmic. Sequencing chromatin-bound RNA greatly increased the sensitivity for detecting lowly expressed lncRNAs and enabled us to discover and localize hundreds of novel regulated liver lncRNAs, including lncRNAs showing sex-biased expression or responsiveness to a xenobiotic agonist ligand of constitutive androstane receptor (Nr1i3). Integration of our findings with prior studies and lncRNA annotations identified candidate regulatory lncRNAs for a variety of hepatic functions based on gene co-localization within topologically associating domains or transcription divergent or antisense to PCGs associated with pathways linked to hepatic physiology and diseases.

genomics↗

bioRxiv2020

Genetic factors contributing to extensive variability of sex-specific hepatic gene expression in Diversity Outbred mice

Sex-specific transcription characterizes hundreds of genes in mouse liver, many implicated in sex-differential drug and lipid metabolism and disease susceptibility. While the regulation of liver sex differences by growth hormone-activated STAT5 is well established, little is known about autosomal genetic factors regulating the sex-specific liver transcriptome. Here we show, using genotyping and expression data from a large population of Diversity Outbred mice, that genetic factors work in tandem with growth hormone to control the individual variability of hundreds of sex-biased genes, including many lncRNA genes. Significant associations between single nucleotide polymorphisms and sex-specific gene expression were identified as expression quantitative trait loci (eQTLs), many of which showed strong sex-dependent associations. Remarkably, autosomal genetic modifiers of sex-specific genes were found to account for more than 200 instances of gain or loss of sex-specificity across eight Diversity Outbred mouse founder strains. Sex-biased STAT5 binding sites and open chromatin regions with strain-specific variants were significantly enriched at eQTL regions regulating correspondingly sex-specific genes, supporting the proposed functional regulatory nature of the eQTL regions identified. Binding of the male-biased, growth hormone-regulated repressor BCL6 was most highly enriched at trans-eQTL regions controlling female-specific genes. Co-regulated gene clusters defined by overlapping eQTLs included sets of highly correlated genes from different chromosomes, further supporting trans-eQTL action. These findings elucidate how an unexpectedly large number of autosomal factors work in tandem with growth hormone signaling pathways to regulate the individual variability associated with sex differences in liver metabolism and disease. Author summaryMale-female differences in liver gene expression confer sex differences in many biological processes relevant to health and disease, including lipid and drug metabolism and liver disease susceptibility. While the role of hormonal factors, most notably growth hormone, in regulating hepatic sex differences is well established, little is known about how autosomal genetic factors impact sex differences on an individual basis. Here, we harness the power of mouse genetics provided by the Diversity Outbred mouse model to discover significant genome-wide associations between genetic variants and sex-specific liver gene expression. Remarkably, we found that autosomal expression quantitative trait loci with a strong sex-bias account for the loss or gain of sex-specific expression of more than 200 autosomal genes seen across eight founder mice strains. Genetic associations with sex-specific genes were enriched for sex-biased and growth hormone-dependent regulatory regions harboring strain-specific genetic variants. Co-regulated gene clusters identified by overlapping regulatory regions included highly correlated genes from different chromosomes. These findings reveal the extensive regulatory role played by autosomal genetic variants, working in tandem with growth hormone signaling pathways, in the transcriptional control of sex-biased genes, many of which have been implicated in sex differential outcomes in liver metabolism and disease susceptibility.

genetics↗

bioRxiv2020

STAT5 regulation of sex-dependent hepatic CpG methylation at distal regulatory elements mapping to sex-biased genes

Growth hormone-activated STAT5b is an essential regulator of sex-differential gene expression in mouse liver, however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5b (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were down regulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were de-repressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG-methylation were seen in STAT5-deficient liver, where sex differences in DNA methylation were abolished at 88% of ~1,500 differentially-methylated regions, largely due to an increase in methylation at the hypomethylated sites. STAT5-dependent CpG-hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (H3K4me1, H3K27ac), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. In conclusion, the sex-dependent binding of STAT5 to liver chromatin is closely linked to sex-dependent demethylation of distal regulatory elements mapping to STAT5-dependent genes important for liver sex bias.

genomics↗