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HDI-STARR-seq library profiling of differential accessible chromatin regions (DARs) in livers of male, female and male treated with continuous growth hormone (cGH) mice.

ABSTRACT: Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models, which are confounded by vector-induced innate immune responses and lack the physiologically relevant cellular and endogenous hormonal context and chromatin environment of complex mammalian tissues. Here, we combine hydrodynamic injection (HDI) with a modified STARR-seq-based MPRA, designated HDI-STARR-seq (PMID:39716078) to determine condition-specific enhancer activity in mouse liver at scale. Analysis of a synthetic STARR-seq library, comprised of 23,912 reporters regions spanning 1839 ATAC regions enabled functional assessment of mouse liver enhancer activity in vivo across three distinct biological conditions: male iver, feamle liver and continuous growth hormone (GH)-infused male liver, which is substantially feminized with regards to gene expression. HDI-STARR-seq reporters representing 305 ATAC regions showed sex-biased and GH-responsive enhancer activity that mirrored the regulation of their chromatin accessibility in hepatocytes, validating these sites as functional, physiologically regulated enhancers. These enhancers were enriched for activating histone marks (H3K4me1, H3K27ac) and for binding sites for GH-activated transcriptional regulator STAT5 and for the STAT5-dependent, sex-specific repressors BCL6 and CUX2, consistent with a cohesive regulatory network controlling hepatocyte lipid and xenobiotic metabolism. Further, de novo motif analysis identified binding sites for HNF4A and for several novel factors specifically enriched at the regulated enhancers. Importantly, sex-biased and GH-regulated functional enhancers were linked to both MASLD (metabolic dysfunction-associated steatotic liver disease)-enabling and MASLD-protective genes, indicating that GH-dependent chromatin remodeling at these loci is a key determinant of sex-differential liver 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE315771 | GEO | 2026/01/07

REPOSITORIES: GEO

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Project description:Sex-dependent pituitary growth hormone (GH) secretory patterns determine the sex-biased expression of >1,000 genes in mouse and rat liver, affecting lipid and drug metabolism, inflammation and disease. A fundamental biological question is how robust differential expression can be achieved for hundreds of sex-biased genes simply based on the GH input signal pattern: pulsatile GH stimulation in males vs. near-continuous GH exposure in females. STAT5 is an essential transcriptional mediator of the sex-dependent effects of GH in the liver, but the mechanisms that underlie its sex-dependent actions are obscure. Here we elucidate the dynamic, sex-dependent binding of STAT5 and the GH/STAT5-regulated repressor BCL6 to mouse liver chromatin, revealing the counteractive interplay between these two regulators of liver sex-specificity. Our findings establish a close correlation between sex-dependent STAT5 binding and sex-biased target gene expression. Moreover, sex-dependent STAT5 binding correlated positively with sex-biased DNase hypersensitivity and H3-K4me1 and H3-K4me3 (activating) marks, correlated negatively with sex-biased H3-K27me3 (repressive) marks, and was associated with sex-differentially enriched motifs for HNF6/CDP factors. Importantly, BCL6 binding was preferentially associated with repression of female-biased STAT5 targets in male liver. Furthermore, BCL6 and STAT5 common targets but not BCL6 unique targets showed strong enrichment for lipid and drug metabolism. These findings provide a comprehensive, genome-wide view of the mechanisms whereby these two GH-regulated transcription factors establish and maintain sex differences affecting liver physiology and disease. The approaches used here to characterize sex-dependent STAT5 and BCL6 binding can be applied to other condition-specific regulatory factors and binding sites and their interplay with co-operative chromatin-binding factors. Mouse livers were excised from individual male and female mice killed at either a peak of STAT5 binding activity, or during the growth hormone (GH) interpulse interval, when STAT5 activity is either low (females) or essentially undetectable (males). Sonicated, cross-linked liver nuclear chromatin was then used to identify STAT5 binding sites by ChIP-Seq.

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HDI-STARR-seq library profiling of differential accessible chromatin regions (DARs) in livers of male, female and male treated with continuous growth hormone (cGH) mice.

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