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.

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.