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. Although GH-regulated sex-specific transcription is well-studied, 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 enhancer activity assessment across different biological conditions. Reporters representing 840 ATAC regions showed sex-biased and/or GH-regulated enhancer activity, in many cases mirroring regulation of their accessibility in hepatocyte chromatin, validating them as functional, physiologically regulated enhancers. The regulated enhancer sequences were enriched for activating histone marks (H3K27ac, H3K4me1), and for binding sites for the STAT5-dependent, sex-specific repressors BCL6 and CUX2; whereas, STAT5 binding was enriched at both regulated and non-regulated enhancers. Motifs for HNF4A and for several novel factors identified de novo were specifically enriched at the regulated enhancers. Sex-biased and GH-regulated 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 MASLD risk.
Chang et al. (Thu,) studied this question.