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

Growth hormone (GH) regulates sexual dimorphic programs in liver gene expression impacting lipid metabolism, bile acid synthesis and xenobiotic processing, which contribute to sex differences in metabolic dysfunction-associated steatotic liver disease (MASLD). Despite extensive knowledge of sex-biased transcriptomes, the functional cis -regulatory elements orchestrating these sex differences in liver metabolic function are largely uncharacterized. We employed an integrative approach combining single nucleus (sn) multiOmics with in vivo functional enhancer profiling using HDI-STARR-seq to identify and validate GH-responsive, sex-biased enhancers in mouse liver. We profiled ∼1,000 differentially accessible regions (DARs) from male, female, and continuous GH-infused (cGH) male livers by snATAC-seq + snRNA-seq, linking chromatin accessibility to sex-biased gene expression. A tiled STARR-seq library comprised of 23,912 unique reporters spanning 1,839 ATAC regions was delivered to mouse liver by hydrodynamic injection (HDI), which enabled functional assessment of enhancer activity under physiologically relevant conditions and with minimal immune response. We identified 4,126 robust active reporters, of which 1,397, representing 840 distinct ATAC regions, showed sex-biased and/or GH-regulated activity, with strong positive correlations between chromatin accessibility and functional enhancer activity. Regulated enhancer regions were significantly enriched for activating histone marks (H3K4me1, H3K27ac) and binding sites for key transcription factors including STAT5, BCL6, HNF4α, and CUX2. Further, de novo motif analysis revealed sex-specific transcription factor binding patterns discriminating regulated from non-regulated enhancers. Finally, many of the functionally active sex-biased, cGH-responsive genes were linked to MASLD-enabling or MASLD-protective sex-biased genes. This work establishes HDI-STARR-seq as a powerful platform for dissecting hormone-regulated enhancer function in vivo and gives mechanistic insights into how hormonal control of chromatin accessibility drives sexual dimorphism in hepatic metabolism and differential metabolic liver disease risk between sexes.