Enhancers regulate gene expression through epigenetic mechanisms influenced by internal and external stimuli. In mouse liver, growth hormone and environmental chemical exposures activate enhancers regulating gene expression in part through changes in chromatin accessibility. Identifying functional enhancers in the complex environment of mouse liver is challenging. This thesis presents HDI-STARR-seq, a massively parallel STARR-seq reporter assay that combines hydrodynamic injection (HDI)-driven plasmid delivery to mouse liver cells in vivo with expression from the liver-specific Albumin promoter to assay functional changes in enhancer-driven transcription induced by sex-dependent hormonal stimulation and xenobiotic exposure. HDI-STARR-seq employs a minimal albumin promoter, shows minimal innate immune response and apparently facilitates plasmid chromatinization recapitulating endogenous liver chromatin states. The assay is robustly reproducible in a small reporter library (~100 regions) and can be scalable for genome-wide analysis using 25,000-50,000 synthetic DNA fragments or enzyme-released mouse liver genomic fragments. Single nucleus-based 10x Genomics Multiome analysis identified accessible mouse liver chromatin regions genome-wide, their linked target genes in hepatocytes, and their differential accessibility between sexes and following exposure to TCPOBOP, a CAR (Nr1i3) agonist ligand. Using HDI-STARR-seq, functional enhancer activity was determined for 1,789 accessible chromatin regions across biological conditions, identifying many sex-biased, GH-regulated enhancers undergoing chromatin changes. The regulated enhancers were associated with enrichment for H3K4me1 and H3K27ac histone marks, showed regulated activities that matched activating histone marks and contained Hnf4a and other enriched motifs in male-biased, GH-repressed enhancer sets. Further, the impact of TCPOBOP exposure for 1 day or 2 weeks on HDI-STARR-seq activity was evaluated in mice of both sexes at 1,834 accessible chromatin regions. Induced H3K27ac and static H3K4me1 were enriched at TCPOBOP-responsive enhancers, as were DR4 motifs bound by CAR/RXR heterodimers. Motifs bound by Tcf, involved in cell proliferation, were enriched in HDI-STARR-seq active enhancers only after 2-week TCPOBOP exposure, indicating activation of a late responding signaling pathway. This research gives novel insights into the relationship between functional enhancer activity in mouse liver and hormonal and xenobiotic regulated epigenetic landscapes, and establishes the utility of HDI-STARR-seq for discovery of biologically relevant enhancer sequences in vivo. / 2026-09-18T00:00:00Z