Dynamic regulation of cystathionine gamma-lyase expression during normoxia, ischemia, and reperfusion in liver: interplay of transcription factors, microRNAs, and histone modifications

Abstract

Cystathionine gamma-lyase (CTH), a key enzyme in hydrogen sulfide (H2S) biosynthesis, has been studied extensively in models of oxidative stress. Dysregulated CTH has been reported in cardiovascular disease, ischemia-reperfusion (I/R) injury, aging, and xenobiotic stress, where reactive oxygen species are central to pathology. Despite this, how CTH is regulated at the molecular level under basal, ischemic, and reperfusion conditions is largely unknown. We observed that CTH expression is dysregulated during ischemia and reperfusion in Huh7 hepatic cells, mouse liver, and human liver, and therefore systematically dissected its transcriptional, post-transcriptional, and epigenetic control. Promoter-deletion reporter assays identified the -275/+91 bp region as the core human CTH promoter. This region was predicted to harbour binding sites for Sp1, CREB, and NFY, which were confirmed through overexpression, knockdown or inhibition, and ChIP assays in Huh7 cells. Computational and experimental screening identified miR-150-5p and miR-510-5p as novel post-transcriptional regulators that bind directly to the CTH 3'-UTR. We also examined histone marks (H3K9ac, H3K9me3, H3K4me3, and H3K27me3) at the CTH promoter and found that H3K9ac, H3K4me3, and H3K27me3 correlate with CTH expression across basal, ischemic, and reperfusion conditions. Mechanistically, ischemia reduces Sp1 occupancy at the CTH promoter, raises miR-150-5p and miR-510-5p levels, lowers H3K9ac, collectively suppressing CTH. Reperfusion reverses this with greater Sp1 and CREB binding, lower miR-150-5p and miR-510-5p levels, and reduced H3K27me3, restoring CTH expression. Together, these findings reveal novel roles for Sp1, CREB, NFY, miR-150-5p, miR-510-5p, and histone modifications in the regulation of CTH during basal, ischemia and reperfusion conditions.