HIF-1a CUT&Tag binding profile reveals a multifaceted regulation of steroidogenesis

The production of steroid hormones by the adrenal cortex is essential for maintaining homeostasis in response to stress. Disruptions to this process have been linked to various diseases, including congenital defects in steroid synthesis, Cushing syndrome, and adrenal tumors. Therefore, proper regulation of steroid hormone levels is essential for maintaining physiological balance and overall health. Hypoxia is a key regulator of adrenal steroidogenesis acting via the stabilization of HIF-1a transcription factors. We recently identified HIF-1a as a regulator of adrenal steroidogenesis through its control of specific microRNAs (miRNAs) that target key steroidogenic enzymes. However, the mechanisms by which HIF-1a influences miRNA expression remain unclear. To address this, we used the Cleavage Under Targets & Tagmentation (CUT&Tag) technique to map HIF-1a binding sites across the genome in a murine adrenocortical cell line. Our analysis of the HIF-1a binding profile revealed putative binding sites not only in classic steroidogenic gene loci, including Cyp11a1, but also in miRNA loci involved in steroid regulation. Crucially, HIF-1a-bound regions included genes involved in miRNA biogenesis and function, such as nuclear microprocessor and cytoplasmic RNA-induced silencing complex (RISC) components. Subsequent analysis of hypoxic transcriptomic profiles demonstrated widespread repression of these miRNA biogenesis and function genes by hypoxia, that could be modulated by HIF-1a. Based on our previous identification of HIF-1a as a regulator of adrenal steroidogenesis, our current findings further support its dual role in directly controlling steroidogenic gene expression via both transcriptional and posttranscriptional mechanisms. These results reinforce the HIF pathway as a master regulator of steroid hormone output under hypoxic conditions. Overall design: HIF-1a CUT&Tag samples prepared from murine adrenocortical Y-1 cells grown under standard culture conditions in control medium for 24 hours (2 replicates) or Y-1 cells grown under standard culture conditions in the presence of 1 mM DMOG (dimethyloxalylglycine) for 24 hours (2 replicates)