The Cistrome Response to Hypoxia in Human Umbilical Vein Endothelial Cells

Hypoxic stress triggers transcriptional signaling mainly through hypoxia-inducible transcription factors (HIFs), which bind hypoxia response elements (HREs) in gene regulatory regions. However, only a small proportion ([~]1%) of known HREs are occupied by HIFs during hypoxia, suggesting the involvement of additional hypoxia-responsive factors. To address this gap, we utilized MOA-seq. This MNase-based assay enables genome-wide, high-resolution (<30 bp) identification of transcription factor (TF) occupancy footprints embedded within larger regions, most of which were previously annotated as open or accessible chromatin. Applying this native cistrome mapping to endothelial cells under normoxia or hypoxia (1, 3, or 24 hours) revealed thousands of hypoxia-responsive genomic sites with dynamic TF footprints. The affected genes were enriched in canonical hypoxia-induced pathways, such as angiogenesis. Motif analysis identified over 100 candidate TFs potentially mediating these multifaceted genomic responses. By grouping the gain/loss footprint patterns over time, we defined 10 distinct TF kinetic clusters, half of which were associated with HIF1A. HIF1A-proximal binding sites suggested co-activators, while non-HIF1A clusters pointed to additional TFs with HIF1A-independent roles. This analysis provides insight into how multiple TF networks coordinate hypoxia responses and highlights the power of cistrome profiling to deepen understanding of genomic regulation under low oxygen conditions. KEY POINTSO_LIMOA-seq mapped TF occupancy at 21,765 sites in normoxia, including 7,444 beyond the known ENCODE cCREs. C_LIO_LIHypoxia for 1, 3, and 24h changes the cistrome occupancy at thousands of genes. C_LIO_LIClustering analysis of hypoxia-responsive footprints consolidated cistrome kinetics into HIF1A-associated and HIF1A-independent TFs. C_LI