The cistrome response to hypoxia in human umbilical vein endothelial cells

Transcriptional signaling during hypoxic stress is primarily governed by hypoxia-inducible transcription factors (HIFs), which bind to 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 pathways.

To address this gap, we utilized MOA-seq, an MNase-based assay that enables genome-wide, high-resolution (<30 bp) identification of transcription factor (TF) occupancy footprints within open chromatin. We applied this cistrome profiling approach to nuclei from cultured endothelial cells exposed to normoxia or hypoxia for 1, 3, or 24 hours, revealing thousands of hypoxia-modulated 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 this complex multifaceted genomic response. We followed up these observations with a quantitative footprint clustering analysis to gain mechanistic insight into how concurrent TF regulatory networks might choreograph these responses. We found 10 distinct MOA-defined kinetic clusters, half of which contained HIF1A motifs.

HIF1A-proximal binding sites revealed candidate co-activators, whereas motifs in non-HIF1A clusters implicated HIF1A-independent pathways. These findings highlight the utility of cistrome analysis in unraveling the genomic response to hypoxia.