The cistrome response to hypoxia in human umbilical vein endothelial cells

The primary goal of this study was to achieve a comprehensive, high-resolution understanding of how human umbilical vein endothelial cells (HUVECs) remodel their chromatin landscape in response to hypoxic stress. While it is well established that hypoxia-inducible factors (HIFs) play a pivotal role in activating hypoxia-responsive genes, the discovery that only a small fraction of known hypoxia response elements (HREs) is bound by HIFs under low-oxygen conditions suggests the involvement of additional, complementary regulatory factors. To investigate these putative co-regulatory elements, we adapted and applied a recently developed technique, MOA-seq (MNase-defined cistrome-Occupancy Analysis), which excels at identifying genome-wide, protein-occupied loci (MOA footprints) with approximately 30-bp resolution. By profiling small MNase-sensitive footprints, MOA-seq offers an orthogonal and more precise view of transcription factor (TF) occupancy compared to traditional methods like ChIP-seq or ATAC-seq. Through a time-course analysis (1, 3, and 24 hours of hypoxia), we generated a "cistrome atlas" of MOA footprints for more than 5,000 candidate regulatory regions undergoing hypoxia-induced changes. Our approach not only recapitulated canonical hypoxia-regulated pathways but also uncovered novel sites and TF motifs that do not co-localize with HIF-binding motifs. These high-resolution footprints revealed new potential avenues for understanding how endothelial cells respond to low-oxygen environments, beyond the classic HIF-centric paradigm. Critically, such a broad scope of regulation underscores the complexity of vascular remodeling, oxidative stress responses, and angiogenic signaling processes vital to both normal physiological adaptation and the development of pathologies such as cancer, atherosclerosis, and other hypoxia-linked conditions. By integrating MOA-seq TF occupancy profiling with differential gene expression analyses, we are expanding the toolkit for dissecting gene regulatory networks over time. This insight has far-reaching implications: the newly uncovered regulatory elements may serve as potential therapeutic targets or biomarkers for diseases where hypoxia is a key driver. Moreover, the single-assay simplicity of MOA-seq provides a scalable strategy for profiling diverse cellular contexts, adding value to fundamental research and translational applications.