Endothelial cell differentiation is encompassed by changes in long range chromatin interactions between inactive chromatin regions. Homo sapiens

Endothelial cells, which constitute the luminal layer of blood and lymphatic vessels, differentiate from mesodermal progenitors during a process called vasculogenesis. By analyzing changes in chromatin interactions in human umbilical vein endothelial cells (HUVEC) compared to human embryonic stem cells (ESC) and mesendoderm cells (MSC), we identified regions exhibiting endothelial-specific compartmentalization and changes in the degree of connectivity within topologically associated domains (TADs). These EC-specific regions of chromatin interactions were characterized by EC-specific transcription, binding of lineage-determining transcription factors and cohesin. In addition, we developed a method that identified for the first time hundreds of cell-type specific long-range interactions between TADs. Most of these interactions were connections between regions enriched for H3K9me3, suggesting that majority of long-range interactions form between regions of heterochromatin involving pericentromeric regions during cellular differentiation. Secondly, we demonstrate that the hierarchy of chromatin domains is already pre-established in ESCs suggesting that higher level chromatin aggregation is conserved throughout cellular differentiation. Finally, we investigated the effect of hypoxia on transcription and chromatin interactions of ECs. Although hypoxic stress altered the expression of hundreds of genes, it had only a minimal effect on the chromatin compartmentalization, on the amount of local interactions within TADs or long range interactions. Collectively our results show that large structural rearrangements establish chromatin architecture required for functional endothelium and this architecture remains mostly unchanged in response to hypoxic stress. Overall design: Chromatin interactions of HUVECs were analyzed genome-wide using tethered conformation capture (TCC) in normoxia and hypoxia (8h, 1% O2). Transcriptional response was studied using global run-on sequencing (GRO-seq), cohesin binding using ChIP-seq for RAD21 and histone modifications using ChIP-seq for H3K9me3 and H3K27me3.