Abstract Body: Each cell type exhibits a unique chromatin organization and transcriptome that define its identity and function. Chromatin architectural proteins like CTCF and the cohesin complex are pivotal in shaping this regulatory landscape. Understanding how they drive cell-type-specific gene expression is essential for decoding complex traits and designing targeted therapies for diseases rooted in cellular identity. Endothelial cells (ECs), which line blood vessels and maintain vascular homeostasis, are key regulators of blood pressure (BP) and possess distinct transcriptional programs and cis-regulatory elements governed by chromatin architecture.
We hypothesized that CTCF and RAD21 (a cohesin subunit) play specialized roles in establishing the EC-specific transcriptome by modulating chromatin organization. To test this hypothesis, we depleted CTCF and RAD21 in human induced pluripotent stem cells (iPSCs) and iPSC-derived ECs (iECs), representing undifferentiated and lineage-committed states, respectively. We performed RNA sequencing to assess transcriptional changes and CUT&Tag to profile CTCF and RAD21 binding in both cell types.
Transcriptomic analysis revealed that both factors regulate EC-specific gene expression through shared and distinct pathways. Consistent with these transcriptional changes, CTCF or RAD21 depletion in iECs led to prominent loss of binding at promoters, suggesting direct regulatory roles, whereas in iPSCs, loss occurred mainly at intronic regions, indicating broader structural functions in the undifferentiated state. We also identified a potential EC-specific association between RAD21 and the ETS transcription factor FLI1, suggesting a novel regulatory axis in vascular gene control. Supporting these distinct roles, integration with Micro-C data showed that CTCF- and RAD21-sensitive loops in iECs were largely promoter-centric, whereas in iPSCs, RAD21-sensitive loops engaged more with non-promoter regions. Notably, we identified disrupted CTCF/Rad21 binding promoter regions that harbor SNPs associated with BP and vascular traits, linking chromatin architecture to noncoding genetic risk.
In conclusion, our study reveals distinct yet cooperative roles of CTCF and RAD21 in shaping EC-specific gene regulation. These findings provide insight into chromatin-mediated vascular identity and highlight regulatory loci with therapeutic relevance to hypertension and vascular disease.