Enhancer hubs govern chromatin topology and Th17 identity

Abstract / SummaryA wealth of noncoding regulatory elements has been described across mammalian cell types, yet determining their functional role remains a challenge. Regulatory control of gene expression is critical during active processes such as the adaptive immune response. Upon antigen presentation, a naive CD4+ T cell undergoes major transcriptional and structural reorganization necessary for establishment of subset identity and immune function. In this study, we systematically measure the regulatory potential of candidate regulatory elements associated with open chromatin across five mouse CD4+ subsets. Using ATAC-STARR-seq, we found that approximately 25% of open chromatin regions demonstrate regulatory activity. Most of these encode a broadly shared latent functional potential, though a subset display activity restricted to specific cellular contexts. To distinguish regulatory potential from endogenous function, we performed CRISPR-based epigenome editing screens at noncoding regions of Th17 cells and identified a set of core elements essential for subset polarization. Integrating Region Capture Micro-C, we resolved precise 3D chromatin topologies that explain functional regulatory networks via physical contacts. We characterize examples of active regulatory hubs formed through multiple CTCF-independent interactions organized in a hierarchical architecture. Furthermore, we discover a critical Batf enhancer that operates these contacts. Using targeted perturbations, we disrupt local chromatin topology and gene expression with profound consequence to downstream Th17 phenotypes. Finally, we confirm the physiological necessity of these functional enhancers in vivo, demonstrating the importance of noncoding elements for Th17 identity. Together, this work reveals how DNA sequence and chromatin cooperate to shape the regulatory logic of immune cells, with implications for understanding cell fate and function.