Systematic functional characterization of CD4+ T cell regulatory elements reveals that while sequence-encoded enhancer activity is broadly shared across subsets, a small core set of essential enhancers at the Batf, Rorc, and Il17a loci governs Th17 cell identity through precise CTCF-independent 3D chromatin hub architectures, where perturbation of a single critical enhancer can disrupt local chromatin topology and produce genome-wide regulatory effects comparable to gene knockout with profound consequences for immune function.

A 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+ T cell subsets. Using ATAC-STARR-seq, we found that approximately one quarter of open chromatin regions demonstrate regulatory activity. Most exhibit shared functional potential across subsets, though we identify enhancers with activity that is 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 via these contacts. Using targeted perturbations, we disrupt local chromatin topology and gene expression with profound consequence to downstream Th17 cell phenotypes. We confirm the physiological necessity of these functional enhancers in vivo, demonstrating the importance of noncoding elements for Th17 cell identity. Together, this work reveals how DNA sequence and chromatin cooperate to shape the regulatory logic of Th17 cells, with implications for cis-regulatory principles beyond the immune system.