RORA proximal enhancer regulates its expression during early human Th17 cell differentiation [ATAC]

Distal regulatory elements, such as enhancers, play a pivotal role in dictating cell identity by controlling the transcriptional status of specific cells. The comprehensive understanding of the epigenetic landscape, encompassing enhancer establishment and chromatin accessibility, during the differentiation of human Th17 cells remains incomplete. Leveraging ATAC-seq based chromatin accessibility profile along with the profiling of key histone marks we identified potential enhancers crucial for fate specification of Th17 cells. We found that 24 single nucleotide polymorphisms (SNPs) associated with autoimmune diseases were located near Th17-enhancers. Interestingly, these SNPs overlapped the binding sites of transcription factor active in Th17 cells. Among the Th17 specific enhancers, we identified an enhancer in the intron of RAR-related orphan receptor alpha (RORA) that was earlier predicted to regulate RORA expression. Functional validation through luciferase reporter assays confirmed that this enhancer positively regulate the transcription. Moreover, employing CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer, we demonstrated its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer modulates Th17 differentiation and into the role of regulatory SNPs within noncoding regions in conferring resistance or susceptibility to Th17 cell-mediated autoimmune pathologies.