Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary, CTCF-mediated chromatin looping and a long-range intergenic enhancer

Abstract

ABSTRACTThe Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease.SIGNIFICANCEThe complement system is a complex network of protein effectors and regulators that play a key role in immunity. Several regulators of complement response are clustered within Regulators of Complement Activation (RCA) gene family. Its members are all functionally, structurally, and genetically related. However, the functional relevance of this close gene organisation is unknown. We show that the clustering of the RCA members is due to shared long-range regulatory elements and physical chromatin looping. We also reveal that the RCA genes are divided into two adjacent chromatin domains and a domain boundary falls within the body of an expressed gene (CR1). Overall, our findings in the RCA cluster offer insights into their evolution, biology and roles in disease.