Mechanisms that affect long-distance activity of the 3′ regulatory region of the immunoglobulin heavy chain gene locus (111.31)

Abstract

Abstract The 3' regulatory region (3' RR) of the Igh locus works at long distances on VH and I region promoters to initiate germline transcription (GT) and promote class switch recombination (CSR). The 3' RR contains multiple elements, including enhancers (hs3a-hs4), and a proposed insulator region containing CTCF binding sites, i.e. hs5/6/7 and the downstream region (“38kb”). To better understand how the 3' RR functions, we used chromatin immunoprecipitation assays to monitor transcription factor binding sites in splenic B cells that undergo GT and CSR (LPS+/-IL4), or are deficient in GT and CSR (p50-/-), or do not undergo CSR despite efficient GT (anti-IgM+IL4). The hs5-7 and 38kb regions were observed to contain multiple Pax5 binding sites and Pax5 was shown to interact with CTCF. We found that the Pax5 binding profile to the 3' RR dynamically changed during CSR. The role of the hs5-7 region was assessed by genomic deletion. B cells from hs5-7 KO mice showed a modest increase in expression of the nearest downstream genes. In addition, Igh alleles in KO mice were in a less contracted configuration compared to WT Igh alleles and showed a two-fold increase in the usage of proximal VH7183 gene families. Hs5-7 KO mice were essentially indistinguishable from wild type mice in B cell development, allelic regulation, class switch recombination, and somatic hypermutation. These observations suggest redundancy and synergism among the multiple modules of the 3′ RR for Igh regulation.