Examining the Function of Elements hs5, 6 and 7 of the Immunoglobulin Heavy Chain 3' Regulatory Region through a Mouse Knockout Model

Abstract

A large regulatory region located downstream of the IgH gene locus (3' RR) contains multiple DNaseI hypersensitive (hs) sites, several of which are associated with four enhancers (hs3a, hs1.2, hs3b and hs4) that are required for class switch recombination (CSR). The adjacent hs5-7 region, the function of which is unknown, contains a high density of binding sites for CTCF, a zinc finger protein associated with mammalian insulator activity. Chromatin conformation capture (3C) assays revealed long-distance interactions involving hs5-7 (and the 3' RR enhancers) with upstream VH and I region sequences. We predicted that hs5-7 insulates the IgH locus from neighboring, non-IgH genes and facilitates interactions that are required for IgH expression. To test this, I have generated a mouse with an 8 kb deletion of hs5-7 and examined the effects of removal of hs5-7 on insulation of the IgH locus and B cell specific processes. Hs5-7-/- mice showed (1) a small increase in expression of some of the neighboring, non-IgH genes, (2) normal hematopoietic stem and lymphoid progenitor cell development, as well as normal B, T and macrophage lineages, (3) a less contracted VH region configuration that was associated with a modest increase in the usage of proximal V H gene families as compared to distal VHJ558 gene segments, (4) a minor increase in DHQ52-JH3 usage, (5) increased pericentromeric recruitment and pairing of IgH alleles, (6) normal IgM allelic expression, (7) normal long range interactions between the 3' RR and upstream regions during switching, (8) normal CSR with a mild increase in switching to IgG1 upon in vitro stimulation with LPS+IL4 and increased IgG2a serum levels, (9) increased histone AcH3:AcH4 ratios during switching, and (10) a small reduction in somatic hypermutation frequency. That general aspects of B cell biology, including IgH rearrangements and expression, remain similar to wild type may reflect the redundancy and synergism previously reported for the 3' RR enhancers and/or the retention of additional CTCF sites downstream of the hs5-7 deletion breakpoint that compensate for the hs5-7 deletion.