Epigenetic Regulation of Individual Modules of the immunoglobulin heavy chain locus 3' Regulatory Region

Abstract

The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3' of the Igh constant region genes. The ~40 kb 3' RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3' RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3' RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3' RR function by interacting with each other and with target sequences of the Igh locus.

Keywords: CTCF; Pax5; class switch recombination; enhancers; immunoglobulin heavy chain gene locus; insulators; somatic hypermutation.

Figure 1

Schematic representation of mouse (A) and human (B) Igh gene loci with emphasis on the 3′ RR. The depicted orientation reflects the centromere (proximal) to telomere (distal) orientation of the genome sequences. DNase I hypersensitive sites hs3A, hs1.2, hs3B, and hs4 (mouse) (red line); and hs3, hs1.2, and hs4 (human) are enhancers of the 3′ RR. (A) Hs1.2 is the center of a palindromic region (double-headed arrow) (see text). The 3′ RR hs5–8 region (gray hexagon) downstream of hs4 contains CTCF sites interspersed with Pax5 sites, and has insulator activity (blue line). Each of the downstream neighboring non-Igh genes, hole (Tmem121), crip1 and 2, and mta-1, has the same transcriptional orientation (demarcated by purple arrows), which is opposite to that of all the immunoglobulin heavy chain genes. Arcs indicate examples of physical interactions that occur in B cells among the elements of the 3′ RR, and between these elements and regulators of germline transcripts (GT) that are located upstream of switch sequences associated with each C_H gene, or with the expressed VDJ gene. Transcription factor binding sites for Pax5, NFκB, octamer-binding proteins, and a G-rich DNA binding protein are present in each of the 3′ RR enhancers (4) (red line). The CTCF-binding region (blue line) has binding sites for Pax5 and cohesin in addition to CTCF (5). (B) The human 3′ Igh enhancers and other features are shown to scale under the scheme of the locus. Numbers represent the actual location within human chromosome 14 (NT_026437.10). This figure has been used with permission from its original publication in Molecular Immunology. Some annotations and modifications have been added.