The role of CTCF binding sites in the 3' immunoglobulin heavy chain regulatory region

Abstract

The immunoglobulin heavy chain locus undergoes a series of DNA rearrangements and modifications to achieve the construction and expression of individual antibody heavy chain genes in B cells. These events affect variable regions, through VDJ joining and subsequent somatic hypermutation, and constant regions through class switch recombination (CSR). Levels of IgH expression are also regulated during B cell development, resulting in high levels of secreted antibodies from fully differentiated plasma cells. Regulation of these events has been attributed primarily to two cis-elements that work from long distances on their target sequences, i.e., an ∼1 kb intronic enhancer, Eμ, located between the V region segments and the most 5' constant region gene, Cμ; and an ∼40 kb 3' regulatory region (3' RR) that is located downstream of the most 3' C(H) gene, Cα. The 3' RR is a candidate for an "end" of B cell-specific regulation of the Igh locus. The 3' RR contains several B cell-specific enhancers associated with DNase I hypersensitive sites (hs1-4), which are essential for CSR and for high levels of IgH expression in plasma cells. Downstream of this enhancer-containing region is a region of high-density CTCF binding sites, which extends through hs5, 6, and 7 and further downstream. CTCF, with its enhancer-blocking activities, has been associated with all mammalian insulators and implicated in multiple chromosomal interactions. Here we address the 3' RR CTCF-binding region as a potential insulator of the Igh locus, an independent regulatory element and a predicted modulator of the activity of 3' RR enhancers. Using chromosome conformation capture technology, chromatin immunoprecipitation, and genetic approaches, we have found that the 3' RR with its CTCF-binding region interacts with target sequences in the V(H), Eμ, and C(H) regions through DNA looping as regulated by protein binding. This region impacts on B cell-specific Igh processes at different stages of B cell development.

Keywords: CTCF; Pax5, chromosome conformation capture (3C) assay; class switch recombination; enhancers; immunoglobulin heavy chain gene locus; insulators.

FIGURE 1

Schematic of 3′ RR. The top line shows relative positions of V, D, and J segments, the intronic enhancer, Eμ, and the C_H genes. The 3′ RR region is located downstream of the Cα gene of the Igh locus and has two major modules: an ∼28 kb region containing four enhancers, that, collectively, are essential for GT and CSR and for high levels of Igh expression in plasma cells. The 5′ 3 enhancers, hs3A, hs1.2, and hs3B, occupy a palindromic region (blue box), with hs3A and hs3B in inverted orientation at the ends of the region. A fourth enhancer hs4 occupies a separate structural and functional unit (red oval). In the 10 kb downstream, there is a high-density of CTCF binding sites associated with DNase I hypersensitive sites hs5, hs6, and hs7, and with a segment 4 kb further downstream, termed “38” because it is located ∼38 kb from the beginning of the 3′ RR (with BAC199 M11 as a reference, Genbank AF450245; purple rectangle). This region also contains interspersed Pax5 sites.

FIGURE 2

The 3′ RR interacts via looping with many different target Igh sequences during B cell development. During VDJ joining in pro- and pre-B cells, the 3′ RR interacts with two CTCF sites upstream of D_H (red arrow). Also in pre-B cells, the 3′ RR is involved in allelic regulation (brown arrow). In B cells, interaction of the 3′ RR with I/switch regions is associated with GT and subsequent CSR (purple arrows). In plasma cells (lower right), the 3′ RR interacts with the expressed VDJ region and the intronic enhancer. Also in malignant plasma cells, the translocated c-myc oncogene interacts in cis by looping with the 3′ RR (Ju et al., 2007). Studies show some evidence that CTCF binding sites in the 3′ RR have some insulation activity (gray box) that is detected to impact as far as the “hole” gene. However, CTCF sites associated with downstream genes appear to provide an over-riding local influence (as discussed in the text).