Pax5 and Linker Histone H1 Coordinate DNA Methylation and Histone Modifications in the 3′ Regulatory Region of the Immunoglobulin Heavy Chain Locus

Abstract

The 3 regulatory region (3 RR) of the murine immunoglobulin heavy chain (IgH) locus contains multiple DNase I-hypersensitive (hs) sites. Proximal sites hs3A, hs1.2, and hs3B are located in an extensive palindromic region and together with hs4 are associated with enhancers involved in the expression and class switch recombination of IgH genes. Distal hs5, -6, and -7 sites located downstream of hs4 comprise a potential insulator for the IgH locus. In pro-B cells, hs4 to -7 are associated with marks of active chromatin, while hs3A, hs1.2, and hs3B are not. Our analysis of DNA methylation-sensitive restriction sites of the 3 RR has revealed a similar modular pattern in pro-B cells; hs4 to -7 sites are unmethylated, while the palindromic region is methylated. This modular pattern of DNA methylation and histone modifications appears to be determined by at least two factors: the B-cell-specific transcription factor Pax5 and linker histone H1. In pre-B cells, a region beginning downstream of hs4 and extending into hs5 showed evidence of allele-specific demethylation associated with the expressed heavy chain allele. Palindromic enhancers become demethylated later in B-cell differentiation, in B and plasma cells.The immunoglobulin heavy chain (IgH) gene locus undergoes a panoply of regulated DNA events during B-cell development, including VDJ joining to construct variable-region genes; class switch recombination (CSR) to achieve the production of non-IgM classes, such as IgG, IgE, and IgA; and somatic hypermutation (reviewed in reference 23). The identification of the specific cis regulatory regions that regulate these processes is of considerable interest. Candidates include promoters that are locally associated with V and D segments and also with I regions upstream of each constant-region gene, except for C␦. In addition, regulators capable of acting at considerable distances from their target sequences flank the C H genes (Fig. 1A). The first of these to be identified was the ϳ1-kb intronic enhancer E, which is situated in the intron between J H and C Targeted deletion studies with mice have revealed that E is important for VDJ joining (1,29,32,36).A 3Ј regulatory region (3Ј RR) extends ϳ35 kb downstream of the C␣ gene and includes multiple DNase I-hypersensitive (hs) sites. The murine 3Ј RR contains modules (Fig. 1A) that are developmentally regulated, as assessed by DNase I hypersensitivity, reporter assays, and engagement with active chromatin marks. The three most proximal sites, i.e., hs3A, hs1.2, and hs3B, form a 25-kb palindromic region by virtue of almost identical hs3A and hs3B sequences in inverted orientation at either end of this segment and by intervening families of locally repetitive sequences flanking the central hs1.2 enhancer (4, 34). hs3A, hs1.2, and hs3B are DNase I hypersensitive at later stages of B-cell development, i.e., in plasma cells (14,20). A second unit contains hs4, which appears to be active beginning in pro-B cells and continuing throughout B-cell development (14,20,24). A third 3Ј RR m...

Polymorphisms of the IgH enhancer HS1.2 and risk of systemic lupus erythematosus

“…The binding of HS1.2 and HS4 enhancers to nuclear protein complexes including NF-κB has previously been demonstrated 29–31. The difference between HS1.2 allele *1 and *2 could affect the three-dimensional structure associated with the region encompassing the specific palindromic structure on both sides of the HS1.2,11 thus affecting spatial localisation of the enhancers in germline transcription and subsequent events of the class switch recombination 38 39. Furthermore, the protein DNA complexes could affect the efficiency of the interaction with different alleles of HS1.2 and reduce or facilitate the looping and positioning of the enhancer near the promoter, as shown by 3C experiments in mice 40 41…”

Developmental Switch in the Transcriptional Activity of a Long-Range Regulatory Element

“…How the 3=RR can mediate its silencing activity is presently unknown and may involve a developmental stage-dependent interplay between the topological reorganization of the IgH locus, which may be modulated by CTCF insulators and transcription factor-mediated loops, posttranslational modifications of factors such as CTCF, and the 3=RR epigenetic modifications and recruitment of transcription factors and corepressors (48)(49)(50)(51). Interestingly, there is some evidence that the human ␤-globin locus control region can in some contexts repress gene expression through transcriptional interference, potentially involving transcripts initiating in flanking repetitive sequences and running through the ␤-globin locus (52).…”

Ig Synthesis and Class Switching Do Not Require the Presence of the hs4 Enhancer in the 3′ IgH Regulatory Region

“…Second, hs3b is absent in other species than the mouse, so that humans could be considered as carrying a natural knockout of hs3b (14,15). Third, the hs4 enhancer was associated with active chromatin (demethylated DNA and acetylated histone H3 and H4) from pro-B cells to plasma cells, while hs3b has become associated with activated chromatin markers later in B cell differentiation (B cell and plasma cell stages) (4,16). In the present study, we have tested the hypothesis that hs4 is of importance for CSR and Ig production.…”