Chromatin remodeling directly activates V(D)J recombination

Abstract

V(D)J recombination substrate choice is regulated to ensure that the appropriate gene segments are rearranged during lymphocyte development. It has been proposed that regulation of substrate usage is determined by changes in accessibility of the DNA targets. We show that Rag-mediated recombination of an episomal substrate in cells is affected by its packaging into chromatin. Chromatinized substrates were inefficiently rearranged, and methylation further reduced recombination. Disruption of nucleosomes by using butyrate on methylated substrates was sufficient to activate recombination, and dexamethasone could activate recombination in the absence of detectable transcription. Therefore, chromatin structure, and its manipulation by altering nucleosome positioning, can directly affect recombination efficiencies.Antigen receptor genes are assembled during lymphoid development from gene segments flanked by recombination signal sequences (RSSs) that are targets for the V(D)J recombination machinery (1). This nonhomologous site-specific recombination process must be precisely controlled because of the potentially severe consequences of aberrant chromosomal recombination events. Coexpression of the lymphoid-specific genes Rag-1 and Rag-2 occurs in developing B and T cells, ensuring that only the appropriate cell types are recombinationally active (2). A second level of regulation exists to control lineage specificity. Complete Ig gene rearrangement occurs only in B cells whereas T cell receptor genes rearrange exclusively in T cells (3). Moreover, within developing B and T cells, there are temporal controls over antigen receptor rearrangement. For example, in early B cells, the Ig heavy chain is rearranged before the light chain, and D to J H gene segments are

Chromatin accessibility and epigenetic modifications differ between frequently and infrequently rearranging VH genes

“…Our data together with a previous report suggest that recombination can take place at V H genes when DNA methylation occurs in the coding region (Johnson et al, 2004). It has been proposed that DNA methylation in the RSS directly inhibits V(D)J cleavage and induces an inaccessible chromatin configuration (Cherry and Baltimore, 1999;Whitehurst et al, 2000;Nakase et al, 2003). In agreement with these observations, we show that a CpG site within the spacer of the V1 RSS element is 80% demethylated.…”

“…Because several studies have shown that DNA methylation can inhibit recombination (Cherry and Baltimore, 1999;Whitehurst et al, 2000;Nakase et al, 2003), we attempted to determine in the V H S107 family if the extent of CpG methylation varied for individual V H genes. We analyzed all three V H S107 family members as well as the V H 81X gene by bisulphite conversion of genomic DNA from freshly isolated pro-B cells and thymocytes of μMT mice.…”

V(D)J recombination is not activated by demethylation of the kappa locus

“…Regulated demethylation could control the specificity of the V(D)J recombinase. Consistent with such a concept, exogenously introduced hypermethylated recombination substrates including both episomes and transgenes are refractory to V(D)J recombination compared with their unmethylated counterparts (15)(16)(17). At a minimum, this suggests that methylation of target sites bound by ubiquitous trans-acting factors inhibits recombination so that demethylation of these loci should be required for the initiation of recombination.…”

“…Methylation also may serve a negative role by repressing the basal level of recombination throughout the loci and cryptic genomic sites. Previous studies have demonstrated that methylated recombination substrates are less recombinationally active (15)(16)(17). Further studies are required to isolate the factors that confer specificity to the recombination process and to determine the mechanism by which they alter the accessibility of loci for recombination.…”

“…Recent data suggests that nucleosomal remodeling may be the critical change required to allow the V(D)J recombinase access to target sites (15,38,39). The role of demethylation may be to alter the chromatin structure of the kappa locus, providing a permanent change in the accessibility of the locus for transcription.…”