B cell–specific loss of histone 3 lysine 9 methylation in the VH locus depends on Pax5

Abstract

Immunoglobulin heavy chain rearrangement (V H -to-DJ H ) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse V H locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non-B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous V H -to-DJ H recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the V H locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell-specific V H -to-DJ H recombination.During hematopoiesis, transcription factors initiate and maintain lineage-specific commitment 1 . In B and T lymphocytes, the unique variable (V), diversity (D) and joining (J) (V(D)J) recombination of immunoglobulin and T cell receptor (TCR) gene segments provides critical developmental checkpoints as well as diverse, clonotypic antigen recognition capability 2 . All immunoglobulin and TCR loci use a common recombinase machinery, including proteins encoded by the recombination activating genes Rag1 and Rag2 that recognize and cut at common DNA recognition elements called recombination signal sequences (RSSs). However, V(D)J recombination proceeds with strict T lineage-B lineage specificity and developmentally determined order. The mechanisms responsible for these aspects of V(D)J regulation remain poorly understood.

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

“…For the V H 81X gene, as previously reported by Johnson et al (2004), we found that the CpG site at position +143 (relative to the start of the coding region) was methylated in all clones analyzed, whereas the second site (+196) was partially methylated (62% of all clones, Fig. 4D).…”

“…Because it has been proposed that removal of H3/K9me2, a maker of repressive chromatin, seems to be required to promote V H-to-DJ H rearrangements (Johnson et al, 2004), we tested the hypothesis that H3/K9me2 was preferentially removed from highly rearranging V H genes but not from poorly rearranging V H genes. Indeed, we find that in pro-B cells, poorly rearranging V H genes show higher association with H3/K9me2.…”

“…It has been shown that V H genes are associated with dimethylated histone H3 at lysine 9 (H3/ K9me2), a marker of repressed chromatin, before the onset of V H -to-DJ H rearrangements (Johnson et al, 2004). Since removal of that modification seems to be required to promote V H rearrangements (Johnson et al, 2004), we wanted to test the hypothesis that H3/K9me2 is more frequently removed from highly rearranging V H genes than from poorly rearranging V H genes.…”

“…Since removal of that modification seems to be required to promote V H rearrangements (Johnson et al, 2004), we wanted to test the hypothesis that H3/K9me2 is more frequently removed from highly rearranging V H genes than from poorly rearranging V H genes. To test our hypothesis, we performed ChIP assays on freshly isolated pro-B cells and thymocytes from μMT mice and on NIH-3T3 fibroblasts.…”

“…DJ H -distal V H gene segments are blocked from undergoing V H -to-DJ H rearrangement in pro-B cells from Pax5-deficient mice (Urbanek et al, 1994;Nutt et al, 1997;Hesslein et al, 2003). Until recently, there was evidence for two roles of Pax5 in controlling V H -to-DJ H rearrangements: (a) facilitating rearrangement of distal V H genes by inducing IgH locus contraction (Fuxa et al, 2004), and (b) removing histone H3 methylated at lysine 9, a marker of repressed chromatin, from the V H locus, making V H genes accessible for V H -to DJ H recombination (Johnson et al, 2004). In addition, we recently reported that Pax5 binds to V H coding regions and to the RAG1/ RAG2 complex, which may facilitate RAG mediated recombination of individual V H genes (Zhang et al, 2006).…”