Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL-7 and Pax5

Abstract

The usage of >100 functional murine Ig heavy chain V(H) genes, when rearranged to D(H)J(H) genes, generates a diverse antibody repertoire. The V(H) locus encompasses 2.5 Mb, and rearrangement of V(H) genes in the D(H)-distal half of the locus are controlled very differently from the V(H) genes in the proximal end of the locus. The rearrangement of distal but not proximal V(H) genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal V(H) rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal V(H) segments and the repressive histone modification H3K27me3 is exclusively present on proximal V(H) segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal V(H) genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the V(H) locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.

Fig. 1.

The reciprocal pattern of histone H3K27me3 and H3K36me2 on Ig V_H genes. (A) Genomic organization of the murine IgH locus, showing position of the different V_H families and the location of the genes analyzed in this work. (B) ChIP assays were performed by using antibodies reactive with H3K27me3 or H3K36me2 on pro-B cells from 4- to 6-week-old RAG1^−/− mice; pro-B, pre-B, immature B, CD4⁻CD8⁻ T (DN T), CD4⁺CD8⁺ T (DP T) thymocytes from 4- to 6-week-old BALB/c mice; MLP cells from 4- to 6-week-old μMT mice, and an ES cell line. The names of the genes assayed are shown at the bottom of the graphs. Distal V_H genes are black bars, the middle V_HS107.1 gene is a gray bar, and proximal V_H7183 genes are white bars. V_HJ558 and V_H7183 are family-specific primers, and other primer sets are specific for individual genes. Analysis was performed by real-time PCR. Data are presented as relative to the positive control of the Neuregulin gene (Neuregulin = 1) for H3K27me3 and CAD gene (CAD = 1) for H3K36me2. Results represent the mean ± SD. (C) ChIP-on-chip analysis was performed on pro-B cells from RAG^−/− mice with anti-H3K27me3 and anti-H3K36me2 antibodies. The bars represent the distribution of the statistically significant regions of these modifications on Ig V_H locus as determined by MAT software (40). The locations of the D_HJ_H, the V_H7183 and the interspersed V_HJ558 and V_H3609 families are marked on the top of the graph.

Fig. 2.

The patterns of histones H3K27me3 and H3K36me2 and Ezh2 in fetal liver are different from adult bone marrow. (A) ChIP assays were performed by using antibodies reactive with H3K27me3 or H3K36me2 on pro-/pre-B cells (CD19⁺IgM⁻) from BALB/c fetal liver, pro-B cells (B220⁺CD19⁺) from μMT fetal liver, and pro-B cells (B220⁺CD19⁺) from 3- to 4-week-old μMT bone marrow. (B) ChIP assays were performed by using Ezh2 antibody on pro-B cells from μMT fetal liver and 3- to 4-week-old μMT bone marrow. Data are presented as relative to the positive control of the Neuregulin gene (Neuregulin = 1).

Fig. 3.

IL-7 signaling modulates the extent of H3K27me3 and H3K36me2 on V_H genes in B lineage cells. (A) ChIP assays were performed on μMT or IL-7^−/− pro-B cells (B220⁺CD19⁺) from 3- to 4-week-old bone marrow. (B) ChIP assays were performed on pro-B cells (B220⁺CD43⁺CD19⁺IgM⁻) from 4- to 7-week-old bone marrow of IL-7 transgenic (Tg) mice or their littermates (B6).

Fig. 4.

Pax5 modulates H3K27me3 on V_H genes. ChIP assays were performed on B220⁺c-Kit⁺ pro-B cells from Pax5^−/− mice and their littermates (WT).