The epigenetic profile of Ig genes is dynamically regulated during B cell differentiation and is modulated by pre-B cell receptor signaling

Abstract

Ag receptor loci poised for V(D)J rearrangement undergo germline transcription (GT) of unrearranged genes, and the accessible gene segments are associated with posttranslational modifications (PTM) on histones. In this study, we performed a comprehensive analysis of the dynamic changes of four PTM throughout B and T cell differentiation in freshly isolated ex vivo cells. Methylation of lysines 4 and 79 of histone H3, and acetylation of H3, demonstrated stage and lineage specificity, and were most pronounced at the J segments of loci poised for, or undergoing, rearrangement, except for dimethylation of H3K4, which was more equally distributed on V, D, and J genes. Focusing on the IgL loci, we demonstrated there are no active PTM in the absence of pre-BCR signaling. The kappa locus GT and PTM on Jkappa genes are rapidly induced following pre-BCR signaling in large pre-B cells. In contrast, the lambda locus shows greatly delayed onset of GT and PTM, which do not reach high levels until the immature B cell compartment, the stage at which receptor editing is initiated. Analysis of MiEkappa(-/-) mice shows that this enhancer plays a key role in inducing not only GT, but PTM. Using an inducible pre-B cell line, we demonstrate that active PTM on Jkappa genes occur after GT is initiated, indicating that histone PTM do not make the Jkappa region accessible, but conversely, GT may play a role in adding PTM. Our data indicate that the epigenetic profile of IgL genes is dramatically modulated by pre-BCR signaling and B cell differentiation status.

Figure 1. The dynamic pattern of histone modifications during lymphocyte development

A. ChIP assays were performed using antibodies reactive with H3K4me2 in pro-B and pro-T cells from 4–6 week old RAG1^−/− mice; lin⁻, pro-B, pre-B and immature B cells from 4–6 week old BALB/c mice; and NIH3T3 cells. The name of the gene(s) assayed is shown at the bottom of the graphs. Analysis was performed by real-time PCR. J_H1 cannot be detected in WT B lineage cells by real-time PCR since most D_H-proximal RSS of J_H1 alleles were deleted by D-J rearrangement. J_H4 and Jβ genes in WT B lineage or DP T cells, respectively, were not shown because they might be in the rearranged transcribed region. Data is presented as relative to the positive control of CAD gene. Results represent the mean ± SD. B. ChIP assays were performed using antibodies reactive with H3K79me2. Data is presented as relative to the positive control of CAD gene. C. ChIP assays were performed using antibodies reactive with H3K4me3. Data is presented as relative to the positive control of CAD gene. D. ChIP assays were performed using antibodies reactive with AcH3. Data is presented as relative to the positive control of GAPDH gene.

Figure 2. Time course of germline transcription, PTMs and gene rearrangement in 103/bcl2/4 cell line after temperature shift to 39 °C

A. Induction of expression of GT (8.4 kb and 4.7 kb Jκ, Jλ2), RAG1 and RAG2 after temperature shift. B. Induction of AcH3, H3K4me2, H3K4me3 and H3K79me2 at Jκ1 after temperature shift. C. Induction of κ and λ rearrangement after temperature shift.

Figure 3. The patterns of PTMs and germline transcripts on Ig light chain genes during B cell development

A. ChIP data from Fig. 1 on IgL genes in pro-B and pro-T cells from 4–6 week old RAG1^−/− mice; pro-B, pre-B and immature B cells from 4–6 week old BALB/c mice. B. Genomic organization of the murine Igκ and Igλ locus. Top: relative positions of the Jκ gene segments and Cκ exon, and two enhancer regions: the intronic (MiEκ) and 3′Eκ enhancers. Bottom: relative positions of the Vλ and Jλ gene segments and Cλ exons. Arrows denote the positions of the 8.4 kb and 4.7 kb germline transcript promoters. C. GT levels of 8.4 kb and 4.7 kb Jκ, and Jλ2 loci. The level of each GT is plotted relative to the level in immature B cells.

Figure 4. Jκ and Jλ germline transcription and PTMs in large and small pre-B cells

A. GT levels of 8.4 kb and 4.7 kb Jκ, Jλ1 and Jλ2 loci in large pre-B and small pre-B cells from 4–6 week old B6 mice. The level of each GT in large pre-B cells is plotted relative to the level in small pre-B cells. B. PTMs patterns on Ig κ and λ genes in large pre-B and small pre-B cells.

Figure 5. Jκ and Jλ germline transcription and PTMs in μ⁺ RAG^−/− cells, RAG^−/−, mb1^−/− and μMT pro-B cells

A. Transcript levels of Jκ and Jλ loci in pre-B cells from μ⁺RAG^−/− mice or in pro-B cells from RAG^−/− or μMT mice. The level of each GT is plotted relative to the level in μ⁺RAG^−/− pre-B cells. B. PTMs patterns on Ig κ and λ genes in pre-B cells from μ⁺RAG^−/− mice or in pro-B cells from RAG^−/−, μMT or mb1^−/− mice.

Figure 6. Germline transcription, rearrangement and PTMs in MiEκ ^−/− pre-B cells

A. Transcript levels of Jκ and Jλ loci in pre-B cells from MiEκ ^−/− or WT mice. The level of each GT in MiEκ ^−/− cells is plotted relative to the level in WT pre-B cells. B. IgL rearrangement levels in pre-B cells from MiEκ^−/− or WT mice. C. PTMs patterns on Igκ and Igλ genes in pre-B cells from MiEκ^−/− or WT mice.