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Histone reader BRWD1 targets and restricts recombination to the Igk locus

Nature Immunology volume 16, pages 1094–1103 (2015)Cite this article

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Abstract

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5′ to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

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Figure 1: STAT5-regulated Brwd1 expression during B cell lymphopoiesis.
Figure 2: Impaired B lymphopoiesis in Brwd1mut mice.
Figure 3: BRWD1 is required for normal small pre-B cell development.
Figure 4: BRWD1 is required for Igk recombination.
Figure 5: BRWD1 is recruited to Igk marked with H3K9Ac and H3S10pK14Ac.
Figure 6: Recruitment of BRWD1 to H3K9Ac and H3S10pK14Ac genome-wide.
Figure 7: BRWD1 regulates chromatin accessibility and nucleosome positioning in vivo.
Figure 8: BRWD1 is required for recruitment of RAG1 and RAG2 to Igk.

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Acknowledgements

We thank M. Olson and D. Leclerc for cell-sorting services and the ImmGen Consortium for data assembly. We thank W.J. Greenleaf (Stanford University) for ATAC-Seq methodology. We also thank O. Kalinina (University of Chicago, Chicago, Illinois, USA) for Igkdel mice. Supported by the US National Institutes of Health (GM088847, GM101090, AI120715 and U19 AI082724 to M.R.C.). D.G.S. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA

    Malay Mandal, Keith M Hamel, Azusa Tanaka & Marcus R Clark

  2. Center for Research Informatics, University of Illinois at Chicago, Chicago, Illinois, USA

    Mark Maienschein-Cline & Neil Bahroos

  3. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Grace Teng & David G Schatz

  4. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

    Grace Teng & David G Schatz

  5. Department of Human Genetics, Institute for Genomics and Systems Biology, University of Chicago, Chicago, Illinois, USA

    Jigyasa H Tuteja

  6. Department of Pathology, University of Chicago, Chicago, Illinois, USA

    Jeffrey J Bunker

  7. Reproductive Biology, The Jackson Laboratory, Bar Harbor, Maine, USA

    John J Eppig

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Contributions

M.M. designed, carried out and analyzed most of the experiments including ChIP-Seq and ATAC-Seq, oversaw the entire project and wrote the first draft of the manuscript. K.M.H. assisted M.M. in flow cytometry in some of the experiments, as well as in confocal microscopy, immunoblotting, shRNA experiments and adoptive-transfer studies. A.T. assisted in flow cytometry in some of the experiments. M.M.-C. performed ChIP-Seq and ATAC-Seq analysis with M.M. N.B. worked with M.M.-C. G.T. generated the RAG1, RAG2 and H3K4me3 ChIP-Seq data. J.H.T. assisted M.M. in development of the ATAC-Seq methodology. J.J.B. assisted M.M. in H3S10p ChIP. J.J.E. generated and provided advice about Brwd1mut mice. D.G.S. collaborated on RAG and H3K4me3 ChIP-Seq data. M.R.C. oversaw the entire project and prepared the final draft of the manuscript.

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Correspondence to Malay Mandal or Marcus R Clark.

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Integrated supplementary information

Supplementary Figure 1 Brwd1 transcripts in Brwd1mut B cells.

(a) Analysis of Brwd1 transcripts in isolated small pre-B (Lin−B220+CD43−IgM−FSClo) and splenic B (B220+) cells from WT and Brwd1mut bone marrow (BM) and spleen by PCR using cDNAs prepared from those cells with several Brwd1 primer sets (Supplementary Table 1) that amplify different regions of Brwd1 transcript. (b) DNA sequence chromatograms from homozygous WT and Brwd1mut animals across the exon 10–intron 10 junction, as indicated. There is a T-to-C change in the Brwd1mut allele (n = 10). (c) Sequencing of aberrant PCR products obtained with primer set 2 (Supplementary Table 1) that amplifies exon 9–11 of cDNA prepared from isolated small pre-B cells of WT and Brwd1mut BM with predicted translated amino acid sequences (n = 5). (d) Immunoblot analysis of BRWD1 on total nuclear cell lysates from splenic B cells isolated from WT and Brwd1mut mice (n = 3).

Supplementary Figure 2 Early common progenitor, myeloid, erythroid and T lymphocyte cell development is unaltered in Brwd1mut (Mut) animals.

(a) Absolute numbers of cells per mouse at different stages of B cell development in the BM of WT and Brwd1mut/WT heterozygous (Het) mice (n = 3). (b) Differential blood counts of WT and Brwd1mut mice (n = 3). (c) Flow cytometry analysis of common myeloid progenitors (CMP; Lin−Sca1−cKithiFcγRloCD34hi), granulocyte and macrophage progenitors (GMP; Lin−Sca1−cKithiFcγRhiCD34hi) and megakaryocyte and erythroid progenitors (MEP; Lin−Sca1−cKithiFcγRloCD34lo) from BM of WT and Brwd1mut mice (n = 3). (d) Cellularity of myeloid progenitor cells in the BM of WT and Brwd1mut mice (n = 3). (e) Flow cytometric analysis of BM erythroid cells of WT and Brwd1mut mice (n = 3). (f) Cellularity of erythroid cells at different developmental stages includes proerythroblasts (population I, Ter119medCd71hi), basophilic erythroblasts (population II, Ter119hiCd71hi), and late erythroblasts (population III–IV, Ter119hiCd71med, Ter119hiCd71lo) (n = 3). (g) Flow cytometric analysis of BM myeloid cells of WT and Brwd1mut mice (n = 3). (h) Cellularity of Gr1+Mac1+ cells in the BM of WT and Brwd1mut mice (n = 3). (i) Flow cytometric analysis identifying LSK (Lineage−Sca1+cKit+), HSC (hematopoietic stem cells), MPP (multipotent progenitors), LMPP (lymphoid restricted multipotent progenitors) and CLP (common lymphoid progenitors) in the BM of WT and Brwd1mut mice (n = 3). (j) Total progenitor cell numbers of LSK, HSC, MPP, LMPP and CLP progenitors in BM of WT and Brwd1mut mice (n = 3). (k) Flow cytometric analysis of thymocytes identifying SP (single-positive, CD4+ or CD8+), DP (double-positive, CD4+CD8+) and DN (double-negative, CD4−CD8−) populations in WT and Brwd1mut mice. The DN population was further analyzed by CD25 and CD44 staining to identify DN1, DN2, DN3 and DN4 populations (right panel of j) (n = 4). (l) Absolute numbers of DNs, CD4 and CD8 populations in the thymus of WT and Brwd1mut mice (n = 4). All data in bar graphs are presented as average ± s.d.

Supplementary Figure 3 BRWD1 in B cell development and immunoglobulin light chain recombination.

(a,b) LSK progenitors from WT (CD45.1) and WT (CD45.2) (a) or WT (CD45.1) and Brwd1mut (CD45.2) (b) mice were flow sorted and mixed 1:1 and transplanted into sublethally irradiated (550 rad) Rag2−/−Il2rg−/− hosts. B cell development in spleen (a) and T cell development in thymi (b) were then analyzed by flow cytometry after 5 weeks of transplantation. Data are representative of four independent experiments. (c) Flow cytometric assay for apoptosis with annexin V and 7-AAD in B cell progenitors of different developmental stages isolated from WT and Brwd1mut mice (n = 2). (d) Cell cycle analysis of B cell progenitors of different developmental stages isolated from WT and Brwd1mut mice. Numbers above bracketed lines indicate the percentage of cells in S-G2M (n = 2). (e) Quantitative RT-PCR analysis of the expression of Ccnd2 and Ccnd3 in flow-sorted large pre-B cells from BM of WT and Brwd1mut mice (n = 2). *P < 0.05 compared with respective controls (unpaired t-test). (f). Frequency (as a percentage) of Jκ usage in rearranged Igk in isolated small pre-B cells of WT and Brwd1mut mice (n = 2). (g) Usage of Vκ gene family in small pre-B cells of WT and Brwd1mut mice (n = 2). (h) Analysis of Igl-expressing immature B cells and splenic IgM-expressing B cells isolated from WT and Brwd1mut mouse BM and spleen, respectively (n = 3).

Supplementary Figure 4 H3K9Ac and H3S10pK14Ac histone marks do not require BRWD1.

(a) ChIP with IgG or BRWD1-specific antibodies in flow-sorted small pre-B cells isolated from WT and Brwd1mut (Mut) mice followed by quantitative PCR for Jκ1–Jκ2 and Jκ4–Jκ5 regions. Background IgG subtracted from each lane. (b,c) ChIP with IgG, H3K9Ac (b) and H3S10pK14Ac (c) specific antibodies in flow-sorted pro-B and small pre-B cells isolated from WT and Brwd1mut mice followed by quantitative PCR with non-overlapping primer sets (Supplementary Table 1) designed to detect various regions of Igk. Data are representative of three independent experiments (average ± s.d.).

Supplementary Figure 5 De novo motifs in BRWD1, H3K9Ac and H3S10pK14Ac ChIP-Seq peaks.

(a) Representative BRWD1, H3K9Ac and H3S10pK14Ac ChIP-Seq alignment (chromosome 1 and chromosome 6). A region (40,000K–110,000K) is zoomed in on (middle panel). Data are representative of two independent experiments. (b) Percentage of ChIP-Seq peaks (P < 10−7) alone or in combination in different genomic regions. Promoter (–5 kb to +500 bp from TSS); Intragenic (in exons or introns); Intergenic (not in promoter, exons and introns). (c) ChIP-qPCR with IgG and BRWD1-specific antibodies in flow-sorted immature B cells from Igkdel animals and for indicated regions of Igl (expressing immunoglobulin λ-chain). Data are representative of two independent experiments (average ± s.d.). (d) De novo DNA sequence motifs identified among BRWD1, H3K9Ac and H3S10pk14Ac-bound regions. (e,f) De novo DNA sequence motifs identified in BRWD1/H3S10pK14Ac (d) and BRWD1/H3K9Ac/H3S10pK14Ac (e) binding regions.

Supplementary Figure 6 Regulation of gene accessibility during B lymphopoiesis.

(a) Total and overlapping accessibility peaks in WT and Brwd1mut small pre-B cells. (b) Accessibility (open chromatin) at Ccnd3 (cyclin D3) locus in WT and Brwd1mut (Mut) small pre-B cells. Y-axis represents tags per million reads. Data are representative of two independent experiments. (c) Accessibility (open chromatin) at entire Igk locus (mm9 chromosome 6: 67,500,000–70,800,00) showing distal and proximal Vκ regions. Data are representative of two independent experiments. (d) Accessibility (open chromatin) at proximal Vκ and Jκ-Cκ regions. Data are representative of two independent experiments. (e) Accessibility (open chromatin) at Jκ-Eκi regions and immediate upstream. Data are representative of two independent experiments.

Supplementary Figure 7 BRWD1 regulates accessibility and nucleosome positioning.

(a) Nucleosome positioning at RSS and Jκs in WT and Brwd1mut (Mut) small pre-B cells. Data are representative of two independent experiments. (b) Accessibility (open chromatin) and nucleosome positioning at 3ʹEκ. Data are representative of two independent experiments. (c) DNA footprinting analysis of ±1 kb region for WT (red) and Brwd1mut (blue) small pre-B cells at H3K9Ac, BRWD1+H3K9Ac, H3S10pK14Ac and BRWD1+H3S10pK14Ac peaks centered at 0. Nucleosome differential (upper panel) and accessibility (lower panel) were demonstrated. For nucleosome differential (Y-axis), values greater than 0 indicate the presence of a nucleosome, whereas values less than 0 tend to be nucleosome free. The X-axis is the distance in bp from the indicated peak or motif. (d) Alignment of BRWD1, H3K9Ac and H3S10pK14Ac enrichment at GAGA motifs. The Y-axis represents the normalized immunoprecipitation signal distribution for GAGA motifs centered at 0. (e) Position of short GAGAG (CTCTC) motifs in Jκ and Eκi regions. (f) Quantitative RT-PCR analysis of the expression of Rag1 and Rag2 in flow-sorted small pre-B cells from BM of WT and Brwd1mut mice (n = 2). (g) Quantitative RT-PCR analysis of the expression of Tcfe2a (encodes E2A), Pax5, Ikzf1 (IKAROS), Irf4, Irf8, Smarca4 and Myc in flow-sorted small pre-B cells from BM of WT and Brwd1mut mice (n = 3).

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Mandal, M., Hamel, K., Maienschein-Cline, M. et al. Histone reader BRWD1 targets and restricts recombination to the Igk locus. Nat Immunol 16, 1094–1103 (2015). https://doi.org/10.1038/ni.3249

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