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The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth

Nature Immunology volume 16, pages 775–784 (2015)Cite this article

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Abstract

Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage–specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.

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Figure 1: Brg1 is essential for specification of the B cell fate.
Figure 2: Genome-wide occupancy by Brg1 across the pro-B cell genome.
Figure 3: Brg1 is required for the establishment of an accessible B lineage–specific enhancer repertoire.
Figure 4: Brg1 is essential for promoting contraction of the Igh locus.
Figure 5: Brg1 acts to induce the expression of Myc and an ensemble of genes encoding products associated with protein synthesis to regulate B cell growth.
Figure 6: Brg1 regulates Myc expression to promote pro-B cell growth.
Figure 7: Brg1 expression acts to maintain the pro-B cell compartment.

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Acknowledgements

We thank L. Edsall, S. Kuan, B. Li and B. Ren for sequencing; the Murre laboratory for discussions; A. Bortnick for comments on the manuscript; C. Benner for help with the Hi-C analysis; B. Bruneau (Gladstone Institute of Cardiovascular Disease) and P. Chambon (Institut Génétique Biologie Moléculaire Cellulaire) for Smarca4fl/fl mice; Y. Zhuang (Duke University) for E2A-GFP mice; and S. Smale (University of California, Los Angeles) for plasmids pRVGP and pRVGP-BB and antibody Ik-C. ATAC-Seq was conducted in part at the IGM Genomics Center of the University of California, San Diego (supported by the US National Institutes of Health (P30CA023100)). Imaging was performed at the microscopy core of the School of Medicine, University of California, San Diego (supported by the US National Institutes of Health (NS047101)). Supported by Deutsche Forschungsgemeinschaft–Sonderforschungsbereich 873 (project B11 to H.-R.R.), the European Molecular Biology Organization (C.B.), the Swiss National Science Foundation (C.B.) and the US National Institutes of Health (RO1 AI109599-25 to C.M.).

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Authors and Affiliations

  1. Department of Molecular Biology, University of California, San Diego, La Jolla, California, USA

    Claudia Bossen, Caroline S Murre & Cornelis Murre

  2. Center for Computational Biology, Institute for Genomic Medicine, University of California, San Diego, La Jolla, California, USA

    Aaron N Chang

  3. Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

    Robert Mansson

  4. Hematology Center, Karolinska University Hospital, Stockholm, Sweden

    Robert Mansson

  5. Division of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany

    Hans-Reimer Rodewald

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  1. Claudia Bossen
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Contributions

C.B. performed and analyzed the majority of the experiments; C.S.M. performed fluorescence in situ hybridization; A.N.C. analyzed RNA-Seq data; R.M. sorted B cells at various developmental stages; H.-R.R. provided Il7rCre/+ mice; C.B. and C.M. wrote the manuscript; and C.M. supervised the study.

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Correspondence to Cornelis Murre.

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Integrated supplementary information

Supplementary Figure 1 Brg1 is required for the orchestration of early B cell development.

Flow cytometric analysis of B-lineage cells derived from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice. BM cells were gated on the Gr1−CD11b−Ter119−CD19+B220+ population and segregated into early pro-B (CD43+CD24intBP1−), late pro-B (CD43+CD24intBP1+), large pre-B (CD43+CD24hiFSChi) and small pre-B (CD43−IgM−) cells as shown on top panel. Cell numbers are indicated for Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice (lower panel). Data representative of two independent experiments. **P < 0.01 (two-tailed unpaired Student’s t test).

Supplementary Figure 2 Brg1 expression is required for the CLP compartment.

(a,b) CD45.1 mice were reconstituted with a mixture of CD45.1+ and CD45.2+ Smarca4+/+ER-Cre or CD45.2 Smarca4fl/flER-Cre BM cells. Transplanted mice were analyzed for the presence of CLPs and developing B cells two weeks after tamoxifen treatment. Flow cytometric analyses of the BM cells derived from transplanted mice are shown for the indicated genotypes. (a) Representative flow cytometry plots of CLPs (Lin−IL7R+Flt3+) and BLPs (Lin−IL7R+Flt3+Ly6D+). (b) Representative flow cytometry plots of developing BM B cells (Gr1−CD11b−Ter119−CD19+IgM−). Data representative of two independent experiments.

Supplementary Figure 3 Brg1 occupancy at super-enhancers in pro-B cells.

(a) Med1, Brg1, E2A, EBF1, Pax5 and Ikaros occupancy at super-enhancers associated with Foxo1 and Innpp5d genes are depicted. (b) The numbers of peaks identified for various proteins in super-enhancers as a percentage of total peaks identified in total enhancers. (c) Med1, Brg1, E2A and Ikaros tag count in typical enhancers (TE) or super-enhancers (SE) normalized by enhancer size. Only Med1 presents increased binding in SE compared to TE. ChIP-Seq data derived from one independent experiment.

Supplementary Figure 4 Brg1 binds at de novo enhancers in pro-B cells.

(a) Clustering centered on Ikaros-bound sites and further analyzed in a window of ± 3 kb for Ikaros occupancy in Tcf3−/− (E2A-deficient) pre-pro-B and Rag1−/− pro-B cells as well as Brg1 occupancy in Rag1−/− pro-B cells. Ikaros peaks were comprised of Ikaros binding sites in pre-pro-B cells and high-affinity binding sites in pro-B cells. (b) Nucleosome depletion was assessed through MNase sequencing. MNase-Seq tag coverage is assessed in Tcf3−/− pre-pro-B and Rag1−/− pro-B cells. MNase reads are plotted as a function of genomic distance from Brg1 bound-sites (identified in pro-B cells). ChIP-Seq and MNase-Seq data derived from one independent experiment.

Supplementary Figure 5 Brg1 regulates the binding of transcription factors in the Igh locus.

(a) Expression of pro-B lineage-specific transcription factors in Rag1−/− cells transduced respectively with a control retrovirus (sh-Ctrl) and a retrovirus expressing a shRNA directed against Smarca4 (sh-Smarca4) determined by RNA-Seq. Expression represented as mean normalized counts. Mean from 3 independent experiments. (b) Igh locus contraction upon depletion of Smarca4 expression. Orthogonal projections derived from 3D-FISH experiments are shown. BAC probe RP23-201H14 is rendered in green and BAC probe RP24-189H12 in red. DAPI staining is shown in blue. (c) RNA-Seq tag counts representing antisense transcription on the Igh locus in sh-Ctrl and sh-Smarca4 Rag1−/− pro-B cells as well as Smarca4+/+ and Smarca4Δ/Δ pro-B cells is depicted and are representative of at least two independent experiments. Right panel, antisense transcription associated with PAIR elements 4 and 6 is downregulated in absence of Brg1.

Supplementary Figure 6 Brg1 expression interferes with the development of small pre-B cells.

(a) Ratios of cell numbers for distinct stages in early B cell development as defined by late pro-B (B220+CD19+CD43+CD24intBP1+), large pre-B (B220+CD19+CD43+CD24hiFSChi) and small pre-B (B220+CD19+CD43−IgM−) for Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice. (b) Flow cytometric analysis of sorted pro-B population derived from Smarca4+/+ER-Cre and Smarca4fl/flER-Cre mice. Purity of the population is shown before and after sorting from in vitro expanded B220+ BM cultures. (c) Depletion of the large pre-B cell population in cells depleted for the expression of Brg1. Cell size as measured by forward scattered light (FSC) of sorted Smarca4+/+ER-Cre and Smarca4fl/flER-Cre pro-B cells after 3 days in culture in presence of tamoxifen is shown. Cells were pre-gated using CD19+IgM−IgD− as markers. Data are representative of two independent experiments.

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Bossen, C., Murre, C., Chang, A. et al. The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth. Nat Immunol 16, 775–784 (2015). https://doi.org/10.1038/ni.3170

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