Articles

The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth © 2015 Nature America, Inc. All rights reserved.

Claudia Bossen1, Caroline S Murre1, Aaron N Chang2, Robert Mansson3,4, Hans-Reimer Rodewald5 & Cornelis Murre1 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. B cell differentiation is controlled by a subset of well-characterized transcription factors that act at distinct checkpoints during hematopoiesis. The first cells in the fetal liver or bone marrow (BM) that are primed for the B cell fate are the common lymphoid progenitors (CLPs). The transcriptional regulators PU.1 and Ikaros are essential for the development of CLPs1,2. The CLP population is heterogeneous and can be segregated into two compartments on the basis of expression of the cell surface marker Ly6D. Ly6D− CLPs, called ‘all-lymphoid progenitors’ (ALPs), display potential to develop into the B cell, T cell and natural killer cell lineages, whereas Ly6D+ CLPs, also called ‘B cell–biased lymphoid progenitors’ (BLPs), give rise mainly to the B cell lineage3,4. The E2A transcriptional regulators control the developmental transition from ALP to BLP3. Once the E2A proteins are activated, they induce expression of the gene encoding the transcriptional regulator Foxo1 (Foxo1), which in turn activates expression of the gene encoding the transcriptional regulator EBF1 (Ebf1)5. EBF1 and Foxo1 then act in a positive intergenic feedback loop to promote the B cell fate. Developmental progression from the pro-B cell stage to the pre-B cell stage is controlled by the precursor of the B cell antigen receptor (pre-BCR). Once the pre-BCR is expressed on the cell surface, it induces the proliferation of large pre-B cells, which in turn differentiate into small resting pre-B cells. Both pro-B cells and large pre-B cells require the transcription factor c-Myc to promote cellular expansion, cell growth and cell survival6,7. The transcriptional regulator Ikaros is essential for promotion of the developmental transition from the large pre-B cell stage to the small pre-B cell stage8–10.

The developmental progress of B cells can also be characterized by the status of the rearrangement of immunoglobulin-encoding genes. The locus encoding the immunoglobulin heavy chain (Igh) undergoes recombination of the diversity (D) region and joining (J) region (DH-JH recombination) starting at the BLP stage, whereas rearrangement of the variable (V), D and J regions (VH-DHJH rearrangement) occurs at the pro-B cell stage. Recombination of the locus encoding the immunoglobulin light chain is initiated and completed at the small pre-B cell stage. Recombination of Igh coincides with commitment to the B lineage and is tightly regulated to ensure that DH-JH recombination precedes VH-DHJH recombination and that the entire VH repertoire is represented. The mouse VH repertoire is composed of over 100 VH segments divided across different families that are segregated into proximal and distal VH segments11. During developmental progression, distal VH segments are brought into closer spatial proximity to JH segments through locus contraction12. Contraction of the Igh locus is controlled by multiple transcription factors, including E2A, YY1 and Pax5 (refs. 13–15). Lineage-specific transcriptional regulators such as E2A, EBF1 and Foxo1 act mainly by binding to distally located enhancer elements that are characterized by DNase I hypersensitivity, active histone marks and non-coding transcription16. On the one hand, enhancers displaying the histone marks of histone H3 monomethylated at Lys4 (H3K4me1), histone H3 dimethylated at Lys4 (H3K4me2) and histone H3 acetylated at Lys27 (H3K27ac) are considered active and are bound by the histone acetyltransferase p300 (ref. 17). On the other hand, enhancers without deposition of H3K27ac are thought to be in a poised state17.

1Department

of Molecular Biology, University of California, San Diego, La Jolla, California, USA. 2Center for Computational Biology, Institute for Genomic Medicine, University of California, San Diego, La Jolla, California, USA. 3Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden. 4Hematology Center, Karolinska University Hospital, Stockholm, Sweden. 5Division of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany. Correspondence should be addressed to C.M. ([email protected]). Received 28 January; accepted 31 March; published online 18 May 2015; doi:10.1038/ni.3170

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Enhancers activate transcription by looping to their cognate promoter regions. Promoter-enhancer interactions are facilitated by the mediator or cohesin complexes18. Super-enhancers, which represent clusters of enhancers, are frequently associated with developmentally regulated genes and are characterized by a high density of binding of mediators and transcription factors19. Enhancer elements need to be established, maintained and/or inactivated during the developmental progression of cells. A key step for enhancer establishment is the removal of nucleosomes to allow occupancy by transcription factors across enhancer regions. Prominent among chromatin remodelers that promote depletion of nucleosomes is the BAF complex20. The BAF complex consists of at least 14 subunits encoded by 28 genes. The polymorphic composition of the BAF complex underlies its specialized functions in a tissue-specific manner. Depletion of nucleosomes requires the ATPase activity of the BAF complex members Brm or Brg1, encoded by Smarca2 or Smarca4, respectively20. Here we demonstrate that Brg1 acts at multiple developmental stages to orchestrate B cell development. Specifically, we found that at the onset of B cell development, Brg1 provided transcriptional regulators closely associated with a B lineage–specific transcriptional signature access to a large enhancer repertoire. In committed pro-B cells, Brg1 was essential for accessibility to transcription factor– binding sites across the Igh locus and concomitant merging of distal and proximal VH regions. Finally, we found that Brg1 controlled pro-B cell growth and prevented premature pre-B cell differentiation by fI/+

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RESULTS Brg1 specifies the B cell fate Published studies have indicated an important role for Brg1 in early B cell development21–24. However, it has remained unclear how Brg1 expression acts to orchestrate B cell fate. As a first approach to addressing this question, we depleted the CLP compartment of Brg1 expression through the use of mice with loxP-flanked alleles encoding Brg1 (Smarca4fl/fl) deleted by Cre recombinase expressed from the gene encoding the interleukin 7 (IL-7) receptor α-chain (Il7rCre/+)25,26. The use of Il7rCre/+ leads to efficient recombination of loxP-flanked alleles in the lymphoid compartment with an onset at the CLP stage in the BM26. To alleviate potential defects due to heterozygous nature of ll7rCre/+, we directly compared Smarca4fl/flIl7rCre/+ mice with their Smarca4fl/+Il7rCre/+ littermates (control mice). Smarca4fl/flIl7rCre/+ mice did not display gross abnormalities, and their BM cellularity was normal (Fig. 1a). B cell cellularity, however, was more than tenfold lower in Smarca4fl/flIl7rCre/+ mice than in the Smarca4fl/+Il7rCre/+ control mice (Fig. 1b). In concordance with published results24, the cellularity of the pro-B cell, pre-B cell, immature B cell and mature B cell compartments was greatly reduced in BM

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Figure 1  Brg1 is essential for specification of the B cell fate. (a) Cellularity of BM derived from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice (two femurs, two tibias and an iliac crest from each mouse). (b) B cell development in BM isolated from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice. Numbers adjacent to outlined areas (left) indicate percent B220 +CD19+ cells. Right, quantification of CD19+B220+ cells. (c) Quantification of B cell progenitors among BM cells obtained from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice, gated on the Gr1−CD11b−Ter119−CD19+B220+ population and segregated as pro-B cells (Pro-B) (IgM−IgD−c-Kit+CD25−), pre-B cells (Pre-B) (IgM−IgD−c-Kit−CD25+), immature B cells (Imm B) (IgM+IgD−) and mature B cells (Mat B) (IgM+IgD+). (d) Quantification of ALPs (Lin−IL-7R+Flt3+MCSFR−c-KitloLy6D−) and BLPs (Lin−IL-7R+Flt3+MCSFR−cKitloLy6D+) (far right) among CLPs obtained from Smarca4fl/flIl7rCre/+ mice and Smarca4fl/+Il7rCre/+ mice and gated as at left. Numbers adjacent to or in outlined areas indicate percent Flt3+IL-7R+ cells among PI− (live) Lin− singlets (far left), c-KitloMCSFR− cells among the cells at left (middle left), and IL-7R+Ly6D− and IL-7R+Ly6D+ cells among the cells at left (middle right). (e) Cre recombinase activity in ALPs (Lin−IL-7R+Flt3+Ly6D−) and BLPs (Lin−IL-7R+Flt3+Ly6D+), assessed as expression of YFP (left), and pro-B and pre-B cells (defined as in c) (right), among cells from Smarca4fl/+Il7rCre/+Rosa26YFP/+ and Smarca4fl/flIl7rCre/+Rosa26YFP/+ mice, gated on Lin−Flt3+IL-7R+ cells (left) or CD19+B220+IgM−IgD− cells (right). Numbers in quadrants indicate percent cells in each throughout. Each symbol (a–d) represents an individual mouse; small horizontal lines indicate the mean. *P < 0.01 (two-tailed unpaired Student’s t-test). Data are representative of four (a,b) or three (c–e) independent experiments.



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Articles

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50 Figure 2  Genome-wide occupancy by Brg1 across the pro-B cell p300 genome. (a) Expression of Smarca4 and Smarca2 transcripts during 0 75 hematopoiesis and early B cell development (horizontal axis); results E2A are presented relative to those of the control gene Actb. (b) Distribution 0 of 13,169 Brg1-binding sites across genomic regions in Rag1−/− pro-B cells. TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region. (c) Heat map of ChIP-Seq data showing distribution of the deposition of H3K4me1, H3K4me2 and H3K4me3 in Rag1−/− pro-B cells, assessed in a 6-kilobase window across Brg1-bound sites, gated on genome-wide Brg1-bound sites. (d) Quantification of Brg1 and p300 tags in Rag1−/− pro-B cells, assessed in a 200–base pair window of combined Brg1- and p300-binding sites. Binding sites present cut-off for tag count as determined by HOMER software. (e) Occupancy by Brg1, p300 and E2A across Pax5 (chromosome 4, positions 44,703,000–44,730,000 in the mm9 NCBI assembly of the mouse genome) and Cd19 (chromosome 7, positions 133,551,000–133,564,000 in mm9) in Rag1−/− pro-B cells. (f) Association of cis-regulatory sequences with Brg1 occupancy; letter size indicates nucleotide frequency. Numbers at left indicate significance of motif occurrence (binomial test). (g) Brg1 ‘reads’ for a 100–base pair window centered on 5,000 Ikaros-, E2A-, EBF1-, Pax5-, PU.1- and CTCF-binding sites. Data are representative of two experiments (a; error bars, s.d. of n = 2 independent cell sorts), or one experiment (b–g).

derived from Smarca4fl/flIl7rCre/+ mice relative to the cellularity of those compartments in the Smarca4fl/+Il7rCre/+ control mice (Fig. 1c and Supplementary Fig. 1). In contrast, the number of ALPs and BLPs was not altered in Smarca4fl/flIl7rCre/+ mice relative to the number of these cells in their Smarca4fl/+Il7rCre/+ littermates (Fig. 1d). To determine whether B cells detected in Smarca4fl/flIl7rCre/+ mice had undergone efficient depletion of Brg1 expression, we crossed Smarca4fl/flIl7rCre/+ mice with mice containing sequence encoding yellow fluorescent protein (YFP) inserted into the ubiquitously expressed Rosa26 locus. In these Rosa26YFP/+ mice, YFP expression is blocked by a loxP-flanked ‘stop’ site, which is removed in cells that express Cre recombinase. On the one hand, large proportion of Ly6D+ CLPs (>90%) from Smarca4fl/flIl7rCre/+Rosa26YFP/+ mice displayed YFP expression, indicative of efficient deletion in the BLP compartment (Fig. 1e). On the other hand, a substantial proportion of proB cells and pre-B cells from Smarca4fl/flIl7rCre/+Rosa26YFP/+ mice lacked YFP expression (Fig. 1e), which suggested that Brg1 expression was not completely abrogated in the pro-B cells and pre-B cells that developed in Smarca4fl/flIl7rCre/+ mice. To determine whether Brg1 expression in hematopoietic progenitor cells is required before and/or at the CLP cell stage, we depleted cells of Brg1 expression through the use of mice with transgenic expression of tamoxifen-inducible Cre linked to the estrogen receptor (ER-Cre). For this, we injected CD45.2+ Smarca4+/+ER-Cre and Smarca4fl/flER-Cre BM cells together with CD45.1+ wild-type BM cells into CD45.1+ recipient mice. At 10 weeks after transplantation, we injected tamoxifen into the recipient mice and assessed the presence of CLPs in these mice. Treatment of the mice with tamoxifen resulted in the generation of considerably fewer CLPs from Smarca4fl/flER-Cre nature immunology  aDVANCE ONLINE PUBLICATION

(Smarca4∆/∆) BM cells than from Smarca4+/+ER-Cre (Smarca4+/+) BM cells (Supplementary Fig. 2a). The abundance of BLPs was further reduced in the absence of Brg1, and developing Smarca4∆/∆ B cells were almost completely undetectable (Supplementary Fig. 2a,b). Together these observations indicated that Brg1 was required for establishment of the B cell fate. Genome-wide occupancy by Brg1 in pro-B cells As a first approach to determining how Brg1 regulates pro-B cell development, we examined the expression of Smarca4 transcripts during hematopoiesis. For this purpose, we isolated RNA from LSK (Lin−Sca-1+c-Kit+) cells, LMPPs (lymphoid-primed multipotent progenitors), ALPs, BLPs, pro-B cells, pre-B cells, immature B cells and mature B cells from wildtype mice and analyzed Smarca4 expression. We found that Smarca4 transcripts were absent or their abundance was low in the majority of hematopoietic progenitors but their abundance was elevated in BLPs relative to that in the rest of the hematopoietic progenitor cells (Fig. 2a). In cells committed to the B cell lineage, Smarca4 expression was highest in pro-B cells but was decreased in pre-B cells (Fig. 2a). To assess the spectrum of Brg1-bound sites across the pro-B cell genome, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) with an antibody to Brg1. We found that Brg1 occupancy was abundant (13,000 bound sites), with the majority of Brg1 binding detected at putative enhancers (Fig. 2b,c). Intergenic and intronic regions showed enrichment for Brg1-bound sites, and Brg1-bound sites were associated predominantly with genomic regions that displayed deposition of H3K4me1 and H3K4me2 (Fig. 2b,c). Brg1 also bound to promoter regions, albeit with much lower frequency than the frequency of its binding to putative enhancer elements (Fig. 2b,c). 

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Figure 3  Brg1 is required for the establishment of an accessible B lineage–specific enhancer repertoire. (a) ATAC-Seq tag coverage in ALPs and BLPs from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice, plotted as a function of genomic distance from E2A-, Ikaros-, EBF1- or CTCF-bound sites (identified in pro-B cells). (b) ATAC ‘reads’ and deposition of H3K4me2 or occupancy by E2A, Ikaros or EBF1 (left margin) across intergenic genomic regions flanking a 3′ Foxo1 enhancer (left; chromosome 3, positions 52,243,000–52,253,000) or a Cd79a regulatory region (right; chromosome 7, positions 25,677,000–25,687,000) in B cells at various stages of development (left margin); pre-pro-B cells are represented by E2A-deficient multipotent progenitors, and pro-B cells were derived from Rag1−/− mice. (c) Abundance of E2A protein in BM Lin+ cells (CD11b+Ter119+CD3+NK1.1+ GR1+), ALPs, BLPs and BM B cells (CD19+) from E2A-GFP mice, presented as mean fluorescence intensity (MFI). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (d) RT-PCR analysis of Tcf3, Ebf1 and Ikzf1 in ALPs and BLPs sorted as in a, presented relative to Actb expression. NS, not significant (P > 0.05); *P < 0.05 and **P < 0.01 (two-tailed unpaired Student’s t-test). Data are from two independent experiments (a,b) or are representative of one experiment (c,d; average and s.d of biological triplicates in d).

To evaluate the degree of overlap between Brg1 occupancy and an active enhancer repertoire, including super-enhancers, we compared Brg1-bound sites with those occupied by p300. Notably, a large fraction of Brg1-bound sites overlapped with those occupied by p300 (Fig. 2d), which indicated that Brg1 associated mainly with an active enhancer repertoire. As expected, we observed Brg1 occupancy across enhancer elements that exhibited binding of E2A and p300 and were associated with the expression of a subset of B lineage–specific genes, including Pax5 and Cd19 (Fig. 2e). At a genome-wide level, sites occupied by Brg1 showed considerable enrichment for consensus binding motifs for the transcription factors E2A, EBF1, IRF, Ikaros, PU.1 and Runx (Fig. 2f). Consistent with that analysis, sites occupied by Brg1 were closely associated with those occupied by Ikaros and, to a lesser degree, with E2A-, EBF-, Pax5- and PU.1-bound sites (Fig. 2g). In contrast, Brg1bound sites were not frequently associated with sites occupied by the transcriptional repressor CTCF (Fig. 2g). We observed binding of Brg1 at super-enhancers, including the Foxo1 and Inpp5d superenhancers (Supplementary Fig. 3a). The proportion of Brg1 bound to 

super-enhancers was comparable to that of transcription factors known to associate with super-enhancers, such as E2A, Pax5 and Med1 (a member of the mediator complex) (Supplementary Fig. 3b). To assess the strength with which Brg1 bound to super-enhancers, we normalized the number of tag counts to the size of the enhancers, since the median size of super-enhancers is approximately tenfold greater than that of typical enhancers19. We found that unlike results obtained for Med1, the strength of Brg1’s binding was not greater at superenhancers than at typical enhancers (Supplementary Fig. 3c). Published studies have established that Ikaros interacts with Brg1 (refs. 27,28). Such findings raised the question of whether Ikaros recruits Brg1 to the pro-B cell enhancer repertoire. Hence, we investigated the binding of Ikaros in progenitors of B cells using E2A-deficient pre-pro-B cells29. We found that Ikaros bound to a spectrum of sites in pre-pro-B cells distinct from that observed in pro-B cells, even though the expression of Ikaros protein was similar in both cell types30 (Supplementary Fig. 4a). Notably, the ‘de novo’ enhancer repertoire associated with pro-B cells (‘new’ compared with that in pre-pro-B cells) displayed overlapping aDVANCE ONLINE PUBLICATION  nature immunology

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patterns of occupancy by Ikaros and occupancy by Brg1 (Supplementary Fig. 4a). Furthermore, pro-B cell–specific Brg1-bound sites were associated with regions that underwent depletion of nucleosomes during the transition from the pre-pro-B cell stage to the pro-B cell stage (Supplementary Fig. 4b). Collectively, these results showed that Brg1 binding was closely associated with a de novo enhancer repertoire that was established during the transition from pre-pro-B cell to pro-B cell. Brg1 facilitates access to enhancer repertoires As Brg1 is a chromatin remodeler and is required for specification of the B cell fate, we considered the possibility that Brg1 might serve nature immunology  aDVANCE ONLINE PUBLICATION

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* 1.0 1.0 Figure 4  Brg1 is essential for promoting contraction of the Igh locus. (a) Meta-analysis of the pro-B cell interactome, showing the deposition of H3K4me2, region of open 1.0 chromatin (determined by ATAC-Seq) and Brg1-bound sites in 0.5 0.5 chromosome 12 (positions 114,300,000–117,400,000) Ctrl-sh 0.5 (refs. 33,34), derived from Hi-C studies and presented in Smarca4-sh circular format (Circos software); thickness of the connecting lines reflects the natural log ratio of observed interaction 0 0 0 frequency versus expected interaction frequency in 0 0.5 1.0 Ctrl-sh Smarca4-sh Hi-C data sets. Bin size, 10 kilobases. Colors indicate Distance (µm) constant (C), J, D and V regions (key, top right). Only significant interactions are presented here: P < 0.001 (binomial test). (b) Occupancy by Brg1, E2A, Pax5, YY1, Ikaros and CTCF across the end point of interactions associated with the Igh locus (chromosome 12, positions 115,825,000–115,875,00). (c) Frequency of the association of lineage-specific transcription factors, CTCF and Med1 with loop-attachment points (node size) and significance and reproducibility of tethering, as P values (line thickness); colors (red-blue) indicate association strength, calculated as the log ratio of observed frequency to expected frequency (obs/exp). (d) ATAC-Seq tag counts associated with Smarca4+/+ and Smarca4∆/∆ pro-B cells, presented as combined ATAC-Seq peaks (57 peaks total) associated with the VH region cluster; colors indicate regions of open chromatin with a twofold greater (red) or twofold lower (blue) tag count in Smarca4∆/∆ pro-B cells than in Smarca4+/+ pro-B cells; numbers in parenthesis indicate number of peaks. (e) Quantification of transcription factor peaks in region of open chromatin in Smarca4+/+ and Smarca4∆/∆ pro-B cells from d, determined by ATAC-Seq; colors indicate transcription factor peaks associated with regions of open chromatin with a twofold greater (red) or twofold lower (blue) tag count in Smarca4∆/∆ pro-B cells than in Smarca4+/+ pro-B cells. (f) Distribution of spatial distances separating two bacterial artificial chromosome probes, located at each end of the Igh locus, in Rag1−/− cells transduced with retrovirus expressing control shRNA (Ctrl-sh) or Smarca4-specific shRNA (Smarca4-sh) (left), and cumulative frequency versus probe distance in pro-B cells transduced in the same way (right). (g) Use of proximal VH7183 segments relative to use of distal VHJ558 segments in Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ pre-B cells, assessed by Southern blot analysis. Each symbol represents an open region on Igh (d) or an individual allele (f) or an individual mouse (g); small horizontal lines indicate the mean (f,g). *P < 0.05 and **P < 0.01 (two-tailed unpaired Student’s t-test). Data are representative of two experiments (Hi-C data and ATAC-Seq, a,d,e) or one experiment (ChIP-Seq, a,b,c,e) or are from one experiment representative of five independent experiments (f, left) or from five independent experiments (f, right) or two independent experiments (g). Distance (µm)

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a crucial role in the generation of a de novo enhancer repertoire by facilitating the depletion of nucleosomes across an ensemble of B lineage–specific enhancers. The expression of genes associated with the establishment of B cell identity is activated in BLPs, which suggests that a pro-B cell–specific enhancer repertoire is established in the BLP compartment. As a first approach to exploring this possibility, we used ATAC (‘assay for transposase-accessible chromatin’) sequencing (ATAC-Seq) to capture open chromatin sites31. ATAC-Seq generates results comparable to those obtained by mapping of DNase I–hypersensitivity sites and involves incubation of nuclei with a transposase. The transposase ‘preferentially’ integrates into active 

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Myc Figure 5  Brg1 acts to induce the expression of Myc and an ensemble of genes encoding products associated with protein synthesis to regulate Smarca4 +/+ Ikaros 40 Brg1 B cell growth. (a) RNA-Seq analysis of gene expression in Smarca4+/+ Smarca4 +/+ IgG 0 ∆ /∆ and Smarca4∆/∆ pro-B cells, presented as mean normalized counts Smarca4 Ikaros 100 (mean of the counts divided by size factors (DESeq software package)); Smarca4 ∆ /∆ IgG EBF1 colors indicate transcripts significantly less abundant (blue) or more 0 0.6 40 abundant (red) in Brg1-deficient pro-B cells than in wild-type pro-B Ikaros cells (difference of over twofold for each). (b) Ontology analysis 0 60 (via the DAVID bioinformatics database) of clusters of genes with 0.4 Pax5 lower transcript abundance in Smarca4∆/∆ pro-B cells than in 0 +/+ Smarca4 pro-B cells. ncRNA, non-coding RNA. (c) Quantitative 50 ATAC PCR analysis of Myc among RNA from Smarca4∆/∆ and Smarca4+/+ 0 0.2 Smarca4+/+ pro-B cells (left) and in Rag1−/− pro-B cells transduced 50 ATAC with retrovirus expressing control or Smarca4-specific shRNA (right); ∆ /∆ Smarca4 0 results were normalized to those of Actb and are presented relative to +/+ those of Smarca4 cells (left) or cells transduced with control shRNA 0 2 10 kb 1 Site 1 Site 2 (right). *P < 0.05 (two-tailed unpaired Student’s t-test). (d) Long-range genomic interactions (>100 kilobases), including deposition of H3K4me2 and H3K4me3, as well as Brg1-, Ikaros-, E2A-, EBF1- and Pax5-bound sites, across a 4.4-megabase region containing the Myc locus (chromosome 15, positions 59,700,000–64,100,000), presented in circular format (connecting line thicknesses and bin size as in Fig. 4a). (e) Occupancy by Brg1, EBF1, Ikaros and Pax5 across the Myc super-enhancer (chromosome 15, positions 63,440,000–63,500,000) in Rag1−/− pro-B cells (top four rows), as well as regions with nucleosome depletion across the same region Smarca4+/+ and Smarca4∆/∆ pro-B cells, as determined by ATAC ‘reads’ (below); below horizontal axis (red bars), sites 1 and 2 of the Myc superenhancer. (f) Occupancy by Ikaros and IgG (control) across the Myc super-enhancer (sites 1 and 2 in e) in Smarca4+/+ and Smarca4∆/∆ pro-B cells. Data are representative of two independent experiments (a,b,f), one experiment (Hi-C data and ChIP-Seq, d,e) or two experiments (ATAC-Seq, e) or are from one experiment (c; average and s.d. of biological triplicates).

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regulatory regions and simultaneously fragments and tags the genome with sequencing adaptors. This technique can be used for small numbers of cells, including progenitor populations31. To identify accessible enhancer repertoires in progenitors of B cells, we sorted ALPs and BLPs from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice. We incubated the sorted cells with transposase and sequenced the results at a global scale. Next, we plotted ATAC-Seq tag counts as a function of genomic separation from E2A-, Ikaros-, EBF1- or CTCF-bound sites (Fig. 3a). As expected, enhancer repertoires associated with occupancy by E2A, Ikaros or EBF1 were more accessible to transposase activity in BLPs than in ALPs (Fig. 3a). These data indicated that at a developmental stage at which progenitor cells become specified to the B lineage, the enhancer repertoire associated with a B lineage–specific program of gene expression became depleted of nucleosomes. In contrast, Brg1-deficient BLPs displayed either no increase in the number ATAC reads for enhancer repertoires associated with occupancy by E2A, Ikaros or EBF1 relative to that in ALPs or a reduced increase compared with that of Brg1-sufficient BLPs (Fig. 3a). Genomic regions associated with CTCF occupancy showed comparable ATAC tag counts in both Brg1-sufficient and Brg1-deficient ALPs and BLPs (Fig. 3a). Since E2A and EBF1 directly regulate Foxo1 expression in the BLP compartment, we investigated whether putative enhancers associated with the Foxo1 locus underwent depletion of nucleosomes5. We found that, indeed, Brg1 expression was essential for the eviction of nucleosomes across a distally located Foxo1 enhancer associated 

with occupancy by E2A and EBF1 (Fig. 3b, left). Likewise, Brg1 expression was required for the depletion of nucleosomes across a regulatory element located within the Cd79a locus (which encodes the immunoglobulin α-chain) (Fig. 3b, right). The increase in accessibility observed in BLPs relative to accessibility in ALPs might have simply reflected increased abundance of the B lineage–specific transcription factors. To exclude this possibility, we examined E2A protein expression in ALPs and BLPs from mice expressing green fluorescent protein (GFP)-tagged E2A (E2A-GFP mice)32. We found that abundance of E2A protein did not change during the ALP-to-BLP transition (Fig. 3c). Likewise, the abundance of transcripts encoding E2A (Tcf3) and Ikaros (Ikzf1) was equivalent in ALPs and BLPs (Fig. 3d). Thus, the enhancement in open chromatin seen at E2A-binding sites in Brg1-sufficient BLPs relative to its presence in ALPs could not readily be explained in terms of elevated protein expression. Instead, we suggest that Brg1 facilitated the depletion of nucleosomes across an enhancer repertoire closely associated with a B lineage–specific program of gene expression. Brg1 is essential for promoting Igh locus contraction Since Brg1 abundance is particularly substantial in pro-B cells and Brg1 binds together with factors that are associated with contraction of the Igh locus, we assessed occupancy of the Igh locus by Brg1. As reported21, Brg1 bound to JH segments (Fig. 4a). However, occupancy by Brg1 was not limited to the JH elements. Instead, binding of Brg1 aDVANCE ONLINE PUBLICATION  nature immunology

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Figure 6  Brg1 regulates Myc expression 15 Tsr1 Gar1 Srr * to promote pro-B cell growth. (a) c-Myc 30 50 occupancy associated with the promoter c-Myc regions of Gar1 and Tsr1 (top), as well as 0 0 10 50 200 tag counts for RNA-Seq ‘reads’ of those +/+ Smarca4 +/+ ∆/∆ regions in Smarca4 and Smarca4 0 0 pro-B cells (below). (b) Abundance of 50 200 5 ∆/∆ transcripts from genes associated with Smarca4 Smarca4 +/+ Smarca4 ∆ /∆ c-Myc occupancy in promoter regions in 0 0 – +/+ ∆/∆ Smarca4 and Smarca4 pro-B cells. Singlets, Live Lin fl/+ Cre/+ *P = 1 × 10−38 (two-tailed paired Smarca4 II7r Student’s t-test). (c) BrdU incorporation Smarca4 fl/flII7r Cre/+ fl/+ Cre/+ 55 Smarca4 II7r in pro-B cells (right) among cells 38 50 25 * fl/fl Cre/+ obtained from Smarca4 Il7r and 40 fl/+ Cre/+ Smarca4 Il7r mice 4 h after 300 injection of BrdU and gated (left) as pro-B 30 105 105 4 4 10 10 cells (live Lin−CD19+B220+IgM−Ly6D−). 200 fl/fl Cre/+ 20 17 Smarca4 II7r 3 3 10 10 25 Numbers adjacent to outlined areas 2 27 2 100 10 10 10 indicate percent B220+CD19+ cells 0 0 0 among live Lin− singlets (left) and 0 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 IgM−Ly6D− cells (middle) among the BrdU CD19 Ly6D cells at left; numbers above bracketed lines (right) indicate percent BrdU + (divided) cells among the cells in middle column. *P < 0.05 (two-tailed unpaired Student’s t-test). Data are representative of one experiment (a, top row) or two independent experiments (a, bottom two rows, b) or are from two independent experiments (c).

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was dispersed throughout the Igh locus and overlapped with the deposition of H3K4me2 and regions depleted of nucleosomes, as revealed by ATAC-Seq (Fig. 4a). Upon inspecting Hi-C data (meta-analysis of chromosome-conformation capture followed by deep sequencing) generated from pro-B cells, we observed an intricate pattern of significant short-range genomic interactions that spanned the entire cluster of VH segments (Fig. 4a), consistent with an organization of bundles of loops33,34. Notably, we identified a giant loop connecting the extreme ends of the Igh locus and thus identified the boundaries that define the Igh topological domain (Fig. 4a). We also identified a spectrum of long-range genomic interactions involving distal V H region clusters and Igh segments across the 3′ end of the locus and found that a subset of these putative anchors showed enrichment for Brg1 occupancy (Fig. 4a,b). Genomic interactions involving Brg1 showed enrichment mainly for binding of lineage-specific transcription factors, including Pax5, EBF1, YY1, Ikaros and E2A, as well as Med1, relative to the general occupancy of the transcription factor on the Igh locus (Fig. 4c). To determine whether Brg1 modulated the accessibility of transcription factor–binding sites across the Igh locus, we assessed ATAC-Seq ‘reads’ in Brg1-sufficient and Brg1-deficient pro-B cells. We isolated pro-B cells from B220+ cell populations obtained from Smarca4+/+ER-Cre and Smarca4fl/flER-Cre BM and expanded these populations in the presence of IL-7 and stem-cell factor. We found that the vast majority of pro-B cells underwent depletion of Brg1 protein upon culture of the cells for 3 d in the presence of tamoxifen (data not shown). As expected, loss of Brg1 expression was closely associated with decreased chromatin accessibility for a large fraction of sites (50%) bound by lineage-specific transcription factors, but not those bound by CTCF (Fig. 4d,e). The abundance of RNA transcripts encoding lineage-specific transcription factors was not affected by depletion of Brg1 expression (Supplementary Fig. 5a). Consistent with reduced binding of Pax5 and YY1, we observed a reduction in antisense transcription associated with distal regulatory regions called ‘PAIR elements’35,36 (Supplementary Fig. 5c). Our findings indicated that Brg1 occupancy across the Igh locus was closely associated with transcription factor–binding sites. The transcription factors that bind to these sites have been linked to contraction of the Igh locus13–15. These observations raised the nature immunology  aDVANCE ONLINE PUBLICATION

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possibility that Brg1 acts to evict nucleosomes positioned across regulatory elements that mediate long-range genomic interactions. To investigate this possibility, we measured the spatial distances separating the distal VH region cluster from a genomic region located 3′ of the Igh locus. Specifically, we obtained pro-B cells from mice deficient in recombination-activating gene 1 (Rag1−/− mice) to look at germline configuration of the Igh locus and depleted the cells of Brg1 expression through the transduction of short hairpin RNA (shRNA) directed against Smarca4. We then examined the topology of the Igh locus in the transduced pro-B cells by threedimensional fluorescence in situ hybridization. Notably, we found that the Igh locus displayed significant long-range decontraction of the locus in the pro-B cell population upon depletion of Brg1 expression (Fig. 4f and Supplementary Fig. 5b). Since reduced contraction should lead to ‘preferred’ use of the proximal VH segments, we assessed the use of proximal and distal VH segments in pre-B cells sorted from Smarca4fl/+Il7rCre/+ or Smarca4fl/flIl7rCre/+ mice. In agreement with the reduction in contraction of the Igh locus in Rag1−/− pro-B cells depleted of Brg1, we found significantly more use of proximal VH7183 segments than distal JH558 segments in Smarca4fl/flIl7rCre/+ pre-B cells than in Smarca4fl/+Il7rCre/+ pre-B cells (Fig. 4g). Together these observations indicated that Brg1 was essential for promoting contraction of the Igh locus. Brg1 regulates Myc expression to promote pro-B cell growth To determine whether in committed pro-B cells Brg1 acts to maintain an active enhancer repertoire, we isolated RNA from Brg1sufficient and Brg1-deficient pro-B cells and analyzed the RNA by high-throughput sequencing technologies for cDNA (RNA-Seq). We found that approximately 1,800 genes displayed significant differences in transcript abundance in pro-B cells depleted of Brg1 expression relative to their abundance in Brg1-sufficient pro-B cells (Fig. 5a). We found that at least 1,000 genes underwent a change in expression of twofold or more upon depletion of Brg1 (Supplementary Table 1). Notably, gene-ontology analysis showed that the absence of Brg1 affected the expression of a spectrum of genes encoding products associated with ribosome biogenesis (Fig. 5b). The abundance of transcripts encoding products associated with the metabolism of rRNA and the synthesis of non-coding RNA was also lower in Brg1-deficient 

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Figure 7  Brg1 expression acts to maintain the pro-B cell compartment. (a) Difference in transcript abundance in Smarca4∆/∆ pro-B cells versus Smarca4+/+ pro-B cells plotted against that in CLPs versus pro-B cells (left), and in Smarca4∆/∆ pro-B cells versus Smarca4+/+ pro-B cells plotted against that in pre-B cells versus pro-B cells (right), as determined by microarray by the Immunological Genome Project Consortium; colors indicate transcripts significantly less abundant (blue) or more abundant (red) in Smarca4∆/∆ pro-B cells pro-B cells than in Smarca4+/+ pro-B cells (difference of over twofold for each). Spearman correlation = −0.05 (left) or 0.37 (right). (b) Flow cytometry of B cells from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice, identifying pro-B cells (IgM−IgD−c-Kit+CD25−) and pre-B cells (IgM−IgD−c-Kit−CD25+) (left) and the ratio of pre-B cells to pro-B cells in those mice (right). Numbers adjacent to outlined areas (left) indicate percent c-Kit +CD25− cells (top left) or c-Kit−CD25+ cells (bottom right) among PI−CD19+IgM−IgD− singlets. *P < 0.05 (two-tailed unpaired Student’s t-test). (c) Expression of transcripts from genes associated with c-Myc occupancy in pro-B cells and pre-B cells. *P = 2 × 10−32 (two-tailed paired Student’s t-test). Data are representative of two experiments (a (Smarca4∆/∆ and Smarca4+/+ pro-B cells)), three experiments (a (CLPs, pro-B cells and pre-B cells), c) or three independent experiments (b).

pro-B cells than in Brg1-sufficient pro-B cells (Fig. 5b). Finally, we found that the abundance of Myc transcripts was significantly lower in Brg1-deficient pro-B cells than in Brg1-sufficient pro-B cells (Fig. 5a). Real-time PCR analysis confirmed the downregulation of Myc expression in Brg1-deficient pro-B cells and also in Rag1−/− pro-B cells transduced with shRNA directed against Smarca4 (Fig. 5c). The decrease in the abundance of Myc transcripts upon depletion of Brg1 expression raised the question of how Brg1 regulates Myc expression. As a first approach to addressing this question, we inspected the Myc locus for Brg1 occupancy. Brg1 occupancy was particularly prominent in a distally located regulatory element that have been shown to modulate Myc expression in leukemic cells37 (Fig. 5d,e). We found that in pro-B cells, the distally located superenhancer showed deposition of H3K4me2 and was associated with occupancy by Brg1, EBF1, Ikaros and Pax5 (Fig. 5d,e). Furthermore, Hi-C analysis showed that in pro-B cells, the super-enhancer contacted the Myc promoter with relatively high frequency (Fig. 5d). To assess whether Brg1 expression was needed to permit B lineage– specific transcription factors access to the Myc super-enhancer, we examined Ikaros occupancy by ChIP in wild-type and Brg1-deficient pro-B cells. Indeed, occupancy by Ikaros at the Myc super-enhancer was much lower in the absence of Brg1 than in the presence of Brg1 (Fig. 5f). To directly assess whether Brg1 expression was required for the eviction of nucleosomes across the Myc super-enhancer, we analyzed chromatin accessibility in Brg1-sufficient and Brg1deficient pro-B cells by ATAC-Seq. As expected, we found that Brg1 expression modulated the accessibility of chromatin to the Myc super-enhancer (Fig. 5e). c-Myc is known to control cell growth by regulating ribosome biogenesis38. To determine whether c-Myc directly regulated the expression of genes encoding products that regulate ribosome biogenesis, we examined c-Myc occupancy in pro-B cells by ChIP-Seq. Notably, in pro-B cells, we observed binding of c-Myc in promoter regions of genes encoding products associated with ribosome biogenesis, including Tsr1 and Gar1 (Fig. 6a). These findings raised the possibility that in pro-B cells, Brg1 activated Myc expression, which in turn, acted to induce the expression of an ensemble of genes encoding products involved in ribosome biogenesis. We found that the expression of genes associated with the binding of c-Myc in pro-B cells was significantly 

lower in Brg1-deficient than in Brg1-sufficient pro-B cells (Fig. 6b). Globally, approximately 12% of the genes with lower transcript abundance in Brg1-deficient pro-B cells than in Brg1-sufficient pro-B cells were direct targets of c-Myc (Supplementary Table 1). To assess directly whether Brg1 acts to regulate pro-B cell growth, we monitored the proliferation of pro-B cells in vivo in Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice through incorporation of the thymidine analog BrdU. Specifically, at 4 h after injection of BrdU, we isolated BM from Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice and analyzed incorporation of BrdU in the pro-B cell compartment. As we expected, BrdU incorporation indicated a significantly lower degree of proliferation in the pro-B cell population isolated from Smarca4fl/flIl7rCre/+ mice than in that from Smarca4fl/+Il7rCre/+ mice (Fig. 6c). Together these observations indicated that in pro-B cells, Brg1 permitted a subset of B lineage–specific transcription factors access to a super-enhancer to directly activate expression of the gene encoding c-Myc, which in turn induced the expression of genes encoding products associated with ribosome biogenesis, to control the growth of pro-B cells. Brg1 enforces a pro-B cell–specific transcription signature To examine in greater detail the spectrum of genes affected by the loss of Brg1 expression, we compared the differences in transcript abundance in Brg1-sufficient pro-B cells versus Brg1-deficient pro-B cells to the differences during the CLP–to–pro-B cell transition. The transcription signatures of pro-B cells depleted of Brg1 expression relative to those of wild-type pro-B cells did not correlate with the changes in expression that occurred during the CLP–to–pro-B cell transition (Fig. 7a, left). However, the transcription profiles of Brg1deficient pro-B cells versus those of wild-type pro-B cells correlated with changes in transcription signatures that accompanied the pro-B cell–to–pre-B cell transition (Fig. 7a, right). For example, the abundance of Cd79b transcripts was greater, whereas the abundance of Dntt transcripts was lower, in the absence of Brg1 than in the presence of Brg1 (data not shown). These data suggested that Brg1 acted in pro-B cells to suppress a pre-B lineage–specific pattern of gene expression. To confirm those findings in vivo, we examined Smarca4fl/+Il7rCre/+ and Smarca4fl/flIl7rCre/+ mice for changes in the ratio of pro-B cells to pre-B cells. As predicted, we found that the ratio of small pre-B cells aDVANCE ONLINE PUBLICATION  nature immunology

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Articles to pro-B cells was significantly increased in mice that had undergone depletion of Brg1 expression (Fig. 7b). Notably, the large pre-B cell compartment was the most severely affected among the B cell compartments from the BM (Supplementary Fig. 6a), consistent with a requirement for Brg1 in the promotion of cell growth. In further support of that notion, we found that sorted pro-B cells derived from Smarca4fl/flER-Cre mice led to a ‘preferential’ loss of large cells and an increase in the fraction of small pre-B cells upon culture of the cells with tamoxifen, compared with their abundance among sorted and treated Smarca4+/+ER-Cre pro-B cells (Supplementary Fig. 6b,c). To determine how these findings mechanistically related to changes in transcription signatures during the pro-B cell–to–pre-B cell transition, we assigned c-Myc-binding sites to the nearest transcription start site and compared c-Myc occupancy with gene-expression patterns in pro-B cells and pre-B cells. Notably, we found that the expression of an ensemble of genes associated with c-Myc-bound sites in pro-B cells was closely associated with a pro-B cell developmental stage– specific program of gene expression and was downregulated at the pre-B developmental stage (Fig. 7c). Together these data indicated that Brg1 acted to prevent the premature differentiation of pro-B cells into pre-B cells by regulating Myc expression. DISCUSSION It is now established that during developmental progression, transcriptional regulators act together to establish de novo enhancer repertoires as cells differentiate along distinct trajectories39,40. The development of early progenitors of B cells is among the best characterized in terms of the transcription factors that modulate the developmental progression. However, it is less clear how lineage-specific transcription factors activate de novo or poised enhancer repertoires to initiate lineage-specific programs of gene expression. Published studies have demonstrated that chromatin remodelers, including the BAF complex, act to promote nucleosome depletion to permit transcription factors access to their cognate binding sites41,42. Here we mapped the spectrum of Brg1-binding sites across the pro-B cell genome. We found that Brg1 occupancy did not overlap with that of one distinct transcription factor but instead overlapped with that of combinations of transcription factors, including E2A, EBF1, Ikaros and members of the IRF family. How, then, is Brg1 recruited to a B lineage–specific enhancer repertoire with different and unique combinations of transcription factor–binding sites? We found that in pro-B cells, occupancy by Brg1 and p300 overlapped across the majority of the pro-B cell enhancer repertoire. p300 is recruited to active enhancer elements by directly interacting with a multitude of transcription factors43. Thus, we propose that in the CLP compartment, E2A, Foxo1, Ikaros and EBF1 bind to a repertoire of primed and partially opened enhancers. This binding results in the recruitment of p300, a histone acetylase, to an ensemble of B lineage–specific enhancers. The sequestration of p300 to poised enhancers leads to the deposition of H3K27ac, which recruits Brg1, which in turn evicts and/or slides nucleosomes away from the repertoire of poised enhancers. The role of Brg1 in B cell development is not restricted to specification of the B cell fate. Our data indicated multiple roles for Brg1 in cells committed to the B lineage, including contraction of the Igh locus. During early B cell development, the Igh locus undergoes large-scale alterations that merge the proximal and distal VH regions into a single domain as progenitor cells differentiate into proB cells12. How does a chromatin remodeler such as Brg1 promote the merging of chromatin domains? We found that Brg1 bound to multiple sites scattered across the entire Igh locus. Published studies have demonstrated that contraction of the Igh locus is regulated nature immunology  aDVANCE ONLINE PUBLICATION

by multiple transcription factors, including E2A, Pax5, Ikaros and YY1 (refs. 13–15). Here we demonstrated that Brg1 expression was essential for the eviction of nucleosomes across regulatory elements of the Igh locus containing clusters of binding sites for Pax5, YY1, E2A and EBF1. We found that a subset of these sites was associated with long-range genomic interactions that connected the distal VH region cluster with more proximally located anchors possibly to promote Igh locus contraction. Our data also indicated that Brg1 acted in committed pro-B cells to maintain Myc expression and promote cell growth. Brg1 was needed to activate Myc expression by associating with a distally located super-enhancer. The distal super-enhancer of Myc has been identified in acute myelogenous leukemia, in which Brg1 is essential for maintaining Myc expression and promoting the development of leukemia37,44. We found that Brg1 was critical for maintaining accessibility of this super-enhancer and allowing binding of transcription factors. Downregulation of Myc expression caused further downregulation of the expression of genes encoding products associated with the control of lymphocyte growth. Specifically, a wide spectrum of genes encoding products associated with ribosome biogenesis, including factors associated with nucleoli and members of the exosome complex, were downregulated in the absence of Brg1 and exhibited c-Myc binding. Consistent with those findings, we found that the large pre-B cell population was ‘preferentially’ reduced in Brg1-deficient mice compared with that in Brg1-sufficient mice. Thus, we suggest that Brg1 expression promotes the population expansion of pro-B cells and large pre-B cells by elevating the abundance of c-Myc. The c-Myc in increased abundance, in turn, associates with a repertoire of promoters of genes encoding products associated with cell growth and protein synthesis. During the transition from the pro-B cell stage to the small resting pre-B cell stage, Brg1 levels decrease, as do c-Myc levels. Interestingly, we found that depletion of Brg1 expression in the pro-B cell compartment led to the aberrant activation of genes upregulated at the pre-B cell developmental stage. Since we found that occupancy by c-Myc was associated with genes with higher expression at the pro-B cell stage than at the pre-B cell stage, we suggest that premature exit from the cell cycle through downregulation of c-Myc expression induces a pre-B cell developmental stage–specific program of gene expression, consistent with published observations connecting exit from the cell cycle with developmental progression45. Methods Methods and any associated references are available in the online version of the paper. Accession codes. GEO: ATAC-Seq data, Brg1 and Ikaros ChIP-Seq data, and RNA-Seq data, GSE66978. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments 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 pRVGPBB 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.),



Articles 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.). AUTHOR 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. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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21. Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D. & Oettinger, M.A. Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4. Proc. Natl. Acad. Sci. USA 100, 11577–11582 (2003). 22. Osipovich, O.A., Subrahmanyam, R., Pierce, S., Sen, R. & Oltz, E.M. Cutting edge: SWI/SNF mediates antisense Igh transcription and locus-wide accessibility in B cell precursors. J. Immunol. 183, 1509–1513 (2009). 23. Gao, H. et al. Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5. Proc. Natl. Acad. Sci. USA 106, 11258–11263 (2009). 24. Choi, J. et al. The SWI/SNF-like BAF complex is essential for early B cell development. J. Immunol. 188, 3791–3803 (2012). 25. Indra, A.K. et al. Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation. Development 132, 4533–4544 (2005). 26. Schlenner, S.M. et al. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus. Immunity 32, 426–436 (2010). 27. Kim, J. et al. Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity 10, 345–355 (1999). 28. O’Neill, D.W. et al. An ikaros-containing chromatin-remodeling complex in adult-type erythroid cells. Mol. Cell. Biol. 20, 7572–7582 (2000). 29. Ikawa, T., Kawamoto, H., Wright, L.Y. & Murre, C. Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20, 349–360 (2004). 30. Lin, Y.C. et al. A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nat. Immunol. 11, 635–643 (2010). 31. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013). 32. Zhuang, Y., Jackson, A., Pan, L., Shen, K. & Dai, M. Regulation of E2A gene expression in B-lymphocyte development. Mol. Immunol. 40, 1165–1177 (2004). 33. Zhang, Y. et al. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell 148, 908–921 (2012). 34. Lin, Y.C. et al. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat. Immunol. 13, 1196–1204 (2012). 35. Ebert, A. et al. The distal VH gene cluster of the Igh locus contains distinct regulatory elements with Pax5 transcription factor-dependent activity in pro-B cells. Immunity 34, 175–187 (2011). 36. Medvedovic, J. et al. Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire. Immunity 39, 229–244 (2013). 37. Shi, J. et al. Role of SWI/SNF in acute leukemia maintenance and enhancermediated Myc regulation. Genes Dev. 27, 2648–2662 (2013). 38. van Riggelen, J., Yetil, A. & Felsher, D.W. MYC as a regulator of ribosome biogenesis and protein synthesis. Nat. Rev. Cancer 10, 301–309 (2010). 39. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010). 40. Lara-Astiaso, D. et al. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014). 41. Fan, H.Y., He, X., Kingston, R.E. & Narlikar, G.J. Distinct strategies to make nucleosomal DNA accessible. Mol. Cell 11, 1311–1322 (2003). 42. Ramirez-Carrozzi, V.R. et al. Selective and antagonistic functions of SWI/SNF and Mi-2beta nucleosome remodeling complexes during an inflammatory response. Genes Dev. 20, 282–296 (2006). 43. Sakamoto, S. et al. E2A and CBP/p300 act in synergy to promote chromatin accessibility of the immunoglobulin κ locus. J. Immunol. 188, 5547–5560 (2012). 44. Buscarlet, M. et al. Essential role of BRG, the ATPase subunit of BAF chromatin remodeling complexes, in leukemia maintenance. Blood 123, 1720–1728 (2014). 45. Clark, M.R., Mandal, M., Ochiai, K. & Singh, H. Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling. Nat. Rev. Immunol. 14, 69–80 (2014).

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ONLINE METHODS

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Mice. Smarca4fl/fl, Il7rCre/+, Rosa26YFP/+, ER-CRE and E2A-GFP mice were bred and housed in specific pathogen–free conditions in accordance with the Institutional Animal Care and Use Committee of University of California, San Diego. Flow cytometry. BM cells were harvested from femur, tibia and crista iliac. Fc receptors in cells were blocked (anti-CD16/CD32 (2.4G2; eBioscience) and cells were stained with the following monoclonal antibodies conjugated to fluorescein isothiocyanate, peridinin chlorophyll protein–cyanine 5.5, phycoerythrin, phycoerythrin–indotricarbocyanine, phycoerythrin-CF594, allophycocyanin, allophycocyanin-indotricarbocyanine, Pacific Blue, eFluor 450 or Brilliant Violet 421 or biotinylated (all from eBioscience except as noted below): anti-Flt3 (A2F10), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Ter119 (Ter119), anti-CD3ε (145-2c11), anti-CD11c (N418), anti-Ly6c (AL-21), anti-NK1.1 (PK136), anti-B220 (RA3-6B2), anti-CD117 (2B8), anti-CD19 (1D3), anti-IL-7R (A7R34), anti-IgD (11-26c), anti-IgM (11/41), anti-CD25 (pc61.5), anti-Ly6d (49-H4), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD115 (AFS98) and antibody to NK cells (DX5). That staining was followed by incubation with streptavidinconjugated QD655 quantum dots (Life Technologies) and propidium iodide. Antibodies not from eBioscience were from vendors as follows: phycoerythrinCF594–anti-CD19 (BD Biosciences), Brilliant Violet 421–anti-CD117 (BD Biosciences), allophycocyanin-indotricarbocyanine–anti-B220 (BioLegend), CD19 APCCy7 (BioLegend) and fluorescein isothiocyanate–anti-Ly6D (BD Biosciences). For sorting, BM was depleted of cells positive for lineage markers before being stained with a ‘cocktail’ of biotinylated antibodies (anti-B220, anti-Ter119, anti-CD19, anti-CD3, anti-Gr-1, anti-CD11c, anti-CD11b, anti-Ly6c, anti-NK1.1 (all identified above) for sorting of CLPs; and antiGr-1, anti-CD11b and anti-Ter119 (all identified above) for sorting of pre-B cells) followed by depletion of biotin-labeled cells with anti-biotin magnetic beads (Miltenyi Biotech) and an autoMACS Separator. For analysis of BrdU incorporation, mice were given intraperitoneal injection of 200 µl BrdU labeling reagent (Life Technologies) 4 h before being killed. Samples were stained for BrdU with a BrdU Staining Buffer Set for Flow Cytometry from eBioscience. Cells were treated with DNase I (Sigma-Aldrich) for 1 h before incubation with fluorescein isothiocyanate–anti-BrdU (BU20A; eBioscience). Cells were analyzed with an LSRII and were sorted on a FACSAria II (BD Biosciences). Cell culture and plasmids. Rag1-deficient pro-B cells were cultured in OPTIMEM in the presence of 10% FSC, 2× penicillin-streptomycin-glutamine, 50 µM β-mercaptoethanol and IL-7 and stem-cell factor (both cytokines prepared in-house). The GFP-encoding cassette in plasmids pRVGP and pRVGP-BB was replaced with a cassette encoding human CD25 to generate plasmids encoding control shRNA and Smarca4-specific shRNA. Retroviral supernatants were obtained through the transfection of 293T human embryonic kidney cells (by the calcium phosphate method) with control shRNA and Smarca4specific shRNA in conjunction with the packaging plasmid pCL-Eco. Two days after infection, samples underwent enrichment for infected cells through the use of anti–human CD25 magnetic beads (Miltenyi biotech) on an autoMACS separator. Smarca4+/+ER-Cre and Smarca4fl/flER-Cre pro-B cells were cultured as the Rag1-deficient pro-B cells were. This Cre recombinase was activated by the addition of 4-OH tamoxifen (Sigma-Aldrich), at a concentration of 0.33 µM, to the medium. RT-PCR. Total RNA was isolated with an RNeasy Mini Kit (Qiagen) or RNeasy Micro Kit (Qiagen). cDNA was generated with a First-Strand Synthesis kit (Life Technologies) and random hexamers. Quantitative PCR was performed with SYBR Green Master Mix (Roche) on Mx3500P cycler (Agilent Technologies). Primers were as follow: Actb, 5′-GAAGCACTTGCGGTGCACGAT-3′ and 5′-TCCTGTGGCATCCATGAAACT-3′; Smarca4, 5′-CACCTAACCTC ACCAAGAAGATGA-3′ and 5′-CTTCTTGAAGTCCACAGGCTTTC-3′; Smarca2, 5′-AGGACGTGGACGACGAATAC-3′ and 5′-AATGGTCTGGA TGGTCTTGC-3′; Myc, 5′-CCTAGTGCTGCATGAGGAGAC-3′ and 5′-CCT CATCTTCTTGCTCTTCTTCA-3′; Tcf3, 5′-GGGAGGAGAAAGAGGATGA-3′ and 5′-CCGGTCCCTCAGGTCCTTC-3′; Ebf1, 5′-GCCTTCTAACCTGCGG

doi:10.1038/ni.3170 

AAATCCAA-3′ and 5′-GGAGCTGGAGCCGGTAGTGGAT-3′; and Ikzf1, 5′-GGAGGCACAAGTCTGTTGAT-3′ and 5′-CATTTCACAGGCACGCCC ATTCT-3′. RNA-Seq. Total RNA was isolated with an RNeasy Mini kit (Qiagen). RNA was treated with TURBO DNase (Life Technologies). mRNA was purified from total RNA with a Dynabeads mRNA purification kit (Life Technologies). cDNA was generated with a First-Strand Synthesis kit (Life Technologies) and a combination of random hexamers and oligo(dT) in presence of actinomycin D. Second-strand synthesis was performed with dUTP instead of dTTP. The double-stranded cDNA was sonicated to a length of 200–400 base pairs with an S220 Focused-ultrasonicator (Covaris). Sonicated cDNA was ligated to adaptors (NEBNext primer kit). The resulting DNA was treated with uracil-Nglycolsylase before amplification by PCR with the indexing primers (NEBNext primer kit). Following PCR, fragments were selected by size. To perform RNASeq analysis, we sequenced at a depth of 10 × 106 to 15 × 106 reads per sample with an Illumina Hiseq2500. Raw sequencing files underwent quality control with the FastQC tool (Babraham Bioninformatics). Alignment and trimming of reads was performed with the Omicsoft Sequence Aligner algorithm against mm10 as a reference (mm10 NCBI assembly of the mouse genome) with Array Studio software (Omicsoft). RNA transcripts were quantified by RSEM methods (RNA-Seq by expectation-maximization) as implemented in Array Studio (Omicsoft). Principal-component analysis was then performed to check for possible batch effects and outliers. Abundance values (counts) were normalized, and values for samples were compared with the DESeq package of software of the R project for statistical computing. ATAC-Seq. ATAC-Seq was performed as described31. Cells were lysed, then nuclei underwent ‘tagmentation’ (transposase-based fragmentation) for 30 min at 37 °C with the Nextera DNA Sample Preparation Kit. The resultant ‘tagmented’ DNA was cleaned up with DNA Clean and Concentrator columns (ZymoResearch). Library fragments were amplified with 1× NEB Next PCR Master Mix and custom Nextera PCR primers 1–6. The number of cycles was determined by quantitative PCR as described31. Libraries were purified on DNA Clean and Concentrator columns. Libraries were sequenced on a HiSeq 2500 system as single reads. Reads were aligned to mm9 with Bowtie software (Johns Hopkins University) with the parameter –m 1. Data were analyzed with the HOMER suite of tools (hypergeometric optimization of motif enrichment). Tag directories were generated with the parameter – tbp 1, which removed most of the ‘reads’ arising from mitochondrial DNA. ChIP and ChIP-Seq. Cells were fixed for 15 min in PBS containing 1.5 mM ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester), followed by incubation for 15 min with 1% formaldehyde. Formaldehyde was quenched by incubation for 10 min with 0.2 M glycine. Cells were washed twice with PBS and pellets were frozen in liquid nitrogen. Nonspecific binding was blocked in 30 µl protein G Dynabeads (Life Technologies) by incubation with 0.5% BSA (wt/vol) in PBS. Magnetic beads were bound with 6 µg of the appropriate antibody. Antibodies used were as follow: anti-Brg1 (07-478; Millipore) and anti-Ikaros (Ik-C; a gift from S. Smale). Crosslinked cells were lysed in lysis buffer 1 (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% TritonX-100) and were washed with lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA and0.5 mM EGTA). Cells were resuspended and then were sonicated (20 W) in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine) for ten cycles of 10 s each on ice with intervals of 50 s on ice between cycles. Lysates were cleared by centrifugation, and Triton X-100 was added at a final concentration of 1%. Lysates were then incubated overnight at 4 °C with the previously prepared magnetic beads. Beads were washed once with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 and 1 mM EDTA), once with RIPA 500 buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 and 1 mM EDTA), once with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate and 1 mM EDTA) and finally twice with TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA). Bound complexes

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were eluted from the beads by incubation for 30 min at 65 °C with shaking in elution buffer (10 mM Tris-HCl, pH 8.0, 0.5% SDS, 300 mM NaCl and 5 mM EDTA). Crosslinks were reversed by incubation overnight at 65 °C. RNA and protein were digested in the supernatants with RNase A and proteinase K, and DNA was purified with ChIP DNA Clean and Concentrator Columns (Zymoresearch). Libraries were prepared with the NEBNext primer set and were selected by size by 8% PAGE. Libraries were run on Illumina HiSeq 2500. Reads were aligned to mm9 with Bowtie software with the parameter –m 1. Data were analyzed with HOMER software. For PCR after ChIP with anti-Ikaros, the primers were as follow: site 1, 5′-CCTGGTTCCGAGAAGTCACT-3′ and 5′-GCACTCTCTGGAAAGGGT CT-3′; site 2, 5′-TCTCCTAGCCCCTAAGACCA-3′ and 5′-GGAGTTGCC CTTGGAGATCC-3′.

channels. Distances between the probes were obtained with the softWoRx program by calculation of the distances between the centers of mass of the probes. Probe RP23-201H14 was labeled with Alexa Fluor 568–5-dUTP and probe RP24-189H12 was labeled with Alexa Fluor 488–5-dUTP.

Hi-C analysis. Reads were aligned to mm9 with Bowtie software with the parameter –m 1. Data were analyzed with HOMER software. Interactions were prepared for presentation with the Circos software package. The results for all pairwise comparisons of the various features at the interactions end-points were visualized with the cytoscape software platform.

GEO accession codes for publicly available data sets. PU.1, GSE21512; CTCF, c-Myc, p300 and H3K4me2, GSE40173; E2A, H3K4me1 and H3K4me3, GSE21978; EBF, GSE53595; Pax5, GSE38046; YY1, GSE43008; Med1, GSE44288; and Hi-C, GSE35519 and GSE40173.

Imaging. Fluorescent in situ hybridization was performed as described12. The bacterial artificial chromosome probes used were RP23-201H14 and RP24-189H12 from the BACPAC Resource Center at Children’s Hospital Oakland Research Institute. Three-dimensional fluorescent images were acquired on a deconvolution microscope (Deltavision). Optical sections (z-stacks) 0.2 µm apart were obtained throughout the cell volume in the appropriate

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PCR analysis of Igh recombination. Genomic DNA was purified from sorted pre-B cells on DNeasy Mini Columns (Qiagen). PCR was performed on 25, 6.75 or 1.2 ng of genomic DNA with forward primers 5′-GAASAMCCTGT WCCTGCAAATGASC-3′ (VH7183) and 5′-CARCACAGCCTWCATGCAR CTCARC-3′ (VHJ558) and reverse primer 5′-CTCACAAGAGTCCGATAGA CCCTGG-3′ (JH3). PCR products were separated by electrophoresis through a 1.5% agarose gel, then were blotted onto nylon membrane and hybridized with radioactive probe. Signals were detected with a Phosphorimager (GE Healthcase) and were analyzed with ImageJ software.

Statistical methods. Data were analyzed with a two-tailed Student t-test for two-group comparisons. P values of less than 0.05 were considered statistically significant. No blinding was used for the animal studies, and no animal was excluded from the analysis. For fluorescence in situ hybridization, the investigator measuring the distance between the probes was unaware of the sample identity. The distance between the probes was measured only in cells in which the probes could be detected for both alleles.

doi:10.1038/ni.3170