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

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.

Figure 1

The chromatin remodeler Brg1 is essential to specify B cell fate. (a) Normal bone marrow (BM) cellularity in Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. BM cells were derived from two femurs, two tibias and crista iliac. (b) B cell development is blocked in Smarca4^fl/flIl7r^Cre/+ mice. BM cells were isolated from Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. Cells were gated as CD19⁺B220⁺. Percentages (left panel) as well as cellularity (right panel) are indicated. (c) Depletion of B cell progenitors in Brg1-deficient mice. BM cells were gated on the Gr1⁻CD11b⁻Ter119⁻CD19⁺B220⁺ population and segregated into pro-B (IgM⁻IgD⁻Kit⁺CD25⁻), pre-B (IgM⁻IgD⁻Kit⁻CD25⁺), immature B (imm B)(IgM⁺IgD⁻) and mature B (mat B) (IgM⁺IgD⁺) cells. Cell numbers are indicated for Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. (d) Normal numbers of CLPs in Smarca4^fl/flIl7r^Cre/+ mice as compared to Smarca4^fl/+Il7r^Cre/+ mice. Left panels show the gating strategy to identify ALPs (Lin⁻IL7R⁺Flt3⁺MCSFR⁻Kit^loLy6D⁻) and BLPs (Lin⁻IL7R⁺Flt3⁺MCSFR⁻Kit^loLy6D⁺). Right panel indicates absolute numbers of ALPs and BLPs. (e) Cre recombinase activity in ALPs (Lin⁻IL7R⁺Flt3⁺Ly6D⁻) and BLPs (Lin⁻IL7R⁺Flt3⁺Ly6D⁺) (left panel) and proB and pre-B cells (as defined in c) (right panel) derived from Smarca4^fl/+Il7r^Cre/+Rosa26^YFP/+ and Smarca4^fl/flIl7r^Cre/+Rosa26^YFP/+ mice. Left panel shows cells gated on Lin⁻Flt3⁺IL7R⁺. Right panel shows cells gated on CD19⁺B220⁺IgM⁻IgD⁻. a,b: Representative of 4 independent experiments. c–e: Representative of 3 independent experiments. **P < 0.01 (two-tailed unpaired Student’s t test).

Figure 2

Genome-wide Brg1 occupancy across the pro-B cell genome. (a) Smarca4 and Smarca2 transcript expression as fold relative to Actb during hematopoiesis and early B cell development. Error bars represent standard deviation from two independent sorts. (b) Distribution of 13169 Brg1 binding sites across genomic regions in Rag1^−/− pro-B cells. (c) Heatmap of ChIP-Seq data gated on genome-wide Brg1-bound sites. The distribution of H3K4me1, H3K4me2 and H3K4me3 deposition in a 6-kb window across Brg1-bound sites is shown in Rag1^−/− pro-B cells. (d) Brg1 and p300 tag count shown in a 200-bp window of Brg1 and p300 combined binding sites in Rag1^−/− pro-B cells. (e) Brg1, p300 and E2A occupancy across Pax5 (chr4: 44,703,000–44,730,000 in the mm9 build) and Cd19 (chr7: 133,551,000–133,564,000) loci in Rag1^−/− pro-B cells. (f) Cis-regulatory sequences associated with Brg1 occupancy. P values reflecting the significance of motif occurrence are shown. (g) Brg1 reads are shown for a 100-bp window centered on respectively 5000 Ikaros, E2A, EBF1, Pax5, PU.1 and CTCF binding sites.

Figure 3

Brg1 is required to establish an accessible B-lineage specific enhancer repertoire. (a) ATAC-Seq tag coverage in ALPs and BLPs derived from Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. ATAC reads are plotted as a function of genomic distance from E2A, Ikaros, EBF1 or CTCF bound-sites (identified in pro-B cells). (b) ATAC reads as well as deposition of H3K4me2, E2A, Ikaros and EBF1 occupancy are shown across the intergenic genomic regions flanking the Foxo1 and Cd79a loci. Left panel indicates the genomic location of a 3′ Foxo1 enhancer (chr3: 52,243,000 - 52,253,000). Right panel indicates the genomic location of a Cd79a regulatory region (chr7: 25,677,000 - 25,687,000). Pre-pro-B cells were represented by E2A-deficient multipotent progenitors. Pro-B cells were derived from Rag1^−/− mice. (c) E2A-GFP mice were used to determine E2A protein abundance. Mean fluorescence intensity was determined in BM lineage⁺ cells (CD11b⁺Ter119⁺CD3⁺NK1.1⁺GR1⁺), ALPs, BLPs and BM B cells (CD19⁺). (d) Transcript expression was determined by RT-PCR in ALPs and BLPs sorted as in a. Fold relative to Actb expression. Average and error bars derived from triplicates. a,b: Data derived from two independent experiments. c,d: **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t test)

Figure 4

Brg1 is essential to promote Igh locus contraction. (a) A meta-analysis for the pro-B cell interactome that were derived from Hi-C studies depicted as a Circos diagram (chr12: 114,300,000 - 117,400,000)^,. Deposition of H3K4me2, region of open chromatin as determined by ATAC-Seq as well as Brg1 bound-sites are indicated. The thicknesses of the connecting lines reflect the natural log ratio of observed versus expected interaction frequency in the Hi-C data sets. Bin size used for the analysis was 10 kb. Constant, joining, diversity and variable regions are represented by different colors. (b) Brg1, E2A, Pax5, YY1, Ikaros and CTCF occupancy across the end point of interactions associated with the Igh locus (chr12: 115,825,000–115,875,00). (c) Relative frequencies by which lineage-specific transcription factors, CTCF and Med1 are associated with loop-attachment points (node size) and the significance and reproducibility of tethering, in terms of P values (line thickness). Color (red-blue) represents the log ratio of observed frequency relative to expected frequency (association strength). (d) ATAC-Seq tag count associated with Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells shown in combined ATAC-Seq peaks (57 peaks total) associated with the V_H region cluster. Regions of open chromatin with a 2-fold reduction and 2-fold increase in tag count in Smarca4^Δ/Δ compared to Smarca4^+/+ pro-B cells are shown respectively in red and blue. Numbers of peaks are indicated in parenthesis. (e) Number of transcription factor peaks in region of open chromatin from d determined by ATAC-Seq in Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells. Transcription factor peaks associated with regions of open chromatin with a 2-fold reduction or 2-fold increase in tag counts associated with Smarca4^Δ/Δ pro-B cells as compared to Smarca4^+/+ pro-B cells are shown in red and blue, respectively. (f) The distribution of spatial distances separating the two BAC probes are shown for Rag1^−/− cells transduced with control retrovirus or virus expressing an shRNA directed against Smarca4. Representative experiment of 5 independent experiments is shown. Right panel indicates the cumulative frequency distribution of the spatial distances that separate both BAC probes in control and Brg1-deficient pro-B cells. Data is derived from 5 independent experiments. (g) Decreased usage of distal V_H as compared to proximal V_H regions in Brg1-deficient pre-B cells. Usage of proximal V_H7183 segments compared to distal V_HJ558 segments was assessed by Southern blot in pre-B cells sorted from Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. Data is derived from two independent experiments. **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t test).

Figure 5

Brg1 acts to induce the expression of Myc and an ensemble of genes associated with protein synthesis to regulate B cell growth. (a) RNA-Seq analysis of Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells. Expression represented as mean normalized counts. Abundance of transcripts significantly decreased (>two fold) in Brg1-deficient as compared to wild-type pro-B cells are highlighted in blue. Red dots indicate transcript levels that were significantly elevated (>two fold) in Brg1-ablated pro-B cells. (b) Brg1 regulates the expression of genes associated with protein synthesis at the pro-B cell stage. Ontology analysis (DAVID) shows clusters of genes that displayed decreased transcript abundance in Smarca4^Δ/Δ pro-B cells compared to Smarca4^+/+ pro-B cells. (c) Brg1 regulates Myc expression in pro-B cells. RNA was isolated from wild-type or Brg1-depleted pro-B cells and analyzed by qPCR. Left panel shows Myc abundance in Smarca4^Δ/Δ pro-B versus Smarca4^+/+ pro-B cells. Right panel shows Myc expression in Rag1^−/− pro-B cells transduced with retrovirus expressing shRNAs directed against Smarca4 versus transduced pro-B cells expressing a control vector. Fold relative to Actb expression. (d) Circos diagram depicting long-range genomic interactions (> 100 kb) across a 4.4 Mb region containing the Myc locus (chr15: 59,700,000 - 64,100,000). Deposition of H3K4me2/3 as well as Brg1, Ikaros, E2A, EBF1 and Pax5 bound-sites are indicated. The thicknesses of the connecting lines reflect the natural log ratio of observed versus expected interaction frequency in the Hi-C data sets. Bin size used for the analysis was 10 kb. Myc gene is highlighted in grey and the distal Myc super-enhancer is indicated by a red box. (e) Brg1 permits a subset of B-lineage transcription factors access to a super-enhancer to activate Myc expression. Brg1, EBF1, Ikaros and Pax5 occupancy in Rag1^−/− pro-B cells across the super-enhancer (chr15: 63,440,000 - 63,500,000) is shown. Indicated are nucleosome-depleted regions as determined by ATAC reads across the super-enhancer for both Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells. (f) Ikaros occupancy across the Myc super-enhancer is significantly decreased in Brg1-depleted pro-B cells. Ikaros-bound sites across the Myc super-enhancer are indicated in Smarca4^+/+ pro-B cells and Smarca4^Δ/Δ pro-B cells (Sites 1 and 2 are shown in red in (e)). c: Average and standard deviation from three independent experiments. f: Representative from two independent experiments. *P < 0.05 (two-tailed unpaired Student’s t test).

Figure 6

Brg1 regulates Myc expression to promote pro-B cell growth. (a) c-Myc occupancy associated with the promoter regions of the Gar1 and Tsr1 genes is depicted. Tag counts derived from the RNA-Seq reads derived from Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells are indicated. RNA-Seq tag counts are representative of two independent experiments. (b) Relative transcript levels of genes associated with c-Myc occupancy in promoter regions in Smarca4^+/+ and Smarca4^Δ/Δ pro-B cells. ***P = 1 × 10⁻³⁸ (two-tailed paired Student’s t test). (c) BrdU incorporation in pro-B cells derived from Smarca4^fl/flIl7r^Cre/+ mice as compared to Smarca4^fl/+Il7r^Cre/+ mice. Mice were sacrificed 4 hours after BrdU injection. Left panels show the gating strategy to identify pro-B cells (Lin⁻Live⁺CD19⁺B220⁺IgM⁻Ly6D⁻). Right panel indicates percentage of BrdU incorporation in pro-B cells. Data is derived from two independent experiments. *P < 0.05 (two-tailed unpaired Student’s t test).

Figure 7

Brg1 expression acts to maintain the pro-B cell compartment. (a) Brg1 acts to suppress the induction of a pre-B lineage specific program of gene expression. Left panel shows changes in transcript abundance in Smarca4^Δ/Δ pro-B cells versus Smarca4^+/+ pro-B cells plotted against changes in transcript abundance in CLPs versus pro-B cells. Spearman correlation = −0.05. Right panel shows changes in transcript abundance in Smarca4^Δ/Δ pro-B cells versus Smarca4^+/+ pro-B cells plotted against changes in transcript abundance in pre-B cells versus pro-B cells. Spearman correlation = 0.37. Transcripts highlighted in blue and red represent significant differences (> 2-fold) between Smarca4^Δ/Δ pro-B cells when compared to Smarca4^+/+ pro-B cells. Gene expression in CLP, pro-B and pre-B cells determined by microarray by the ImmGen Consortium (b) Flow cytometric analysis of the pro-B (IgM⁻IgD⁻Kit⁺CD25⁻) and pre-B (IgM⁻IgD⁻Kit⁻CD25⁺) cell compartments in Smarca4^fl/+Il7r^Cre/+ and Smarca4^fl/flIl7r^Cre/+ mice. Right panel indicates the ratios of pre-B versus pro-B cells. Representative of 3 independent experiments. (c) Transcript expression of 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).