Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5

Abstract

Transcriptionally silent genes are maintained in inaccessible chromatin. Accessibility of these genes requires their modification by chromatin remodeling complexes (CRCs), which are recruited to promoters by sequence-specific DNA-binding proteins. Early B-cell factor (EBF), which is crucial for B-cell lineage specification, reprograms mb-1 (Ig-alpha) promoters by increasing chromatin accessibility and initiating the loss of DNA methylation. In turn, this facilitates promoter activation by Pax5. Here, we investigated the roles of ATP-dependent CRCs in these mechanisms. Fusion of EBF and Pax5 with the ligand-binding domain of ERalpha allowed for 4-hydroxytamoxifen-dependent, synergistic activation of mb-1 transcription in plasmacytoma cells. Knock-down of the SWI/SNF ATPases Brg1 and Brm inhibited transcriptional activation by EBF:ER and Pax5:ER. In contrast, knock-down of the Mi-2/NuRD complex subunit Mi-2beta greatly enhanced chromatin accessibility and mb-1 transcription in response to the activators. The reduction of Mi-2beta also propagated DNA demethylation in response to EBF:ER and Pax5:ER, resulting in fully unmethylated mb-1 promoters. In EBF- or EBF/Pax5-deficient fetal liver cells, both EBF and Pax5 were required for efficient demethylation of mb-1 promoters. Together, our data suggest that Mi-2/NuRD is important for the maintenance of hypermethylated chromatin in B cells. We conclude that SWI/SNF and Mi-2/NuRD function in opposition to enable or limit the reprogramming of genes by EBF and Pax5 during B-cell development.

Fig. 1.

Tamoxifen-dependent activation of mb-1 gene expression. (A) Pax5:ER increases the display of mIgM on μM.10 cells. Cells were infected with Pax5:ER-YFP retroviruses. YFP⁺ cells were sorted and 0.5 μM 4-OHT was added at 48 h posttransduction. 48 h after the addition of 4-OHT, cell surface expression of mIgM (which directly reflects mb-1 expression) was analyzed by labeling cells with biotinylated anti-IgM and streptavidin-conjugated APC. mIgM was detected using a FACScalibur™ flow cytometer. (B) Synergistic activation of mb-1 transcription by EBF:ER and Pax5 in μM.2 cells. Cells were stably transfected to express EBF:ER (μM.2+EBF:ER cells), then infected with MSCV-Pax5-GFP retroviruses. Cells are shown following incubation with or without 4-OHT and mIgM staining as in A.

Fig. 2.

mb-1 gene activation in the context of EBF, Pax5 and knock-down of chromatin remodeling complexes (CRCs). (A) Western detection of Brg1, Brm, EBF:ER, and Pax5:ER in μM.2 or μM.2+EBF:ER cells. Cells were infected sequentially with Pax5:ER-YFP or YFP control retroviruses and Brg1/Brm-specific shRNA-GFP or control GFP retroviruses. GFP⁺YFP⁺ cells were purified on day 2 postinfection with GFP/shRNA-GFP viruses. 0.5 μM 4-OHT was added for the final 48 h. Western blotting was performed on the sorted cells at day 3 or 4 of shRNA expression. EBF:ER and Pax5:ER were detected using anti-ERα serum. USF1 served as a loading control. (B) Western detection of Mi-2β, EBF:ER and Pax5:ER in μM.2 or μM.2+EBF:ER cells. Other aspects of this experiment are similar to (A). (C) mIgM expression in response to EBF:ER, Pax:ER without or with Brg1/Brm shRNA. In this and all subsequent experiments, cells were labeled with Cy5-conjugated anti-IgM and detected using a CyAn™ flow cytometer. (D) mIgM expression in response to Mi-2β shRNA without or with EBF:ER, Pax:ER or both factors. (E) Quantitative RT-PCR analysis of mb-1 transcripts for the cells in (C and D). All data were obtained from sorted YFP⁺GFP⁺ cells and normalized relative to β-actin transcripts (n = 3) (mean ± SEM.). (F) Accessibility of mb-1 promoter chromatin in response to EBF:ER, Pax5:ER and shRNA. Purified YFP⁺GFP⁺ cells were incubated 48 h with 4-OHT. (C and D). Relative cleavage by Sau96I (test; see Fig. 3A for location) was detected using LM-PCR. Total cleavable DNA was measured following digestion of samples to completion (control). Exposure of control gel was one-fifth that of test gel exposure.

Fig. 3.

Knockdown of Mi-2β increases and propagates EBF- and Pax5-dependent DNA demethylation. (A) Positions of EBF, Pax5/Ets and proximal promoter factor binding sites are indicated relative to CpGs1–8. Binding sites of other factors present in early B cells (Runx1/CBFβ, E2A), but not plasmacytoma cells, are not shown. The location of the Sau96I site used to measure chromatin accessibility is indicated. (B) DNA methylation of mb-1 promoters is decreased synergistically by EBF:ER, Pax5:ER, and Mi-2β shRNA in μM.2 cells. All cells were cultured 48 h in the presence of 4-OHT. Genomic DNA was recovered from μM.2 cells without EBF:ER (−EBF:ER) or μM.2 +EBF:ER cells as shown and converted with sodium bisulfite before amplification and subcloning of mb-1 promoters. The frequency of methylated CpGs at each of the eight CpGs is shown for 20 sequenced clones (black bars). (C) The lack of DNA methylation correlates with high levels of mIgM. μM.2 cells expressing EBF:ER, Pax:ER, and Mi-2β shRNA were sorted to recover the highest and lowest 5% of mIgM⁺ cells. Bisulfite converted mb-1 promoters were amplified and twenty individual clones were sequenced as in B. Methylated (black circles) and unmethylated (open circles) CpGs are indicated in each individual sequence.

Fig. 4.

Reconstitution of EBF expression initiates mb-1 promoter demethylation in EBF KO and EBFKO-Pax5KO cells. (A) DNA demethylation in response to EBF. Cells were infected with FLAG-tagged EBF (18–429) or empty GFP control virus. At day 4 postinfection, GFP positive cells were sorted for bisulfite treatment and isolation of mb-1 promoter clones for sequencing. Each bar graph represents 20 independent clones. (B) Quantitative PCR of gene expression in uninfected versus EBF (18–429)-infected (+EBF) cells at day 4 postinfection. mb-1 transcripts levels were normalized to those of β-actin. Expression of mb-1 transcripts in EBFKO cells and Pax5 transcripts in EBFKO-Pax5KO cells were each set to 1. Data were generated in triplicate for statistical analysis (mean ± SEM).