NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression

Abstract

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.

Figure 1

Identification of direct gene targets for NuRD and PRC2. (A) Quantitative RT–PCR comparing transcript levels in Mbd3^−/− ES cells to those in wild-type ES cells. Results are plotted as log₁₀ fold change relative to wild-type levels. Error bars indicate standard error of the mean (s.e.m.). (B) Chromatin IP in wild-type cells for either Mi2β or IgG control, with qPCR for proximal promoter regions of genes shown. Results are plotted as percentage of input DNA. Histone signatures (according to Mikkelsen et al, 2007) are indicated underneath. Asterisks denote loci at which ChIP for Mi2β is significant with respect to IgG control (P<0.005). (C) Chromatin IP in wild-type cells for either Suz12 or IgG control, plotted as percentage of input DNA. Histone signatures are indicated underneath. Asterisks denote loci where ChIP for Suz12 is significant with respect to IgG control (P<0.005). Error bars indicate s.e.m.

Figure 2

Comparison of multiple histone modifications between Mbd3^−/− and wild-type ES cells. (A) ChIP for histone modifications. Enrichment at promoter regions of genes for histone modifications, bulk histone H3 levels or IgG control plotted as null value relative to wild type. Error bars indicate s.e.m. (B) Whole histone extracts from wild-type, Mbd3^−/− and Eed mutant ES cells blotted with antibodies for histone H3 (loading control), H3K27me3 and H3K27ac to show relative levels.

Figure 3

ChIP-seq analysis, reciprocal changes of H3K27me3 and H3K27ac levels in the absence of NuRD. (A) ChIP-seq for H3K27me3 or H3K27ac in wild-type or Mbd3^−/− cells: average signal profiles, normalised to input, are shown for upregulated, bivalent genes (solid line) relative to all RefSeq (Pruitt et al, 2007) genes in each data set (dotted line) for a 6-Kb region spanning the transcription start site (TSS). Distance from TSS is indicated in base pairs. (B) ChIP for H3K27me3 and H3K27ac in wild-type and Mbd3-null cells and for Mi2β in wild-type cells at one target gene (Htra1) and one non-target gene (T). Data are shown relative to IgG control, distance from TSS is measured in base pairs as the mid point of PCR product. Outlines of the gene structures are indicated, with the total length of the transcriptome indicated below (not to scale). Error bars indicate s.e.m.

Figure 4

Interdependency between NuRD and PRC2 chromatin interactions. (A) Western blot showing relative levels of NuRD and PRC2 components in wild-type, Mbd3^−/− and Eed mutant cells. Alpha tubulin is used as loading control. (B) ChIP for Suz12 in Mbd3-null cells relative to wild type. (C) ChIP for Mi2β in Eed mutant cells relative to wild type. Association of Mi2β and Suz12 in wild-type cells at each gene is indicated below each panel. (D) ChIP for Jarid2 in Mbd3-null cells relative to wild type. (E) ChIP for Mi2β in Jarid2^−/− cells relative to wild type. ChIP data shown is for bivalent genes only. Error bars indicate s.e.m.

Figure 5

Effect of acetylation on recruitment of PRC2 to chromatin. (A) ChIP for H3K27ac and IgG control in wild-type cells treated with TSA and in Mbd3^−/− cells without TSA treatment. (B) Relative enrichment for Mi2β and IgG control in wild-type cells treated with TSA compared with untreated. (C) Relative enrichment for Suz12 and IgG control in wild-type cells treated with TSA compared with untreated. Error bars show s.e.m. (D) Western blot of nuclear extracts prepared from wild-type ES cells with or without TSA treatment. Alpha tubulin is used as a loading control.

Figure 6

Responsiveness of PRC2 recruitment to presence of NuRD. (A) Western blot of nuclear extracts prepared from wild-type, Mbd3^−/− and MER-Mbd3b-MER Mbd3^−/− cells after addition of 4-hydroxytamoxifen for indicated time. Alpha tubulin acts as a loading control. (B) Expression levels of Htra1 in wild-type, Mbd3^−/− and MER-Mbd3b-MER Mbd3^−/− cells over the indicated 4-hydroxytamoxifen time course relative to Mbd3^−/− levels. (C) Occupancy levels for H3K27ac, H3K27me3, Jarid2 and Mer-Mbd3b-MER at the Htra1 locus over the 4-hydroxytamoxifen time course in MER-Mbd3b-MER Mbd3^−/− ES cells. Levels are shown relative to highest point for each protein.

Figure 7

Overlap of NuRD and PRC function in gene regulation. (A) Expression patterns of genes differentially expressed between wild-type and Mbd3^−/− ES cells (P<0.05) were assessed in data from Leeb et al (2010) describing gene expression changes in PRC mutant ES cells. A total of 2907 probe sets were found to be differentially expressed between the reference and Mbd3 mutant samples, mapping to 1967 unique genes using the re-annotated array information provided by Leeb et al. Of these, 1879 genes were also present on the Leeb microarray platform, although not necessarily differentially expressed. The log₂ fold change of these genes relative to their respective wild-type samples is illustrated in the figure. Genes showing reduced expression compared with wild-type cells are indicated in blue; those showing increased expression are indicated in yellow. Mbd3_1, Mbd3_2 and Mbd3_3 indicate results from three different Mbd3^−/− ES cell samples corresponding to two independently derived ES cell lines. Ring1B_1-3, Eed_1-3 and Ring1B/Eed_1-3 represent results from three replicates each of Ring1B-null ES cells, Eed-null ES cells and Ring1B/Eed-double null ES cells, respectively (Leeb et al, 2010). Where multiple probes map to the same gene, only the probe displaying the largest deviation from the reference sample is included here. The four main gene clusters are indicated on the right hand side (I–IV). (B) Two-step model for control of transcription illustrating relationship between acetylation status, here controlled by NuRD, and methylation of H3K27 by PRC2. Top panel: in wild-type cells, PRC2 and NuRD function fully. NuRD directs deacetylation of specific genes, which then become available for trimethylation by PRC2. Middle panel: in cells lacking functional PRC2, for example, Eed^−/− cells, H3K27 trimethylation is lost genome-wide, the balance moves towards the acetylation/deacetylation cycle and transcription of bivalent genes increases overall. Bottom panel: in the absence of NuRD, deacetylation fails to occur at specific loci only. At these genes, there is an increase in acetylation, a subsequent reduction in trimethylation at H3K27 through loss of substrate for PRC2 and an increase in transcription.