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The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network - PubMed

  • ️Sun Jan 01 2012

The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network

Xiangzhi Li et al. Cell Stem Cell. 2012.

Abstract

Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that the histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining, and differentiation potential. They also have aberrant expression of the core transcription factors Nanog, Oct4, and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting the idea that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP-sequencing and transcriptome analyses further demonstrate that Mof is an integral component of the ESC core transcriptional network and that Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3K4 methylation at key regulatory loci, highlighting the complexity and interconnectivity of various chromatin regulators in ESCs.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1. Mof is down regulated during ESC differentiation

(A) Immunoblots for proteins from mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) as indicated on top. Antibodies were indicated on left. (B) Real-time PCR and (C) Immunoblot analyses for RA-induced ESC differentiation. (D) Real-time PCR and (E) Immunoblot analyses for ESC differentiation during EB formation. In (B, D), fold changes of each transcript relative to its expression in day 0 EB formation were presented. For C, E, β-actin was used as the loading control.

Figure 2
Figure 2. Mof is essential for ESC self-renewal

(A) Left, schematic for wild type and Mof knockout alleles. Genotyping primers (red arrows) were indicated. Right, genotyping results for wild type, floxed Mof alleles as well as Cre-ER™ by PCR. (B) Immunoblots for Mof and H4 K16ac in Mof +/+, Mof flox/+ and Mof flox/flox cells after 4-OHT treatment. Immunoblot for β-actin was used as the loading control. (C) Electron microscopy images of wild type (left) and Mof knockout nuclei (right). Densely stained heterochromatin was indicated by arrow. Scale bars, 2 micron. (D) Alkaline phosphatase staining of Mof +/+, Mof +/− and Mof −/− ESCs. (E) Light microscopy images of day 4 EB for Mof +/+, Mof +/− and Mof −/− ESCs. Scale bars, 0.5mm. Also see Supplementary Fig 1.

Figure 3
Figure 3. Mof regulates ESC core transcription network

(A) Heat map of expression of conserved Nanog and Oct4 joint targets (Loh et al., 2006) in WT and Mof −/− ESCs. Fold change of gene expression relative to WT ESCs was indicated at bottom. (B, C) Real-time PCR analyses for pluripotency (B) and differentiation genes (C) in Mof −/− and Mof +/+ ESCs as indicated. All mRNA levels were normalized against β-actin and were presented as relative expression in Mof null versus wild type ESCs. (D) ChIP for pluripotency genes that were down regulated in Mof −/− ESCs. (E) ChIP for differentiation genes that were up regulated in Mof −/− ESCs. For (D–E), primer sets were designed corresponding to Mof binding peaks identified by ChIP-seq (indicated in Supplementary Figure 6). The antibody was indicated on top. Signals for each experiment were normalized to 5% input. For (B–E), Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see Supplementary Fig 6.

Figure 4
Figure 4. ChIP-seq analysis of Mof binding sites in ESCs

(A) Chromosome-distribution of Mof binding peaks in mouse ESCs. Y-axis, count of ChIP-seq reads per kilo base. X-axis, chromosome name. (B) Distribution of Mof binding sites relative to nearest Refseq genes. Top, schematic for eight counting categories. Bottom, pie chart for percentage distribution of Mof peaks in each category. (C) Distribution of Mof peaks in a 12kb region from −2kb to +10kb around TSS (indicated by red arrow). Y-axis, percentage of Mof peaks relative to total Mof peaks within the defined region. X-axis, bin numbers, with each represents a 500bp region. Mof peaks were indicated as class I and class II peaks on bottom. (D) Comparison of Mof distribution within the defined 12kb region in mESCs (blue) and human CD4+ cells (red). TSS and Class I and II sites were indicated on bottom. Also see Supplementary Fig 4

Figure 5
Figure 5. Mof regulates Nanog specific ESC core transcription network

(A) Venn diagram for overlap of Mof bound genes (yellow) and genes that were either up regulated (blue) or down regulated (orange) in Mof −/− ESCs. Fisher’s exact test (p< 2.2×10−16) was performed to test for statistical significance of enrichment of up or down regulated genes with direct Mof binding. (B) GO term analyses for Mof down regulated genes (top) and Mof up regulated genes (bottom). Selected developmental pathways were presented and log p-value was used to rank the enrichment. (C) Top, Venn diagram for overlap of Mof targets with class I (green) or class II (yellow) binding sites. Bottom, a table for number of genes that were up or down regulated in each category. (D, E) GSEA of Mof targets with class I binding sites (D) or class II binding sites (E) and Nanog (left) or Oct4 (right) transcriptome (Ang et al., 2011). NES, normalized enrichment score; FDR (p value), false discovery rate. Also see Supplementary Figure 4 and 5.

Figure 6
Figure 6. Nanog overexpression rescues Mof null phenotypes in ESCs

(A) Immunoblots for Nanog, Mof and H4 K16ac in WT or Mof −/− ESCs rescued with control vector, wild type Mof, mutant Mof or Nanog. Antibodies used in the experiments were indicated on right. Both exogenous wild type and mutant Mof were Myc-tagged. (B) AP staining of wild type ESCs and Mof −/− ESCs expressing exogenous wild type Mof, Mof mutant or Nanog as indicated. Top, percentage of AP positive clones of Mof −/− and three rescue ESCs relative to wild type ESCs was presented. Means and standard deviations (as error bars) from two independent experiments were presented. Bottom, images (20×) for each cell lines as indicated on bottom. (C) Real-time PCR analyses for pluripotency (left) and differentiation genes (right) in Mof −/− and three rescue cell lines as indicated. All mRNA levels were normalized against β-actin and were presented as relative fold changes to wild type ESCs. Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see Supplementary Figure 3.

Figure 7
Figure 7. Mof regulates Wdr5 binding at key ESC loci

(A) Top, Venn diagram for direct physical overlap of binding peaks for Mof (blue), Wdr5 (pink) and H3 K4me3 (orange) (Ang et al., 2011). Total number and percentage of overlapping peaks relative to Wdr5, or H3 K4me3 peaks were summarized in the table below. (B) Distribution of Mof and Mof/Wdr5 joint peaks (top) or Mof/H3K4me3 joint peaks (bottom) as class I or class II peaks. Red arrow, TSS. Y-axis, % peaks relative to total peaks within the defined region. X-axis, each bin represents a 500bp region. (C) The box plots for fold changes in expression of total (white), Mof/Wdr5 (pink) and Mof only (blue) target genes. (D) The box plots for fold changes in expression of total (white), Mof/H3K4me3 (orange) and Mof only (blue) target genes. For (C, D), bottom and top of the boxes correspond to the 25th and 75th percentiles and the internal band is the 50th percentile (median). The plot whiskers extending outside the boxes correspond to the lowest and highest datum within 1.5 interquartile ranges. p-values were calculated using non-paired Wilcoxon tests as indicated. The number of genes in each category was indicated on bottom. Left, down regulated gene set. Right, up regulated gene set. (E) ChIP experiments for Wdr5 (top) and H3 K4me3 (bottom) at selected joint target genes in WT and Mof −/− ESCs. The antibodies used for ChIP were indicated on top. Signals for each experiment were normalized to 5% input. Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see Supplementary Figure 7.

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