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. 2011 May 31;108(22):E149-58.
doi: 10.1073/pnas.1102223108. Epub 2011 May 5.

Genome-wide remodeling of the epigenetic landscape during myogenic differentiation

Patrik Asp  1 , Roy Blum, Vasupradha Vethantham, Fabio Parisi, Mariann Micsinai, Jemmie Cheng, Christopher Bowman, Yuval Kluger, Brian David Dynlacht
Affiliations

Genome-wide remodeling of the epigenetic landscape during myogenic differentiation

Patrik Asp et al. Proc Natl Acad Sci U S A. .

Abstract

We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Dynamic changes in Pol II binding and epigenetic marks associated with differentiation. The average ChIP-seq enrichment per 50 bp bin for the total population of genes in the four dynamic expression groups (SI Materials and Methods) were plotted +/-3 kb of the TSS and as a percentage of gene body length (see also Figs. S3 and S4). The y-axis shows the average log 2 of the enrichment.
Fig. 2.
Fig. 2.
HDM of Pol II and histone marks in myoblasts and myotubes. Density maps were generated for the region -3 kb to +9 kb of the TSS, as described in SI Materials and Methods, for (A) genes up-regulated during myogenesis and (B) permanently repressed genes.
Fig. 3.
Fig. 3.
Characterization H3K27me3 localization and its role in myogenesis (A, B) Patterns of H3K27me3 and its anticorrelation with H3K36me3 across a region of chromosome 10 (Left) and the Myh loci (Right). The y-axis shows the log 2 of the enrichment. (C) ChIP-seq localization of H3K27me3, p300, H3K4me1/3, and Pol II on Myog. Blue bars at the bottom indicate fragments tested for enhancer activity. The y-axis shows the log 2 of the enrichment. (D) Luciferase assay testing the enhancer activity of fragments up- and downstream of Myog coding region. (E) Analysis of extracts and chromatin after miRNA-mediated Suz12 depletion in myoblasts and myotubes showing effect on indicated proteins and global H3K27me3 levels by Western blotting (Left) and recruitment to chromatin by ChIP (Right). (F) Quantitative chromatin IP (qChIP) showing the effect of Suz12 depletion on H3K37me3 levels at Myog genomic regions shown in panel C.
Fig. 4.
Fig. 4.
Dynamic versus stable H3K27 deposition and transcriptional repression (A) RT-qPCR showing the effect of Suz12 depletion on expression of Myog and muscle differentiation genes. Relative expression of each gene is plotted with respect to untreated myoblasts. (B) Impact of miRNA-mediated Suz12 depletion on myogenic differentiation. After depletion, cells were stained for MHC (red) and DAPI, and numbers indicate the fold-difference in total MHC fluorescence normalized to DAPI staining for untreated cells (set to unity) as compared to Luc miRNA (control) and Suz12 miRNA-transduced cells. The size bar in the lower right figure represents 295 μm. (C) H3K27me3 and Bmi1 qChIP on the Myog -1.5 kb region before and after Suz12 knock-down. (D) RT-qPCR showing the effect of Suz12 depletion on mRNA levels of three H3K27me3 Class 1 genes that are up-regulated during differentiation. (E) qChIP for H3K27me3 and Bmi1 occupancy before and after Suz12 knock-down: class I genes up-regulated during differentiation (Left) and permanently repressed (class II) genes (Right) are shown. Error bars for all qChIP and RT-qPCR data shown in all figures represent the standard error of the mean (SEM). Student’s t test was performed to indicate significance: * indicates p-values < 0.05 and ** < 0.01, respectively.
Fig. 5.
Fig. 5.
PRC1 and PRC2 depletion have opposite effects on myogenic differentiation. Western blots showing miRNA-mediated Bmi1 (A) and combined Bmi1/Suz12 (C) depletion in myoblasts and myotubes and the effect on global H3K27me3 levels. (B) The effect of miRNA-mediated depletion of Bmi1 and combined Bmi1/Suz12 depletion on myogenic differentiation. (−) indicates uninfected control. Numbers in white represent the extent of differentiation as calculated in Fig. 4A. The size bar in the lower right figure represents 295 μm. (D) qRT-PCR showing suppression of Ring1B expression. (E) The effect of miRNA-mediated Ring1B depletion on myogenic differentiation. Numbers in white represent the extent of differentiation as calculated in Fig. 4B. The size bar in the lower right figure represents 295 μm. MB, myoblasts; MT, myotubes.
Fig. 1.
Fig. 1.
We performed a genome-wide examination of the epigenetic changes involved in converting precursor cells (myoblasts) to differentiated muscle, and found that cells undergoing terminal differentiation underwent large-scale loss of one particular histone modification, acetylation. Other genes lost a second type of modification, methylation, whereas other genes gained this mark. We identified distinct classes of genes that could be activated as cells differentiate to myotubes (Left) whereas another class of genes (Right) that specify nonmuscle fates recruited the Bmi1 component of PRC1 and remained in a permanently “locked” position, preventing their activation.
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