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. 2013 Jul 11;4(1):189-204.
doi: 10.1016/j.celrep.2013.05.043. Epub 2013 Jun 27.

Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging

Ling Liu  1 , Tom H Cheung, Gregory W Charville, Bernadette Marie Ceniza Hurgo, Tripp Leavitt, Johnathan Shih, Anne Brunet, Thomas A Rando
Affiliations

Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging

Ling Liu et al. Cell Rep. .

Abstract

The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.

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Figures

Figure 1
Figure 1. Microarray Analysis of QSCs and ASCs during Muscle Regeneration
(A) Clustered heatmap of the gene expression profiles of QSCs and ASCs prior to and 36, 60, and 84 hr after muscle injury. Three replicates were used for each sample. (B–D) Box and whisker plots of the expression value (Log2 intensity) and GO analysis of genes specific to QSCs and ASCs 36 and 60 hr postinjury. (E) Transcription network in QSCs. Pathway analysis of the transcription factors that expressed at a substantially higher level in QSCs than ASCs was performed with the Ingenuity software package. Interactions were found between 59 transcription factors. In the map, transcription factors with the most interactions were placed in the center, and those with the least interactions were placed at the periphery. See also Figures S1 and S2 and Table S1.
Figure 2
Figure 2. The Global Profiles of H3K4me3 and H3K27me3 in QSCs and ASCs
(A) Immunofluorescence of Pax7 and H3K4me3 on freshly isolated single fibers. The arrows indicate a fiber-associated QSC that is positive for both Pax7 and H3K4me3. (B) Distribution of H3K4me3 around the TSSs in QSCs and ASCs. Normalized tag intensity of H3K4me3 3 kb upstream and downstream of the TSSs across the genome is shown in the plot. (C) Venn diagram of genes marked by H3K4me3 at their TSSs in QSCs and ASCs. (D) H3K4me3 and H3K27me3 distribution at the TSS of genes that expressed at high levels in QSCs but were downregulated in ASCs. The H3K4me3 and H3K27me3 profiles of representative genes are shown in the top panels, and the level of changes in their expression upon SC activation revealed by microarray analysis is shown in the bottom bar graphs. Error bars represent SDs. (E) Distribution of H3K27me3 around the TSSs in QSCs and ASCs. Normalized tag intensity of H3K27me3 3 kb upstream and downstream of the TSSs across the genome is shown in the plot. (F) Immunofluorescence of freshly isolated myofibers with Pax7 and H3K27me3 antibodies (top panels) and fibers cultured for 2 days ex vivo with MyoD and H3K27me3 antibodies (bottom panels). Images were acquired with the same exposure and gain. Fiber-associated SCs are indicated by the arrows, and all other DAPI+ nuclei are myonuclei within the fiber. See also Figures S3 and S4 and Table S2.
Figure 3
Figure 3. Identification of H3K4me3 and H3K27me3 Bivalent Chromatin Domains in QSCs
(A) Comparison of the proportion of genes marked by one of the four H3K4me3 and H3K27me3 patterns, H3K4me3 only (K4), H3K27me3 only (K27), both (bivalent), and neither (none), in ESCs and QSCs. (B) Box and whisker plot of the expression level of genes that were found bivalent at the TSS in comparison to all genes in QSCs. (C) Venn diagram of genes with bivalent domains at the TSS in QSCs and ASCs. Among the 1,892 genes marked by bivalent domains in QSCs, 1,760 were also found in ASCs. (D) Box and whisker plot of the expression level of genes bivalent at the TSS in QSCs and ASCs of different time points in muscle regeneration. (E) Venn diagram of genes with bivalent domains at TSSs in ESCs and QSCs. (F) H3K4me3 and H3K27me3 patterns in QSCs of genes that are marked by bivalent domains in ESCs. The middle pie chart depicts the proportion of bivalent ESC genes that exhibited different H3K4me3 and H3K27me3 marks in QSCs. Among all genes that are bivalent in ESCs, 46% were found bivalent in QSCs (orange), and another 46% were found to be H3K4me3 only (blue). GO analysis of genes that were found bivalent in QSCs is shown by the bar graph on the left panel. The heatmap on the right panel depicts the expression level in QSCs and ASCs of representative genes that are bivalent at TSS in ESCs but H3K4me3 only in QSCs. The top panel of the heatmap shows genes known as QSC markers, and the bottom panel shows 15 of all 411 genes that encode glycoproteins (p = 10−39). See also Figure S5.
Figure 4
Figure 4. The Hox Loci Exhibit a Mosaic Pattern of Genes Marked by Bivalent Domains
(A–D) The distribution of H3K4me3 and H3K27me3 at the four Hox loci in QSCs and ESCs. (E) Box and whisker plot of the expression levels of Hox genes that are bivalent at the TSSs and those that are marked by H3K4me3 only in QSCs. See also Table S3.
Figure 5
Figure 5. Chromatin Patterns of Myogenic Genes
(A) The distribution of H3K4me3 and H3K27me3 at the TSSs of myogenic transcription factors Pax3, Pax7, Myf5, MyoD, Myogenin (Myog), and Myf6. (B) GO analysis of genes that were neither H3K4me3 nor H3K27me3 in QSCs but acquired H3K4me3 in ASCs. (C) Box and whisker plot of the expression levels of all genes associated with the GO terms listed in (B). (D) The distribution of H3K4me3 and H3K36me3 on representative genes associated with the GO terms listed in (B).
Figure 6
Figure 6. Changes in the H3K4me3 and H3K27me3 Profiles in QSCs with Age
(A and B) FACS isolation of QSCs from hindlimb muscles of (A) 2-month-old and (B) 24-month-old mice. QSCs are shown in orange. The number indicates the percentage of QSCs among the total population of mononucleated cells in the muscle. (C) Distribution of H3K4me3 around the TSSs in young and old QSCs. Normalized tag intensity of H3K4me3 3 kb upstream and downstream of the TSSs across the genome is shown in the plot (p < 0.0001). (D) H3K4me3 intensity plot at TSSs in young and old QSCs. (E) Distribution of H3K27me3 around the TSSs in young and old QSCs. Normalized tag intensity of H3K27me3 3 kb upstream and downstream of the TSSs across the genome is shown in the plot (p < 0.0001). (F) H3K27me3 intensity plot at TSSs in young and old QSCs. See also Figure S6.
Figure 7
Figure 7. Histone Genes Acquire H3K27me3 in QSCs with Age and Exhibit a Reduced Level of Expression
(A and B) Distribution of H3K4me3 and H3K27me3 in histone genes on (A) chromosome 3 and (B) chromosome 13 in young and old QSCs. (C) Box and whisker plot of the expression levels of all histone genes in QSCs from young and old mice. (D) The histone genes that exhibited a reduction in expression with age. Error bars represent SDs. See also Table S4.
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