Regulation of adult stem cell function
The maintenance of tissue mass in postnatal animals and regeneration following normal ‘wear and tear’ in large part depends upon appropriate adult stem cell function. Activated adult stem cells have two alternative fates: i, differentiation to regenerate damaged tissue and ii, self-renewal to replenish the stem cell population. The correct balance of these is essential; cell fate ‘decisions favouring self-renewal can lead to tumourigenesis whereas insufficient differentiation will induce tissue loss. Projects in our lab focus on the signalling pathways that regulate adult stem cell fate and control epigenetic mechanisms; these change chromatin structure and hence potential for gene expression. Adult muscle stem cells provide an excellent paradigm for many adult stem cell lineages and we have recently developed elegant experimental systems that can combine the strengths of genetically modified animals and the isolation of primary cells with sophisticated cell signalling/imaging techniques, next generation sequencing and regeneration models in order to recapitulate in vivo physiology as faithfully as possible.
Muscle consists of postmitotic multinucleated myofibres, the number of which is determined before birth. All postnatal muscle growth and repair depends on the hypertrophy or replacement of the existing fibre population, provided in large part by adult muscle stem cells (called satellite cells). In common with many other stem cell lineages, satellite cells occupy a specific location or niche, which lies between (and in close contact with) the basal lamina and the muscle fibre membrane. The niche provides a mechanism to regulate access of signals to the satellite cell or provide unique signals.
Activated satellite cells undergo a strictly orchestrated process of differentiation to induce growth or repair/regenerate damaged muscle. Mechanisms for satellite cell self-renewal are controversial and may occur via symmetric or asymmetric cell division of stem cells, or dedifferentiation of partially committed myoblasts.
Satellite cells are investigated in vivo or are isolated satellite cells via FACS and studied in vitro.
The importance of the gene silencing complex, polycomb repressive complex 2 (PRC2) in satellite cells
Cell lineage determination requires progressive and step-wise silencing of gene expression, such that only those loci that encode tissue-specific genes are permissive for transcriptional regulation. Reversible histone N-terminal modifications provide sophisticated epigenetic regulation of genomic reprogramming, ultimately via changes in chromatin structure, to enable the formation of compact and inaccessible heterochromatin or relaxed euchromatin. H3K27me3 (histone 3 lysine27 trimethylation), has a key role in gene silencing and acts as a mark to induce further mechanisms of repression. H3K27me3 is indirectly counteracted by H3K4me3 (catalysed by Trithorax family proteins) and H3K27 acetylation. Even though additional histone variants have a function in gene regulation, H3K27me3 and H3K4me3 have a fundamental role in controlling lineage-specifying gene expression and the maintenance of appropriate patterns of gene expression throughout development.
H3K27me3 is mediated by PRC2, a multiprotein complex whose catalytic subunit is Ezh2. In order to investigate the importance of H3K27me3-mediated silencing in adult muscle stem cells, we generated conditional muscle-specific Ezh2-null mice using the Cre-loxP system; Pax7-Cre mice were crossed with Ezh2 floxed mice to generate a strain that was Ezh2-null in a Pax7 expression-dependent manner, i.e. in satellite cells, and all subsequent stages of muscle formation (termed Ezh2P). In addition, Ezh2 floxed mice were crossed with a myogenin-Cre strain to generate mice with deleted muscle Ezh2 from the onset of terminal differentiation, i.e. satellite cells expressed Ezh2 but it was deleted in myoblasts from the onset of myogenin expression (termed Ezh2M).
The Ezh2P mice were significantly growth retarded, due to decreased muscle mass. Interestingly, their muscle phenotype resembled that of aged mice, notably with reduced satellite cell number and proliferative ability, and an impaired inability to regenerate. In contrast, Ezh2M mice were apparently normal (Woodhouse et al 2013). This implies that PRC2-mediated silencing is essential for early satellite cell fate decisions but is not important during differentiation.
This conclusion was supported by ChIP-seq analysis in wild type mice for H3K27me3 and H3K4me3. Non-muscle lineage genes were marked by H3K27me3 but not myogenic genes, such as myogenin and myosin heavy chain. However, genes known to be expressed in satellite cells were marked by H3K4me3 e.g. Pax7.
Intriguingly, the cell cycle inhibitor p16Ink4 (for simplicity, subsequently called p16), which is heavily marked by H3K27me3 in wild type mice, had reduced H3K27me3 in Ezh2P mice, and was significantly upregulated. This may in large part account for the reduced proliferative ability of Ezh2P satellite cells.
p38α is a member of the mitogen activated protein kinase family of signalling molecules. Originally its activity was associated with cell stress but it is now known to have a much wider role in cell biology. p38α MAPK activity is substantially upregulated during myogenesis, and regulates the expression of key myogenic genes; it is also associated with cell cycle exit, essential for myogenic progression; it may thus represent a pivotal ‘gateway’ to muscle stem cell fate decisions. Germline knockout of p38α is embryonic lethal and in order to investigate its importance in adult satellite cells, we generated a conditional null strain by crossing Pax7-Cre with p38 floxed mice, termed p38αKO (control mice termed p38αfl).
p38αKO mice were slightly growth retarded compared with littermates, had the same number of myofibres but of decreased cross-sectional area; however they had significantly more satellite cells. When challenged to regenerate, their proliferative response was enhanced, generating more satellite cells; differentiation was delayed but ultimately not reduced. Thus, p38α has a role in dictating the balance of stem cell self-renewal and differentiation in the early stages of satellite cell activation towards self-renewal (Brien et al 2013). This may have significance during ageing, when satellite cell number can decrease, and their activation/proliferation are impaired.
RNA-seq was performed to define transcriptional profiles in satellite cells from resting and regenerating muscle. p38αKO exhibited a different transcriptional profile compared with the control p38αfl mice.
A & B. Scatter plots comparing global gene expression profiles between p38αfl and p38αKO satellite cell RNA isolated from resting muscle (A) and regenerating muscle (B). R-value represents coefficient of correlation. Differentially expressed genes (assessed by intensity difference, p <0.05) are circled. C. Representation of differentially expressed genes in p38α-null satellite cells isolated from resting muscle (top) and regenerating muscle (bottom). Each bar represents one gene, a bar under the axis implies downregulation and above the axis implies upregulation. D. Heat maps of differentially expressed genes from resting (left) and regenerating (right) muscle. Each row represents one gene, with red representing high expression through green to blue representing low expression. Upregulated and downregulated genes are separated by a black line. E. Gene ontology analysis of genes downregulated in p38αKO satellite cells isolated from resting muscle. Fold enrichment and gene count under each term shown. F. Gene ontology analysis of genes downregulated in p38αKO satellite cells isolated from regenerating muscle. Fold enrichment and gene count under each term shown.
- 1. Importance of p16 in ageing and ‘pre-senescence’.
- 2. Function of the Tis11 family of RNA binding proteins in muscle homeostasis.
- 3. Signalling to chromatin; modulating role of H3S28 phosphorylation on H3K27me3 function.