Twist-1 Induces Ezh2 Recruitment Regulating Histone Methylation along the Ink4A/Arf Locus in Mesenchymal Stem Cells

Abstract

eThe main impairment to tissue maintenance during aging is the reduced capacity for stem cell self-renewal over time due to senescence, the irreversible block in proliferation. We have previously described that the basic helix-loop-helix (bHLH) transcription factor Twist-1 can greatly enhance the life span of bone marrow-derived mesenchymal stem/stromal cells (MSCs). In the present study, we show that Twist-1 potently suppresses senescence and the Ink4A/Arf locus with a dramatic decrease in the expression of p16 and to some extent a decrease in p14. Furthermore, the polycomb group protein and histone methyltransferase Ezh2, which suppresses the Ink4A/Arf locus, was found to be induced by Twist-1, resulting in an increase in H3K27me3 along the Ink4A/Arf locus, repressing transcription of both p16/p14 and senescence of human MSCs. Furthermore, Twist-1 inhibits the expression of the bHLH transcription factor E47, which is normally expressed in senescent MSCs and induces transcription of the p16 promoter. Reduced Twist-1 wild-type expression and function in bone cells derived from SaethreChotzen patients also revealed an increase in senescence. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferases as well as the expression of other bHLH factors. C lonogenic bone marrow (BM)-derived mesenchymal stem/ stromal cells (BMSCs) are a heterogeneous mix of stem cells and committed progenitors that vary in their morphology, proliferation, and differentiation potential (9-12, 18, 23). This is attributed to the existence of a developmental hierarchy of stromal cellular differentiation, comprised largely of committed progenitor cells and a minor population of self-renewing multipotent stem cells capable of differentiating into adipocytes, osteoblasts (OB), chondrocytes, and myocytes (6, 21).Adult somatic stem cells including BMSCs exhibit an increased propensity for cellular senescence during ex vivo expansion, which is accompanied by a reduction in self-renewal and multidifferentiation potential. Senescence is a "fail-safe" mechanism which is activated in response to various stresses such as DNA damage, oxidative damage, and oncogene activation (13, 24). During senescence, cells fail to respond to mitogenic stimuli, undergo dramatic changes in chromatin structure and gene expression, become enlarged and flattened, and remain viable yet nondividing. The Ink4A/Arf locus (the locus of p14Arf and p16) is central to cellular senescence as it signals via p53 and retinoblastoma (Rb) to inhibit proliferation. p14ARF activates p53 by sequestering Mdm2, an E3 ubiquitin ligase, to the nucleolus, thereby preventing the Mdm2-mediated targeting of p53 to proteolytic degradation (16). p53 subsequently activates p21 CIP1/WAF1, which inhibits the cell cycle. Moreover, when activated, p16 inhibits the cyclin-dependent kinases CDK4/6 and leads to the hypophosphorylation of Rb, which in turn represses E2F-mediated cell cycle progression (2,22).Transcr...

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2

“…The upregulation of such noncollagenous ECM proteins is expected from accelerated maturation of preosteoblastic progenitors, and these findings corroborate our data obtained with human AMSCs. Other prominently up-regulated non-ECM genes are known Ezh2 target genes, including the senescence-associated cyclin-dependent kinase (CDK) inhibitory protein p16/ CDKN2A (26) and the CDK inhibitor p57/CDKN1C (27) (Fig. 9e), as well as homeobox transcription factors (51) (Fig.…”

Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

“…DNA was sheared with probe sonication on ice. Sonicated samples were used for immunoprecipitation [36].…”

Significance of Epigenetic Landscape in Cartilage Regeneration from the Cartilage Development and Pathology Perspective

“…A basic helix-loop-helix transcription factor, Twist-1, can also induce Ezh2. This induction resulted in an increase in H3K27me3, repression of the transcription of p16/p14, and the senescence of human BMSCs [99]. DNMTs regulated the cellular senescence of human umbilical cord MSCs through the histone marks at genomic regions of Ezh2-targeting miRNAs and p16 and p21 promoter regions [100].…”