RESUMO
Histone modifications impact final splicing decisions. However, there is little evidence of the driving role of these marks in inducing cell-specific splicing changes. Using CRISPR epigenome editing tools, we show in an epithelial-to-mesenchymal cell reprogramming system (epithelial-to-mesenchymal transition [EMT]) that a single change in H3K27ac or H3K27me3 levels right at the alternatively spliced exon is necessary and sufficient to induce a splicing change capable of recapitulating important aspects of EMT, such as cell motility and invasiveness. This histone-mark-dependent splicing effect is highly dynamic and mediated by direct recruitment of the splicing regulator PTB to its RNA binding sites. These results support a role for H3K27 marks in inducing a change in the cell's phenotype via regulation of alternative splicing. We propose the dynamic nature of chromatin as a rapid and reversible mechanism to coordinate the splicing response to cell-extrinsic cues, such as induction of EMT.
Assuntos
Processamento Alternativo/genética , Transição Epitelial-Mesenquimal/genética , Código das Histonas/genética , Acetilação , Sequência de Bases , Cateninas/metabolismo , Linhagem Celular , Cromatina/metabolismo , Éxons/genética , Feminino , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Tempo , delta CateninaRESUMO
Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.