The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST

The repressor element silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). Effector proteins of REST are C-terminal domain small phosphatases (CTDSPs), which reduce polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA remain unknown. Here we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. We further show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes including miRs required for the induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.

Telomere dysfunction cooperates with epigenetic alterations to impair murine embryonic stem cell fate commitment

The precise relationship between epigenetic alterations and telomere dysfunction is still an extant question. Previously, we showed that eroded telomeres lead to differentiation instability in murine embryonic stem cells (mESCs) via DNA hypomethylation at pluripotency-factor promoters. Here, we uncovered that telomerase reverse transcriptase null (Tert-/-) mESCs exhibit genome-wide alterations in chromatin accessibility and gene expression during differentiation. These changes were accompanied by an increase of H3K27me3 globally, an altered chromatin landscape at the Pou5f1/Oct4 promoter, and a refractory response to differentiation cues. Inhibition of the Polycomb Repressive Complex 2 (PRC2), an H3K27 tri-methyltransferase, exacerbated the impairment in differentiation and pluripotency gene repression in Tert-/- mESCs but not wild-type mESCs, whereas inhibition of H3K27me3 demethylation led to a partial rescue of the Tert-/- phenotype. These data reveal a new interdependent relationship between H3K27me3 and telomere integrity in stem cell lineage commitment that may have implications in aging and cancer.

ATRX Contributes to MeCP2-Mediated Pericentric Heterochromatin Organization during Neural Differentiation

Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2, mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH.