RESUMO
The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.
Assuntos
Histonas , RNA Longo não Codificante , Acetilação , Acetiltransferases/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos , Histonas/genética , Histonas/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Smad7 has been identified as a negative regulator of the transforming growth factor TGF-ß pathway by direct interaction with the TGF-ß type I receptor (TßR-I). Although Smad7 has also been shown to play TGF-ß unrelated functions in the cytoplasm and in the nucleus, a comprehensive analysis of its nuclear function has not yet been performed. Here, we show that in ESCs Smad7 is mainly nuclear and acts as a general transcription factor regulating several genes unrelated to the TGF-ß pathway. Loss of Smad7 results in the downregulation of several key stemness master regulators, including Pou5f1 and Zfp42, and in the upregulation of developmental genes, with consequent loss of the stem phenotype. Integrative analysis of genome-wide mapping data for Smad7 and ESC self-renewal and pluripotency transcriptional regulators revealed that Smad7 co-occupies promoters of highly expressed key stemness regulators genes, by binding to a specific consensus response element NCGGAAMM. Altogether, our data establishes Smad7 as a new, integral component of the regulatory circuitry that controls ESC identity.
Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Proteína Smad7/genética , Ativação Transcricional , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Deleção de Genes , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.
Assuntos
Endoderma , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Endoderma/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Diferenciação Celular , Linhagem da Célula , Metilação de DNARESUMO
There is increasing evidence that long noncoding RNAs (lncRNAs) are among the main regulatory factors of stem cell maintenance and differentiation. They act through various mechanisms and interactions with proteins, DNA, and RNA. This heterogeneity in function increases the capabilities of the lncRNome toolkit but also makes it difficult to predict the function of novel lncRNAs or even rely on biological information produced in animal models. As lncRNAs are species- and tissue-specific, the recent technical advances in self-renewal and differentiation of human embryonic stem cells (ESCs) make these cells the ideal system to identify key regulatory lncRNAs and study their molecular functions. Here we provide an overview of the functional versatility of lncRNA mechanistic heterogeneity in regulating pluripotency maintenance and human differentiation.