RESUMO
DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome-wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue-specific manner. The isoform-specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5-hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform-specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3-mediated regulation of developmental genes.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismoRESUMO
Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.
Assuntos
Retículo Endoplasmático/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Argonautas/análise , RNA Helicases DEAD-box/análise , Retículo Endoplasmático/química , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Ligação a RNA/análise , Ribonuclease III/análiseRESUMO
Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.
Assuntos
Cromatina , Histonas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Células-Tronco Embrionárias , Histonas/química , Histonas/genética , Histonas/metabolismo , CamundongosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.