Regulación génica por REX1 en células troncales. Epigenética de las dianas genómicas.

Los mecanismos moleculares que están detrás de las funciones reguladoras del factor de transcripción REX1 durante el desarrollo embrional son en muchos aspectos todavía una incógnita. Recientes descubrimientos revelaron que numerosas dianas genómicas de REX1 poseen marcas epigenéticas indicativas de improntas genéticas. En el presente trabajo se llevó a cabo la puesta a punto de ensayos que permiten estudiar el nivel de metilación asociado a dinucleótidos CpG en loci diana de REX1: Nespas, Chd2, PisD y Ly6c1. Para ello, DNA genómico de células madre embrionarias (ES) de ratón fue tratado con bisulfito y se diseñaron experimentos de amplificación para cada locus. Un primer intento de amplificación mediante el uso de un primer de secuencia universal (M13) produjo productos poco o no aptos para la cuantificación mediante pirosecuenciación. Como solución se decide utilizar uno de los primers directamente biotinilado. Resultados de un primer ensayo en el locus Nespas mostró aproximadamente la mitad de metilación en células ES carentes de Rex1 que en células ES wild type (wt). A falta del estudio de loci adicionales para una posible generalización, esta observación sugiere para REX1 un papel protector de la metilación de sus sitios de unión

SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability

Summary: During genome duplication, replication forks (RFs) can be stalled by different obstacles or by depletion of replication factors or nucleotides. A limited number of histone post-translational modifications at stalled RFs are involved in RF protection and restart. Provided the recent observation that the SIN3A histone deacetylase complex reduces transcription-replication conflicts, we explore the role of the SIN3A complex in protecting RFs under stressed conditions. We observe that Sin3A protein is enriched at replicating DNA in the presence of hydroxyurea. In this situation, Sin3A-depleted cells show increased RF stalling, H3 acetylation, and DNA breaks at stalled RFs. Under Sin3A depletion, RF recovery is impaired, and DNA damage accumulates. Importantly, these effects are partially dependent on the MUS81 endonuclease, which promotes DNA breaks and MRE11-dependent DNA degradation of such breaks. We propose that chromatin deacetylation triggered by the SIN3A complex limits MUS81 cleavage of stalled RFs, promoting genome stability when DNA replication is challenged

DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops

Summary: Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of γH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms