RESUMO
While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Edição de Genes , Humanos , Masculino , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Estrutura Quaternária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.
Assuntos
Linhagem da Célula , Histonas/metabolismo , Lisina/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Diferenciação Celular/genética , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Genoma , Metilação , Camundongos , Neurônios/citologia , Edição de RNA , Sequências Reguladoras de Ácido Nucleico/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Microbial community profiles of recently formed hot spring systems ranging in temperatures from 57°C to 100°C and pH values from 2 to 4 in Hveragerði (Iceland) were analyzed with PhyloChip G3 technology. In total, 1173 bacterial operational taxonomic units (OTUs) spanning 576 subfamilies and 38 archaeal OTUs covering 32 subfamilies were observed. As expected, the hyperthermophilic (â¼100°C) spring system exhibited both low microbial biomass and diversity when compared to thermophilic (â¼ 60°C) springs. Ordination analysis revealed distinct bacterial and archaeal diversity in geographically distinct hot springs. Slight variations in temperature (from 57°C to 64°C) within the interconnected pools led to a marked fluctuation in microbial abundance and diversity. Correlation and PERMANOVA tests provided evidence that temperature was the key environmental factor responsible for microbial community dynamics, while pH, H2S, and SO2 influenced the abundance of specific microbial groups. When archaeal community composition was analyzed, the majority of detected OTUs correlated negatively with temperature, and few correlated positively with pH.
Assuntos
Archaea/genética , Bactérias/genética , DNA Arqueal/genética , DNA Bacteriano/genética , Fontes Termais/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Ribossômico 16S/genética , Acidobacteria/genética , Actinobacteria/genética , Alphaproteobacteria/genética , Betaproteobacteria/genética , Biodiversidade , Crenarchaeota/genética , Deltaproteobacteria/genética , Euryarchaeota/genética , Gammaproteobacteria/genética , Islândia , Filogenia , Proteobactérias/genética , Análise de Sequência de DNARESUMO
As part of an undergraduate microbiology course, a yellow-orange-pigmented, Gram-staining negative, rod-shaped, non-motile bacterial strain was isolated from a glass tank housing several red-spotted newts (Notophthalmus viridescens). The sequence of the 16S rRNA gene of this strain, designated KM(T), was 97.4-98.0â% similar to those of the type strains of Chryseobacterium luteum, C. shigense and C. vrystaatense, while the similarity levels for protein-coding genes were less than 94.7â% for rpoB, less than 92.1â% for groEL and less than 87.1â% for gyrB. These values are lower than for many other established distinct species. Polyphasic characterization and comparison to these relatives revealed that strain KM(T) was similar to other Chryseobacterium strains in that it contained MK-6 as its major respiratory quinone and phosphatidylethanolamine as the most abundant polar lipid, produced flexirubin-type pigments, oxidase and catalase and primarily contained the fatty acids iso-C15â:â0, iso-C17â:â1ω9c, iso-C17â:â0 3-OH and summed feature 3 (comprising C16â:â1ω6c and/or C16â:â1ω7c). Based on the results of this study, strain KM(T) represents a novel species, for which the name Chryseobacterium angstadtii sp. nov. is proposed. The type strain is KM(T) (â=âATCC BAA-2160(T)â=âNRRL B-59516(T)â=âKCTC 23297(T)).
