RESUMO
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
Assuntos
Linfócitos B/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Ativação Linfocitária , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos B/imunologia , Linhagem Celular , Cromatina/química , Cromatina/genética , Metilação de DNA , Epigênese Genética , Genótipo , Histonas/química , Imunidade Humoral , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação de Ácido Nucleico , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Imagem Individual de Molécula , Relação Estrutura-Atividade , Fatores de Tempo , Transcrição GênicaRESUMO
BACKGROUND: Ectodysplasin-A appears to be a critical component of branching morphogenesis. Mutations in mouse Eda or human EDA are associated with absent or hypoplastic sweat glands, sebaceous glands, lacrimal glands, salivary glands (SMGs), mammary glands and/or nipples, and mucous glands of the bronchial, esophageal and colonic mucosa. In this study, we utilized EdaTa (Tabby) mutant mice to investigate how a marked reduction in functional Eda propagates with time through a defined genetic subcircuit and to test the proposition that canonical NFkappaB signaling is sufficient to account for the differential expression of developmentally regulated genes in the context of Eda polymorphism. RESULTS: The quantitative systems analyses do not support the stated hypothesis. For most NFkappaB-regulated genes, the observed time course of gene expression is nearly unchanged in Tabby (EdaTa) as compared to wildtype mice, as is NFkappaB itself. Importantly, a subset of genes is dramatically differentially expressed in Tabby (Edar, Fgf8, Shh, Egf, Tgfa, Egfr), strongly suggesting the existence of an alternative Eda-mediated transcriptional pathway pivotal for SMG ontogeny. Experimental and in silico investigations have identified C/EBPalpha as a promising candidate. CONCLUSION: In Tabby SMGs, upregulation of the Egf/Tgfalpha/Egfr pathway appears to mitigate the potentially severe abnormal phenotype predicted by the downregulation of Fgf8 and Shh. Others have suggested that the buffering of the phenotypic outcome that is coincident with variant Eda signaling could be a common mechanism that permits viable and diverse phenotypes, normal and abnormal. Our results support this proposition. Further, if branching epithelia use variations of a canonical developmental program, our results are likely applicable to understanding the phenotypes of other branching organs affected by Eda (EDA) mutation.
Assuntos
Ectodisplasinas/genética , Receptor Edar/genética , NF-kappa B/metabolismo , Glândulas Salivares/embriologia , Transdução de Sinais/genética , Animais , Ectodisplasinas/metabolismo , Receptor Edar/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Biológicos , Morfogênese/fisiologia , NF-kappa B/genética , Fenótipo , Glândulas Salivares/crescimento & desenvolvimentoRESUMO
We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min(-1), much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.
Assuntos
RNA Polimerase II/genética , Transcrição Gênica , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Cinética , Fosforilação , Fotoquímica , RNA Polimerase II/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebra Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase I/antagonistas & inibidores , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Nucléolo Celular/genética , Nucléolo Celular/patologia , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/efeitos da radiaçãoRESUMO
BACKGROUND: A quantitative understanding of human folate metabolism is needed. OBJECTIVE: The objective was to quantify and interpret human folate metabolism as it might occur in vivo. DESIGN: Adults (n = 13) received 0.5 nmol [(14)C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. (14)C was measured in plasma, erythrocytes, urine, and feces for >/=40 d. Kinetic modeling was used to analyze and interpret the data. RESULTS: According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of (14)C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 + 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (>99%), most of the visceral folate was pteroylpolyglutamate (>98%), total body folate was 225 micromol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate. CONCLUSIONS: Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing.