RESUMEN
TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB-TFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed 'bootprints'. Global analysis reveals that the TFIIIB-TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.
Asunto(s)
ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción TFIII/metabolismo , Sitios de Unión , Centrómero/metabolismo , Cromatina/química , Huella de ADN/métodos , Nucleosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Regiones Terminadoras GenéticasRESUMEN
Alterations in DNA methylation have been reported to occur during development and aging; however, much remains to be learned regarding post-natal and age-associated epigenome dynamics, and few if any investigations have compared human methylome patterns on a whole genome basis in cells from newborns and adults. The aim of this study was to reveal genomic regions with distinct structure and sequence characteristics that render them subject to dynamic post-natal developmental remodeling or age-related dysregulation of epigenome structure. DNA samples derived from peripheral blood monocytes and in vitro differentiated dendritic cells were analyzed by methylated DNA Immunoprecipitation (MeDIP) or, for selected loci, bisulfite modification, followed by next generation sequencing. Regions of interest that emerged from the analysis included tandem or interspersed-tandem gene sequence repeats (PCDHG, FAM90A, HRNR, ECEL1P2), and genes with strong homology to other family members elsewhere in the genome (FZD1, FZD7 and FGF17). Our results raise the possibility that selected gene sequences with highly homologous copies may serve to facilitate, perhaps even provide a clock-like function for, developmental and age-related epigenome remodeling. If so, this would represent a fundamental feature of genome architecture in higher eukaryotic organisms.
Asunto(s)
Envejecimiento/genética , Metilación de ADN , Genoma Humano , Adulto , Células Cultivadas , Femenino , Genes , Humanos , Inmunoprecipitación , Recién Nacido , Masculino , Familia de Multigenes , Análisis de Secuencia de ADNRESUMEN
The centromeres of budding yeast are ~120 bp in size and contain three functional elements: an AT-rich region flanked by binding sites for Cbf1 and CBF3. A specialized nucleosome containing the H3 variant Cse4 (CenH3) is formed at the centromere. Our genome-wide paired-end sequencing of nucleosomal DNA reveals that the centromeric nucleosome contains a micrococcal nuclease-resistant kernel of 123-135 bp, depending on the centromere, and is therefore significantly shorter than the canonical nucleosome. Unlike canonical nucleosomes, the centromeric nucleosome is essentially perfectly positioned. The entire centromere is included, together with at least 1 bp of DNA upstream of the Cbf1 site and at least 4 bp downstream of the CBF3 site. The fact that the binding sites for Cbf1 and CBF3 are included within the centromeric nucleosome has important implications for models of the centromeric nucleosome and for kinetochore function.
Asunto(s)
Centrómero/genética , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Sitios de Unión/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Fúngico/genética , Genómica/métodos , Histonas/genética , Histonas/metabolismo , Cinetocoros/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
We have used paired-end sequencing of yeast nucleosomal DNA to obtain accurate genomic maps of nucleosome positions and occupancies in control cells and cells treated with 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. In control cells, 3AT-inducible genes exhibit a series of distinct nucleosome occupancy peaks. However, the underlying position data reveal that each nucleosome peak actually consists of a cluster of mutually exclusive overlapping positions, usually including a dominant position. Thus, each nucleosome occupies one of several possible positions and consequently, different cells have distinct local chromatin structures. Induction results in a major disruption of nucleosome positioning, sometimes with altered spacing and a dramatic loss of occupancy over the entire gene, often extending into a neighbouring gene. Nucleosome-depleted regions are generally unaffected. Genes repressed by 3AT show the same changes, but in reverse. We propose that yeast genes exist in one of several alternative nucleosomal arrays, which are disrupted by activation. We conclude that activation results in gene-wide chromatin remodelling and that this remodelling can even extend into the chromatin of flanking genes.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Genes Fúngicos , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fosfatasa Ácida/genética , Isomerasas Aldosa-Cetosa/genética , Amitrol (Herbicida)/farmacología , Arginasa/genética , Cromatina/química , Ensamble y Desensamble de Cromatina , Regulación Fúngica de la Expresión Génica , Sitios Genéticos , Regiones Promotoras Genéticas , Origen de Réplica , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Activación TranscripcionalRESUMEN
In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His-->Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.
Asunto(s)
Diferenciación Celular/fisiología , Histona Desacetilasas/fisiología , Isoenzimas/fisiología , Células Madre Pluripotentes/citología , Animales , Dominio Catalítico , Separación Celular , ADN Complementario , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Humanos , Inmunohistoquímica , Ratones , Mutagénesis , Transducción Genética , Células Tumorales CultivadasRESUMEN
The receptor encoded by the human TLR3 gene recognizes double-strand RNAs (dsRNAs) associated with viral infection. TLR3 expression is strongly activated upon differentiation of monocytes to dendritic cells, and can be further stimulated by the dsRNA analog polyinosine:polycytosine (PI:C). We report evidence for developmental regulation of the TLR3 gene. In dendritic cells derived from cord blood, both differentiation- and PI:C-associated TLR3 transcriptional activation are impaired as compared to cells from adults. Consistent with relative expression patterns, chromatin states and remodeling differ between newborn and adult samples. TLR3 expression in newborn dendritic cells exhibits heterocellularity and allelic imbalance (skewing), features characteristic of cis-acting epigenetic control. These findings reveal a new source for variability in innate immune system function and provide a model for further study of perinatal epigenetic transitions during development.
Asunto(s)
Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Receptor Toll-Like 3/genética , Adulto , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Alelos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Inmunoprecipitación de Cromatina , Cromatografía Líquida de Alta Presión , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Epigénesis Genética/efectos de los fármacos , Sangre Fetal/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Heterocigoto , Histonas/metabolismo , Humanos , Recién Nacido , Poli I-C/farmacología , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Toll-Like 3/metabolismoRESUMEN
Substantial evidence has accumulated linking epigenome change to alterations in stem cell function during postnatal development and aging. Yet much remains to be learned about causal relationships, and large gaps remain in our understanding of epigenome-transcriptome interactions. Here we investigate structural features of large histone H3K27me3-enriched regions in human stem cell-like monocytes and their dendritic cell derivatives, where the H3K27me3 modification is considered to demarcate Polycomb (PcG) domains. Both differentiation- and postnatal development-related change are explored, initially by confirming expected reciprocal relationships between transcript abundance and span of PcG domains overlapping transcribed regions. PcG-associated postnatal transcriptome change specific to the stem cell-like monocytes is found to be incompletely explained by conventional measures of PcG region structure. To address this, we introduce algorithms that quantify local nucleosome-scale conservation of PcG-region topology. It is shown that topology-based comparisons can reveal broad statistical linkage between postnatal gene down-regulation and epigenome remodeling; further, such comparisons provide access to a previously unexplored dimension of epigenome architecture.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Adulto , Algoritmos , Diferenciación Celular/genética , Senescencia Celular/genética , Metilación de ADN , Células Dendríticas/citología , Células Dendríticas/metabolismo , Epigénesis Genética , Sangre Fetal/citología , Sangre Fetal/metabolismo , Expresión Génica , Humanos , Técnicas In Vitro , Recién Nacido , Modelos Genéticos , Monocitos/citología , Monocitos/metabolismo , Proteínas del Grupo Polycomb/genéticaRESUMEN
Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.
Asunto(s)
Acetiltransferasas/metabolismo , Histona Desacetilasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/química , Proteínas de Ciclo Celular , Diferenciación Celular , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Histona Acetiltransferasas , Histona Desacetilasa 1 , Histona Desacetilasas/química , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Complejos Multiproteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transactivadores/química , Transactivadores/metabolismo , Factores de Transcripción , Células U937 , Factores de Transcripción p300-CBPRESUMEN
Diminished ovarian function occurs early and is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated the roles that genome and epigenome structure play in age-related changes in gene expression and ovarian function, using human ovarian granulosa cells as an experimental system. DNA methylomes were compared between two groups of women with distinct age-related differences in ovarian functions, using both Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) and Reduced Representation Bisulfite Sequencing (RRBS); their transcriptomes were investigated using mRNA-seq. Significant, non-random changes in transcriptome and DNA methylome features are observed in human ovarian granulosa cells as women age and their ovarian functions deteriorate. The strongest correlations between methylation and the age-related changes in gene expression are not confined to the promoter region; rather, high densities of hypomethylated CpG-rich regions spanning the gene body are preferentially associated with gene down-regulation. This association is further enhanced where CpG regions are localized near the 3'-end of the gene. Such features characterize several genes crucial in age-related decline in ovarian function, most notably the AMH (Anti-Müllerian Hormone) gene. The genome-wide correlation between the density of hypomethylated intragenic and 3'-end regions and gene expression suggests previously unexplored mechanisms linking epigenome structure to age-related physiology and pathology.
Asunto(s)
Metilación de ADN , ADN/genética , ADN/metabolismo , Células de la Granulosa/fisiología , Ovario/fisiología , Adulto , Factores de Edad , Islas de CpG , Femenino , Expresión Génica , Estudio de Asociación del Genoma Completo , Células de la Granulosa/metabolismo , Humanos , Ovario/citología , Ovario/patología , TranscriptomaRESUMEN
Histone deacetylases are required for transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox transcription factor, HOXA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Sirtuinas/metabolismo , Animales , Línea Celular , Sistema Libre de Células , Proteínas de Unión al ADN/genética , Proteínas Homeobox A10 , Proteínas de Homeodominio , Humanos , Ratones , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sirtuina 2 , Sirtuinas/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
With completion of the human genome project, patterns of higher order chromatin structure can be easily related to other features of genome organization. A well-studied aspect of chromatin, histone H4 acetylation, is examined here on the basis of its role in setting competence for gene activation. Three applications of a new hybrid genome sampling-chromatin immunoprecipitation strategy are described. The first explores aspects of epigenome architecture in human fibroblasts. A second focuses on chromatin from HL-60 promyelocytic leukemia cells before and after differentiation into macrophage-like cells. A third application explores age-related epigenome change. In the latter, acetylation patterns are compared in human skin fibroblast chromatin from donors of various ages. Two sites are reported at which observed histone H4 acetylation differences suggest decreasing acetylation over time. The sites, located in chromosome 4p16.1 and 4q35.2 regions, appear to remodel during late fetal-early child development and from preadolescence through adult life, respectively.
Asunto(s)
Envejecimiento/genética , Ensamble y Desensamble de Cromatina , Genoma Humano , Acetilación , Especificidad de Anticuerpos , Diferenciación Celular , Células Cultivadas , Islas de CpG , Histonas/metabolismo , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Common to numerous differentiation pathways in vertebrate organisms is the regulation of key genes through epigenetic mechanisms. Less well studied is to what extent cells of a given differentiation state, but examined at different points within the life history of an organism, are distinct at the level of the epigenome. A few instances of such variation have been reported, and it would be of considerable value to have at hand a means to characterize additional examples more efficiently. We describe an integrated approach to this task, and further present evidence for regions of age-related histone H4 acetylation change extending over tens to hundreds of kilobases. Broad similarity between two distinct regions of such change suggests a previously unsuspected link between developmental programs and aging.
Asunto(s)
Envejecimiento/genética , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina/métodos , Mapeo Cromosómico , Acetilación , Cromatografía Líquida de Alta Presión , Genoma Humano , Histonas/metabolismo , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The DNA of eukaryotic cells is packaged into chromatin by histone proteins, which play a central role in regulating access to genetic information. The nucleosome core is the basic structural unit of chromatin: it is composed of an octamer of the four major core histones (two molecules each of H2A, H2B, H3, and H4), around which are wrapped â¼1.75 negative superhelical turns of DNA, a total of 145-147bp. Nucleosome cores are regularly spaced along the DNA in vivo, separated by linker DNA. Nucleosomes are compact structures capable of blocking access to the DNA that they contain. For example, they may prevent the binding of transcription factors to their cognate sites. It is therefore very important to obtain quantitative information on the positions of nucleosomes with respect to regulatory regions in vivo. The advent of high-throughput sequencing methods has revolutionized this field. We describe the use and advantages of paired-end sequencing to map nucleosomal DNA obtained by micrococcal nuclease digestion of budding yeast nuclei. This approach provides high-quality genome-wide nucleosome occupancy and position maps.