RESUMO
Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility1. The X chromosome is vital for reproduction and cognition2. Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species.
Assuntos
Hominidae , Cromossomo X , Cromossomo Y , Animais , Feminino , Masculino , Gorilla gorilla/genética , Hominidae/genética , Hominidae/classificação , Hylobatidae/genética , Pan paniscus/genética , Pan troglodytes/genética , Filogenia , Pongo abelii/genética , Pongo pygmaeus/genética , Telômero/genética , Cromossomo X/genética , Cromossomo Y/genética , Evolução Molecular , Variações do Número de Cópias de DNA/genética , Humanos , Espécies em Perigo de Extinção , Padrões de ReferênciaRESUMO
Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE1 and RefSeq2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.
Assuntos
Biologia Computacional , Bases de Dados Genéticas , Genômica , Genoma , Humanos , Disseminação de Informação , Anotação de Sequência Molecular , National Library of Medicine (U.S.) , Estados UnidosRESUMO
The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community.
Assuntos
Sequência Consenso , Bases de Dados Genéticas , Fases de Leitura Aberta , Animais , Curadoria de Dados/métodos , Curadoria de Dados/normas , Bases de Dados Genéticas/normas , Guias como Assunto , Humanos , Camundongos , Anotação de Sequência Molecular , National Library of Medicine (U.S.) , Estados Unidos , Interface Usuário-ComputadorRESUMO
The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Bovinos , Perfilação da Expressão Gênica , Genoma Fúngico , Genoma Humano , Genoma Microbiano , Genoma de Planta , Genoma Viral , Genômica/normas , Humanos , Invertebrados/genética , Camundongos , Anotação de Sequência Molecular , Nematoides/genética , Filogenia , RNA Longo não Codificante/genética , Ratos , Padrões de Referência , Análise de Sequência de Proteína , Análise de Sequência de RNA , Vertebrados/genéticaRESUMO
The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of annotated genomic, transcript and protein sequence records derived from data in public sequence archives and from computation, curation and collaboration (http://www.ncbi.nlm.nih.gov/refseq/). We report here on growth of the mammalian and human subsets, changes to NCBI's eukaryotic annotation pipeline and modifications affecting transcript and protein records. Recent changes to NCBI's eukaryotic genome annotation pipeline provide higher throughput, and the addition of RNAseq data to the pipeline results in a significant expansion of the number of transcripts and novel exons annotated on mammalian RefSeq genomes. Recent annotation changes include reporting supporting evidence for transcript records, modification of exon feature annotation and the addition of a structured report of gene and sequence attributes of biological interest. We also describe a revised protein annotation policy for alternatively spliced transcripts with more divergent predicted proteins and we summarize the current status of the RefSeqGene project.
Assuntos
Bases de Dados Genéticas , Genômica , Mamíferos/genética , Animais , Eucariotos/genética , Éxons , Genoma , Genômica/normas , Humanos , Internet , Anotação de Sequência Molecular , Proteínas/química , Proteínas/genética , RNA/química , Padrões de ReferênciaRESUMO
The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.
Assuntos
Bases de Dados Genéticas , Proteínas/genética , Animais , Éxons , Genômica , Humanos , Internet , Camundongos , Anotação de Sequência Molecular , Análise de SequênciaRESUMO
Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.
Assuntos
Cromatina/metabolismo , Metilação de DNA , Genes Supressores de Tumor , Células-Tronco/metabolismo , Adulto , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Histonas/metabolismo , Humanos , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Células Tumorais CultivadasRESUMO
Complete and accurate annotation of the mouse genome is critical to the advancement of research conducted on this important model organism. The National Center for Biotechnology Information (NCBI) develops and maintains many useful resources to assist the mouse research community. In particular, the reference sequence (RefSeq) database provides high-quality annotation of multiple mouse genome assemblies using a combinatorial approach that leverages computation, manual curation, and collaboration. Implementation of this conservative and rigorous approach, which focuses on representation of only full-length and non-redundant data, produces high-quality annotation products. RefSeq records explicitly link sequences to current knowledge in a timely manner, updating public records regularly and rapidly in response to nomenclature updates, addition of new relevant publications, collaborator discussion, and user feedback. Whole genome re-annotation is also conducted at least every 12-18 months, and often more frequently in response to assembly updates or availability of informative data. This article highlights key features and advantages of RefSeq genome annotation products and presents an overview of NCBI processes to generate these data. Further discussion of NCBI's resources highlights useful features and the best methods for accessing our data.
Assuntos
Sequência de Aminoácidos/genética , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Genoma , Animais , Internet , CamundongosRESUMO
Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Variação Genética , Metagenoma , Microbiologia do Solo , Antibacterianos/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Biblioteca Gênica , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream ( approximately 30 kb) of and extending approximately 60 kb around one such gene, GATA-4, is organized-in Tera-2 undifferentiated embryonic carcinoma (EC) cells-in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)-mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a approximately 60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.
Assuntos
Cromatina/fisiologia , Metilação de DNA/fisiologia , Fator de Transcrição GATA4/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras/fisiologia , Adulto , Carcinoma Embrionário , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/ultraestrutura , Neoplasias do Colo , Ilhas de CpG , Proteínas de Ligação a DNA/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética/fisiologia , Fator de Transcrição GATA4/genética , Inativação Gênica , Humanos , Conformação de Ácido Nucleico , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/fisiologia , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Fatores de Transcrição/fisiologia , Tretinoína/farmacologiaRESUMO
The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Regiões Promotoras Genéticas , Sirtuínas/antagonistas & inibidores , Sirtuínas/metabolismo , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias do Colo/metabolismo , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Epigênese Genética , Feminino , Inativação Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Células HCT116 , Humanos , Dados de Sequência Molecular , Sirtuína 1 , Sirtuínas/genética , Células Tumorais CultivadasRESUMO
Recent work suggests a link between the polycomb group protein EZH2 and mediation of gene silencing in association with maintenance of DNA methylation. However, we show that whereas basally expressed target cancer genes with minimal DNA methylation have increased transcription during EZH2 knockdown, densely DNA hypermethylated and silenced genes retain their methylation and remain transcriptionally silent. These results suggest that EZH2 can modulate transcription of basally expressed genes but not silent genes that are densely DNA methylated.
Assuntos
Neoplasias Ósseas/genética , Neoplasias do Colo/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Inativação Gênica , Osteossarcoma/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteína 1 Homóloga a MutL , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Osteossarcoma/metabolismo , Complexo Repressor Polycomb 2 , RNA Interferente Pequeno , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica , TransfecçãoRESUMO
Histone H3 lysine 9 (H3K9) and lysine 27 (H3K27) trimethylation are properties of stably silenced heterochromatin whereas H3K9 dimethylation (H3K9me2) is important for euchromatic gene repression. In colorectal cancer cells, all of these marks, as well as the key enzymes which establish them, surround the hMLH1 promoter when it is DNA hypermethylated and aberrantly silenced, but are absent when the gene is unmethylated and fully expressed in a euchromatic state. When the aberrantly silenced gene is DNA demethylated and reexpressed following 5-aza-2'-deoxycytidine treatment, H3K9me1 and H3K9me2 are the only silencing marks that are lost. A series of other silenced and DNA hypermethylated gene promoters behave identically even when the genes are chronically DNA demethylated and reexpressed after genetic knockout of DNA methyltransferases. Our data indicate that when transcription of DNA hypermethylated genes is activated in cancer cells, their promoters remain in an environment with certain heterochromatic characteristics. This finding has important implications for the translational goal of reactivating aberrantly silenced cancer genes as a therapeutic maneuver.
Assuntos
Cromatina/genética , Neoplasias Colorretais/genética , Metilação de DNA , Inativação Gênica , Genes Supressores de Tumor , Proteínas Adaptadoras de Transdução de Sinal , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Neoplasias Colorretais/metabolismo , Decitabina , Células HCT116 , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Regiões Promotoras GenéticasRESUMO
Keratins represent a large protein family with essential structural and functional roles in epithelial cells of skin, hair follicles, and other organs. During evolution the genes encoding keratins have undergone multiple rounds of duplication and humans have two clusters with a total of 55 functional keratin genes in their genomes. Due to the high similarity between different keratin paralogs and species-specific differences in gene content, the currently available keratin gene annotation in species with draft genome assemblies such as dog and horse is still imperfect. We compared the National Center for Biotechnology Information (NCBI) (dog annotation release 103, horse annotation release 101) and Ensembl (release 87) gene predictions for the canine and equine keratin gene clusters to RNA-seq data that were generated from adult skin of five dogs and two horses and from adult hair follicle tissue of one dog. Taking into consideration the knowledge on the conserved exon/intron structure of keratin genes, we annotated 61 putatively functional keratin genes in both the dog and horse, respectively. Subsequently, curators in the RefSeq group at NCBI reviewed their annotation of keratin genes in the dog and horse genomes (Annotation Release 104 and Annotation Release 102, respectively) and updated annotation and gene nomenclature of several keratin genes. The updates are now available in the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene).
Assuntos
Bases de Dados Genéticas , Cães/genética , Genoma , Cavalos/genética , Queratinas/genética , Animais , Éxons , Íntrons , Anotação de Sequência Molecular , Especificidade da EspécieRESUMO
Epigenetic silencing of genes in association with aberrant promoter DNA hypermethylation has emerged as a significant mechanism in the development of human cancers. Such genes are also often targets of the polycomb group repressive complexes in embryonic cells. The polycomb repressive complex 2 (PRC2) has been best studied in this regard. We now examine a link between PRC1 and cancer-specific gene silencing. Here, we show a novel and direct association between a constituent of the PRC1 complex, CBX7, with gene repression and promoter DNA hypermethylation of genes frequently silenced in cancer. CBX7 is able to complex with DNA methyltransferase (DNMT) enzymes, leading us to explore a role for CBX7 in maintenance and initiation of gene silencing. Knockdown of CBX7 was unable to relieve suppression of deeply silenced genes in cancer cells; however, in embryonal carcinoma (EC) cells, CBX7 can initiate stable repression of genes that are frequently silenced in adult cancers. Furthermore, we are able to observe assembly of DNMTs at CBX7 target gene promoters. Sustained expression of CBX7 in EC cells confers a growth advantage and resistance to retinoic acid-induced differentiation. In this setting, especially, there is increased promoter DNA hypermethylation for many genes by analysis of specific genes, as well as through epigenomic studies. Our results allow us to propose a potential mechanism through assembly of novel repressive complexes, by which the polycomb component of PRC1 can promote the initiation of epigenetic changes involving abnormal DNA hypermethylation of genes frequently silenced in adult cancers.
Assuntos
Metilação de DNA , Inativação Gênica , Neoplasias/genética , Proteínas Repressoras/genética , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Complexo Repressor Polycomb 1 , Regiões Promotoras Genéticas , Tretinoína/farmacologia , DNA Metiltransferase 3BRESUMO
We describe construction of a novel modification, "6C," of chromatin looping assays that allows specific proteins that may mediate long-range chromatin interactions to be defined. This approach combines the standard looping approaches previously defined with an immunoprecipitation step to investigate involvement of the specific protein. The efficacy of this approach is demonstrated by using a Polycomb group (PcG) protein, Enhancer of Zeste (EZH2), as an example of how our assay might be used. EZH2, as a protein of the PcG complex, PRC2, has an important role in the propagation of epigenetic memory through deposition of the repressive mark, histone H3, lysine 27, tri-methylation (H3K27me3). Using our new 6C assay, we show how EZH2 is a direct mediator of long-range intra- and interchromosomal interactions that can regulate transcriptional down-regulation of multiple genes by facilitating physical proximities between distant chromatin regions, thus targeting sites within to PcG machinery.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Linhagem Celular Tumoral , Cromatina/química , Cromossomos Humanos/química , Citocinas , Regulação para Baixo/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Conformação ProteicaRESUMO
Epigenetic gene regulation is a key determinant of heritable gene expression patterns and is critical for normal cellular function. Dysregulation of epigenetic transcriptional control is a fundamental feature of cancer, particularly manifesting as increased promoter DNA methylation with associated aberrant gene silencing, which plays a significant role in tumor progression. We now globally map key chromatin parameters for genes with promoter CpG island DNA hypermethylation in colon cancer cells by combining microarray gene expression analyses with chromatin immunoprecipitation-on-chip technology. We first show that the silent state of such genes universally correlates with a broad distribution of a low but distinct level of the PcG-mediated histone modification, methylation of lysine 27 of histone 3 (H3K27me), and a very low level of the active mark H3K4me2. This chromatin pattern, and particularly H3K4me2 levels, crisply separates DNA-hypermethylated genes from those where histone deacetylation is responsible for transcriptional silencing. Moreover, the chromatin pattern can markedly enhance identification of truly silent and DNA-hypermethylated genes. We additionally find that when DNA-hypermethylated genes are demethylated and reexpressed, they adopt a bivalent chromatin pattern, which is associated with the poised gene expression state of a large group of embryonic stem cell genes and is characterized by an increase in levels of both the H3K27me3 and H3K4me2 marks. Our data have great relevance for the increasing interest in reexpression of DNA-hypermethylated genes for the treatment of cancer.
Assuntos
Cromatina/química , Neoplasias do Colo/genética , Metilação de DNA , DNA/química , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Neoplasias do Colo/patologia , Ilhas de CpG , Progressão da Doença , Epigênese Genética , Inativação Gênica , Histonas/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/citologiaRESUMO
It is increasingly apparent that cancer development not only depends on genetic alterations but on an abnormal cellular memory, or epigenetic changes, which convey heritable gene expression patterns critical for neoplastic initiation and progression. These aberrant epigenetic mechanisms are manifest in both global changes in chromatin packaging and in localized gene promoter changes that influence the transcription of genes important to the cancer process. An exciting emerging theme is that an understanding of stem cell chromatin control of gene expression, including relationships between histone modifications and DNA methylation, may hold a key to understanding the origins of cancer epigenetic changes. This possibility, coupled with the reversible nature of epigenetics, has enormous significance for the prevention and control of cancer.