RESUMO
During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Motivos de Nucleotídeos , CoesinasRESUMO
Nucleosomes cover most of the genome and are thought to be displaced by transcription factors in regions that direct gene expression. However, the modes of interaction between transcription factors and nucleosomal DNA remain largely unknown. Here we systematically explore interactions between the nucleosome and 220 transcription factors representing diverse structural families. Consistent with earlier observations, we find that the majority of the studied transcription factors have less access to nucleosomal DNA than to free DNA. The motifs recovered from transcription factors bound to nucleosomal and free DNA are generally similar. However, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many transcription factors preferentially bind close to the end of nucleosomal DNA, or to periodic positions on the solvent-exposed side of the DNA. In addition, several transcription factors usually bind to nucleosomal DNA in a particular orientation. Some transcription factors specifically interact with DNA located at the dyad position at which only one DNA gyre is wound, whereas other transcription factors prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals notable differences in the binding of transcription factors to free and nucleosomal DNA, and uncovers a diverse interaction landscape between transcription factors and the nucleosome.
Assuntos
Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , Humanos , Camundongos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Motivos de Nucleotídeos , Ligação Proteica , Rotação , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/química , Fatores de Transcrição/classificaçãoRESUMO
Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.
Assuntos
DNA/genética , DNA/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Reprodutibilidade dos Testes , Especificidade por Substrato/genéticaRESUMO
Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.
Assuntos
Adenocarcinoma/genética , Neoplasias Intestinais/genética , Mutação , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Exoma , Feminino , Humanos , Neoplasias Intestinais/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-2/genéticaRESUMO
BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Linfócitos/imunologia , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/imunologia , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Estudos de Casos e Controles , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Exoma/genética , Exoma/imunologia , Feminino , Humanos , Linfócitos/patologia , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Neoplasias Primárias Múltiplas/patologiaRESUMO
Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether, 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.
Assuntos
DNA Helicases/genética , Leiomiossarcoma/genética , Complexo Mediador/genética , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Uterinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas Correpressoras , Exoma , Feminino , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leiomiossarcoma/mortalidade , Pessoa de Meia-Idade , Chaperonas Moleculares , Taxa de Mutação , Homeostase do Telômero , Neoplasias Uterinas/mortalidade , Proteína Nuclear Ligada ao XRESUMO
Hereditary factors are presumed to play a role in one third of colorectal cancer (CRC) cases. However, in the majority of familial CRC cases the genetic basis of predisposition remains unexplained. This is particularly true for families with few affected individuals. To identify susceptibility genes for this common phenotype, we examined familial cases derived from a consecutive series of 1514 Finnish CRC patients. Ninety-six familial CRC patients with no previous diagnosis of a hereditary CRC syndrome were included in the analysis. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. Exome sequencing was utilized to search for genes harboring putative loss-of-function variants, because such alterations are likely candidates for disease-causing mutations. Eleven genes with rare truncating variants in two or three familial CRC cases were identified: UACA, SFXN4, TWSG1, PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, and AKR1C4. Loss of heterozygosity was examined in all respective cancer samples, and was detected in seven occasions involving four of the candidate genes. In all seven occasions the wild-type allele was lost (P = 0.0078) providing additional evidence that these eleven genes are likely to include true culprits. The study provides a set of candidate predisposition genes which may explain a subset of common familial CRC. Additional genetic validation in other populations is required to provide firm evidence for causality, as well as to characterize the natural history of the respective phenotypes.
Assuntos
Neoplasias Colorretais/genética , Predisposição Genética para Doença , Perda de Heterozigosidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Exoma , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
BACKGROUND: Classic Kaposi sarcoma (cKS) is an inflammatory tumor caused by human herpesvirus 8 (HHV-8) commonly observed in elderly men of Mediterranean origin. We studied a Finnish family of 5 affected individuals in 2 generations. Except for atypical mycobacterial infection of the index case, the affected individuals did not have notable histories of infection. METHODS: We performed genome and exome sequencing and mapped shared chromosomal regions to identify genetic predisposition in the family. RESULTS: We identified 12 protein-coding candidate variants that segregated in the 3 affected cousins from whom we had samples. The affected mother of the index case was an obligatory carrier. Among the 12 candidates was a rare heterozygous substitution rs141331848 (c.1337C>T, p.Thr446Ile) in the DNA-binding domain of STAT4. The variant was not present in 242 Finnish control genomes or 180 additional regional controls. Activated T-helper cells from the HHV-8-negative variant carriers showed reduced interferon γ production, compared with age and sex matched wild-type individuals. We screened STAT4 in additional 18 familial KS cases and the variant site from 56 sporadic KS cases but detected no pathogenic mutations. CONCLUSIONS: Our data suggest that STAT4 is a potential cKS-predisposition gene, but further functional and genetic validation is needed.
Assuntos
Predisposição Genética para Doença/genética , Fator de Transcrição STAT4/genética , Sarcoma de Kaposi/genética , Idoso , Sequência de Aminoácidos , Feminino , Ligação Genética , Genoma , Humanos , Interferon gama/imunologia , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Linfócitos TRESUMO
Insect flight is one of the most energetically demanding activities in the animal kingdom, yet for many insects flight is necessary for reproduction and foraging. Moreover, dispersal by flight is essential for the viability of species living in fragmented landscapes. Here, working on the Glanville fritillary butterfly (Melitaea cinxia), we use transcriptome sequencing to investigate gene expression changes caused by 15 min of flight in two contrasting populations and the two sexes. Male butterflies and individuals from a large metapopulation had significantly higher peak flight metabolic rate (FMR) than female butterflies and those from a small inbred population. In the pooled data, FMR was significantly positively correlated with genome-wide heterozygosity, a surrogate of individual inbreeding. The flight experiment changed the expression level of 1513 genes, including genes related to major energy metabolism pathways, ribosome biogenesis and RNA processing, and stress and immune responses. Males and butterflies from the population with high FMR had higher basal expression of genes related to energy metabolism, whereas females and butterflies from the small population with low FMR had higher expression of genes related to ribosome/RNA processing and immune response. Following the flight treatment, genes related to energy metabolism were generally down-regulated, while genes related to ribosome/RNA processing and immune response were up-regulated. These results suggest that common molecular mechanisms respond to flight and can influence differences in flight metabolic capacity between populations and sexes.
Assuntos
Borboletas/genética , Voo Animal , Expressão Gênica , Caracteres Sexuais , Transcriptoma , Animais , Borboletas/fisiologia , Metabolismo Energético/genética , Feminino , Finlândia , Masculino , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNARESUMO
ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 [16%] samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Exoma/genética , Repetições de Microssatélites/genética , Mutação/genética , Proteínas Nucleares/genética , Proteína 1 de Ligação ao Retinoblastoma/genética , Fatores de Transcrição/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Neoplasias Colorretais/patologia , Feminino , Seguimentos , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
Microsatellite instability can be found in approximately 15% of all colorectal cancers. To detect new oncogenes we sequenced the exomes of 25 colorectal tumors and respective healthy colon tissue. Potential mutation hot spots were confirmed in 15 genes; ADAR, DCAF12L2, GLT1D1, ITGA7, MAP1B, MRGPRX4, PSRC1, RANBP2, RPS6KL1, SNCAIP, TCEAL6, TUBB6, WBP5, VEGFB, and ZBTB2; these were validated in 86 tumors with microsatellite instability. ZBTB2, RANBP2, and PSRC1 also were found to contain hot spot mutations in the validation set. The form of ZBTB2 associated with colorectal cancer increased cell proliferation. The mutation hot spots might be used to develop personalized tumor profiling and therapy.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Oncogenes , Idoso , Estudos de Casos e Controles , Feminino , Marcadores Genéticos , Humanos , Masculino , Análise de Sequência de DNARESUMO
OBJECTIVE: Early-onset colorectal cancer (CRC), defined here as age of onset less than 40 years, develops frequently in genetically predisposed individuals. Next-generation sequencing is an increasingly available option in the diagnostic workup of suspected hereditary susceptibility, but little is known about the practical feasibility and additional diagnostic yield of the technology in this patient group. MATERIALS AND METHODS: We analyzed 38 young CRC patients derived from a set of 1514 CRC cases. All 38 tumors had been tested in our laboratory for microsatellite instability (MSI), and Sanger sequencing had been used to screen for MLH1 and MSH2 mutations in MSI cases. Also, gastrointestinal polyposis had been diagnosed clinically and molecularly. Family histories were acquired from national registries. If inherited syndromes had not been diagnosed in routine diagnostic efforts (n = 23), normal tissue DNA was analyzed for mutations in a comprehensive set of high-penetrance genes (MLH1, MSH2, MSH6, PMS2, APC, MUTYH, SMAD4, BMPR1A, LKB1/STK11, and PTEN) by exome sequencing. RESULTS: CRC predisposition syndromes were confirmed in 42% (16/38) of early-onset CRC patients. Hereditary nonpolyposis colorectal cancer was diagnosed in 12 (32%) patients, familial adenomatous polyposis in three (7.9%), and juvenile polyposis in one (2.6%) patient. Exome sequencing revealed one additional MLH1 mutation. Over half of the patients had advanced cancers (Dukes C or D, 61%, 23/38). The majority of nonsyndromic patients had unaffected first-degree relatives and microsatellite-stable tumors. CONCLUSIONS: Microsatellite instability positivity or gastrointestinal polyposis characterized all patients with unambiguous highly penetrant germline mutations. In our series, exome sequencing produced little added value in diagnosing the underlying predisposition conditions.
Assuntos
Polipose Adenomatosa do Colo/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Exoma/genética , Predisposição Genética para Doença , Polipose Intestinal/congênito , Síndromes Neoplásicas Hereditárias/diagnóstico , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal/genética , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/genética , Adenosina Trifosfatases/genética , Adulto , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Testes Genéticos , Humanos , Polipose Intestinal/diagnóstico , Polipose Intestinal/genética , Masculino , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Síndromes Neoplásicas Hereditárias/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Serina-Treonina Quinases/genética , Análise de Sequência de DNA , Proteína Smad4/genética , Adulto JovemRESUMO
DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.
RESUMO
Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, here we analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, we also targeted genes involved in protein phosphorylation simultaneously with their homologues. We identify genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38betaMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, we identify a translational regulator, eIF-3p66, that specifically affects the Cyclin/Cdk pathway activity.
Assuntos
Ciclo Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Interferência de RNA , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Tamanho Celular , Sequência Conservada/genética , Drosophila melanogaster/metabolismo , Biblioteca Gênica , Genes de Insetos/genética , Humanos , Fenótipo , Fosforilação , Transdução de SinaisRESUMO
Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis.
Assuntos
Aterosclerose/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Haploinsuficiência , Doença de Hodgkin/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Aterosclerose/patologia , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Feminino , Finlândia , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Hematopoese/genética , Doença de Hodgkin/sangue , Doença de Hodgkin/patologia , Humanos , Masculino , Fenótipo , Cultura Primária de Células , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequenciamento Completo do GenomaRESUMO
No existing method to characterize transcription factor (TF) binding to DNA allows genome-wide measurement of all TF-binding activity in cells. Here we present a massively parallel protein activity assay, active TF identification (ATI), that measures the DNA-binding activity of all TFs in cell or tissue extracts. ATI is based on electrophoretic separation of protein-bound DNA sequences from a highly complex DNA library and subsequent mass-spectrometric identification of the DNA-bound proteins. We applied ATI to four mouse tissues and mouse embryonic stem cells and found that, in a given tissue or cell type, a small set of TFs, which bound to only â¼10 distinct motifs, displayed strong DNA-binding activity. Some of these TFs were found in all cell types, whereas others were specific TFs known to determine cell fate in the analyzed tissue or cell type. We also show that a small number of TFs determined the accessible chromatin landscape of a cell, suggesting that gene regulatory logic may be simpler than previously appreciated.
Assuntos
Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Diferenciação Celular , Cromatina/genética , DNA/genética , DNA/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade da Espécie , Distribuição TecidualRESUMO
Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Instabilidade de Microssatélites , Mutação Puntual , Redes Reguladoras de Genes , Humanos , Anotação de Sequência Molecular , Análise de Sequência de DNARESUMO
To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells. In contrast, depletion of CASP8AP2 in normal cells triggers a response that arrests viable cells in S-phase. The arrest is dependent on p53, and preceded by accumulation of markers of DNA damage, indicating that nucleosome depletion is sensed in normal cells via a DNA-damage -like response that is defective in tumor cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Genoma , Neoplasias/metabolismo , Neoplasias/patologia , Nucleossomos/metabolismo , Interferência de RNA , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , DNA/biossíntese , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação , RNA Interferente Pequeno/metabolismo , Fase S , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
Approximately 15% of colorectal cancers exhibit microsatellite instability (MSI), which leads to accumulation of large numbers of small insertions and deletions (indels). Genes that provide growth advantage to cells via loss-of-function mutations in microsatellites are called MSI target genes. Several criteria to define these genes have been suggested, one of them being simple mutation frequency. Microsatellite mutation rate, however, depends on the length and nucleotide context of the microsatellite. Therefore, assessing the general impact of mismatch repair deficiency on the likelihood of mutation events is paramount when following this approach. To identify MSI target genes, we developed a statistical model for the somatic background indel mutation rate of microsatellites to assess mutation significance. Exome sequencing data of 24 MSI colorectal cancers revealed indels at 54 million mononucleotide microsatellites of three or more nucleotides in length. The top 105 microsatellites from 71 genes were further analyzed in 93 additional MSI colorectal cancers. Mutation significance and estimated clonality of mutations determined the most likely MSI target genes to be the aminoadipate-semialdehyde dehydrogenase AASDH and the solute transporter SLC9A8 Our findings offer a systematic profiling of the somatic background mutation rate in protein-coding mononucleotide microsatellites, allowing a full cataloging of the true targets of MSI in colorectal cancer. Cancer Res; 77(15); 4078-88. ©2017 AACR.
Assuntos
Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , Instabilidade de Microssatélites , Modelos Estatísticos , Humanos , MutaçãoRESUMO
The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.