RESUMO
Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance.
Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Estradiol/fisiologia , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Código das Histonas , Humanos , Células MCF-7 , Receptores de Estrogênio/metabolismo , Transcrição GênicaRESUMO
Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.
Assuntos
Sítios de Ligação , Metilação de DNA , DNA/metabolismo , Ensaios de Triagem em Larga Escala , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Cromatina , Desmetilação do DNA , Replicação do DNA , Epigenômica , Expressão Gênica , Camundongos , Nucleossomos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Células Sf9 , Fatores de Transcrição/metabolismoRESUMO
GSTA1 encodes a member of a family of enzymes that function to add glutathione to target electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. GSTA1 has several functional SNPs within its promoter region that are responsible for a change in its expression by altering promoter function. This study aims to investigate distributions of GSTA1 promoter haplotypes across different human populations and to assess their impact on the expression of GSTA1. PHASE 2.1.1 was used to infer haplotypes and diplotypes of six GSTA1 promoter SNPs on 2501 individuals from 26 populations classified by the 1000 Genomes Project into five super-populations that included Africa (N = 660), America (N = 347), East Asia (N = 504), Europe (N = 502), and South Asia (N = 488). We used pairwise FST analysis to compare sub-populations and luciferase reporter assay (LRA) to evaluate the impact of each SNP on activation of transcription and interaction with other SNPs. The distributions of GSTA1 promoter haplotypes and diplotypes were significantly different among the different human populations. Three new promoter haplotypes were found in the African super-population. LRA demonstrated that SNPs at -52 and -69 has the most impact on GSTA1 expression, however other SNPs have a significant impact on transcriptional activity. Based on LRA, a new model of cis-elements interaction is presented. Due to the significant differences in GSTA1 diplotype population frequencies, future pharmacogenomics or disease-related studies would benefit from the inclusion of the complete GSTA1 promoter haplotype based on the newly proposed metabolic grouping derived from the LRA results.
Assuntos
Genética Populacional , Genoma Humano , Glutationa Transferase/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , África , América , Ásia , Sítios de Ligação , Europa (Continente) , Regulação da Expressão Gênica , Genes Reporter , Glutationa Transferase/metabolismo , Haplótipos , Células Hep G2 , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD-derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders.
RESUMO
Infections are thought to trigger CD8+ cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpoint sensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Homeodomínio/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Adulto , Idoso , Animais , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape.
Assuntos
Linfócitos B/fisiologia , Cromatina/metabolismo , Células Progenitoras Linfoides/fisiologia , Transativadores/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Metilação de DNA/genética , Redes Reguladoras de Genes/genética , Linfopoese , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Estrutura Terciária de Proteína/genética , Transativadores/genéticaRESUMO
For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2)) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.
Assuntos
Ilhas de CpG/genética , Metilação de DNA/genética , Genoma , Animais , Linhagem Celular , DNA/genética , Mamíferos/genética , Análise de Sequência de DNARESUMO
Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.
Assuntos
Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Transcrição Gênica , 5-Metilcitosina/análogos & derivados , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Diferenciação Celular , Ilhas de CpG/genética , Citosina/análogos & derivados , Citosina/farmacologia , Células-Tronco Embrionárias , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genéticaRESUMO
In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG-rich sequences that lack DNA methylation. Residual nucleosomes are largely composed of the histone H3.3 variant and are trimethylated at Lys4 of histone H3 (H3K4me3). Canonical H3.1 and H3.2 histones are also enriched at CpG-rich promoters marked by Polycomb-mediated H3K27me3, a modification predictive of gene repression in preimplantation embryos. Histone variant-specific nucleosome retention in sperm is strongly associated with nucleosome turnover in round spermatids. Our data show evolutionary conservation of the basic principles of nucleosome retention in mouse and human sperm, supporting a model of epigenetic inheritance by nucleosomes between generations.
Assuntos
Ilhas de CpG , Nucleossomos/fisiologia , Espermatozoides/metabolismo , Animais , Metilação de DNA , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Espermátides/metabolismoRESUMO
Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Embrionárias/citologia , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Regulação para Baixo , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras GenéticasRESUMO
Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIß (Top2ß) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2ß binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2ß-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2ß protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2ß deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2ß. These findings suggest a chromatin-based targeting of Top2ß to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.
Assuntos
Cromatina/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Neurônios/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Dicetopiperazinas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Piperazinas/farmacologia , Ligação Proteica , Interferência de RNA , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Inibidores da Topoisomerase II/farmacologiaRESUMO
Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.
Assuntos
Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Animais , Diferenciação Celular , Ilhas de CpG , Células-Tronco Embrionárias/citologia , Camundongos , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Células-Tronco/citologia , Fatores de Transcrição/metabolismoRESUMO
Cellular functions including transcription regulation, DNA repair, and DNA replication need to be tightly regulated. DNA sequence can contribute to the regulation of these mechanisms. This is exemplified by the consensus sequences that allow the binding of specific transcription factors, thus regulating transcription rates. Another layer of regulation resides in modifications that do not affect the DNA sequence itself but still results in the modification of chromatin structure and properties, thus affecting the readout of the underlying DNA sequence. These modifications are dubbed as "epigenetic modifications" and include, among others, histone modifications, DNA methylation, and small RNAs. While these events can independently regulate cellular mechanisms, recent studies indicate that joint activities of different epigenetic modifications could result in a common outcome. In this chapter, I will attempt to recapitulate the best known examples of collaborative activities between epigenetic modifications. I will emphasize mostly on the effect of crosstalks between epigenetic modifications on transcription regulation, simply because it is the most exposed and studied aspect of epigenetic interactions. I will also summarize the effect of epigenetic interactions on DNA damage response and DNA repair. The involvement of epigenetic crosstalks in cancer formation, progression, and treatment will be emphasized throughout the manuscript. Due to space restrictions, additional aspects involving histone replacements [Park, Y. J., and Luger, K. (2008). Histone chaperones in nucleosome eviction and histone exchange. Curr. Opin. Struct. Biol.18, 282-289.], histone variants [Boulard, M., Bouvet, P., Kundu, T. K., and Dimitrov, S. (2007). Histone variant nucleosomes: Structure, function and implication in disease. Subcell. Biochem. 41, 71-89; Talbert, P. B., and Henikoff, S. (2010). Histone variants-Ancient wrap artists of the epigenome. Nat. Rev. Mol. Cell Biol.11, 264-275.], and histone modification readers [de la Cruz, X., Lois, S., Sanchez-Molina, S., and Martinez-Balbas, M. A. (2005). Do protein motifs read the histone code? Bioessays27, 164-175; Grewal, S. I., and Jia, S. (2007). Heterochromatin revisited. Nat. Rev. Genet.8, 35-46.] will not be addressed in depth in this chapter, and the reader is referred to the reviews cited here.
Assuntos
Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Neoplasias/genética , Transcrição Gênica , Animais , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA , Reparo do DNA , Histonas/genética , Humanos , Neoplasias/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Coenzimas/imunologia , Células-Tronco Hematopoéticas/imunologia , Histona Acetiltransferases/imunologia , Proteínas Nucleares/imunologia , Proteína Supressora de Tumor p53/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/imunologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Coenzimas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Acetiltransferases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Wnt pathway is a key regulator of embryonic development and stem cell self-renewal, and hyperactivation of the Wnt signalling is associated with many human cancers. The central player in the Wnt pathway is beta-catenin, a cytoplasmic protein whose function is tightly controlled by ubiquitination and degradation, however the precise regulation of beta-catenin stability/degradation remains elusive. Here, we report a new mechanism of beta-catenin ubiquitination acting in the context of chromatin. This mechanism is mediated by the histone acetyltransferase (HAT) complex component TRRAP and Skp1, an invariable component of the Skp-Cullin-F-box (SCF) ubiquitin ligase complex. TRRAP interacts with Skp1/SCF and mediates its recruitment to beta-catenin target promoter in chromatin. TRRAP deletion leads to a reduced level of beta-catenin ubiquitination, lower degradation rate and accumulation of beta-catenin protein. Furthermore, recruitment of Skp1 to chromatin and ubiquitination of chromatin-bound beta-catenin are abolished upon TRRAP knock-down, leading to an abnormal retention of beta-catenin at the chromatin and concomitant hyperactivation of the canonical Wnt pathway. These results demonstrate that there is a distinct regulatory mechanism for beta-catenin ubiquitination/ destruction acting in the nucleus which functionally complements cytoplasmic destruction of beta-catenin and prevents its oncogenic stabilization and chronic activation of the canonical Wnt pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinação , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/metabolismo , Humanos , Modelos Biológicos , RNA Interferente Pequeno , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de SinaisRESUMO
Cancer is traditionally viewed as a primarily genetic disorder, however it is now becoming accepted that cancer is also a consequence of abnormal epigenetic events. Genetic changes and aneuploidy are associated with alterations in DNA sequence, and they are a hallmark of the malignant process. Epigenetic alterations are universally present in human cancer and result in heritable changes in gene expression and chromatin structure over many cell generations without changes in DNA sequence, leading to functional consequences equivalent to those induced by genetic alterations. Importantly, intriguing evidence emerged suggesting that epigenetic changes may precede and provoke genetic changes. In this scenario, epigenetic events are primary events while genetic changes (such as mutations) may simply be a consequence of disrupted epigenetic states. This fact may explain why many genetic screens proved to be limited with regard to cancer causality and pathogenesis. Aberrant epigenetic events affect multiple genes and cellular pathways in a non-random fashion and this can predispose to induction and accumulation of genetic changes in the course of tumour initiation and progression. These considerations are critical for a better understanding of tumourigenesis and molecular events underlying the acquisition of drug resistance, as well as development of novel strategies for cancer therapy and prevention.
Assuntos
Epigênese Genética , Neoplasias/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , Inativação Gênica , Histonas/metabolismo , Humanos , Modelos Genéticos , Mutação , Processamento de Proteína Pós-TraducionalRESUMO
PR-Set7/SET8 is a histone H4-lysine 20 methyltransferase required for normal cell proliferation. However, the exact functions of this enzyme remain to be determined. In this study, we show that human PR-Set7 functions during S phase to regulate cellular proliferation. PR-Set7 associates with replication foci and maintains the bulk of H4-K20 mono- and trimethylation. Consistent with a function in chromosome dynamics during S phase, inhibition of PR-Set7 methyltransferase activity by small hairpin RNA causes a replicative stress characterized by alterations in replication fork velocity and origin firing. This stress is accompanied by massive induction of DNA strand breaks followed by a robust DNA damage response. The DNA damage response includes the activation of ataxia telangiectasia mutated and ataxia telangiectasia related kinase-mediated pathways, which, in turn, leads to p53-mediated growth arrest to avoid aberrant chromosome behavior after improper DNA replication. Collectively, these data indicate that PR-Set7-dependent lysine methylation during S phase is an essential posttranslational mechanism that ensures genome replication and stability.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica/genética , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fase S/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno , Proteínas Supressoras de Tumor/genéticaRESUMO
Epigenetic changes are important etiological factors of human cancer. Epigenetic information in chromatin (known as 'histone code') is a fascinating feature used by cells to extend and modulate the genetic (DNA) code. The histone code is thus proposed to be 'read' by cells to regulate accessibility to, and functions of, chromatin DNA. While the role of the epigenetic code involving chromatin modifying/remodeling complexes in transcriptional regulation is well established, it is only recently that these mechanisms have been implicated in DNA damage detection and DNA repair. However, how the components of the DNA damage sensing and repair machinery gain access to broken DNA in compacted chromatin remains a mystery. Recent studies provide important insights into DNA damage- and repair-specific modifications to histones and shed light on how the epigenetic code controls DNA repair.
Assuntos
Cromatina/genética , Reparo do DNA/genética , Epigênese Genética/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
DNA is packaged into chromatin, a highly compacted DNA-protein complex; therefore, all cellular processes that use the DNA as a template, including DNA repair, require a high degree of coordination between the DNA-repair machinery and chromatin modification/remodelling, which regulates the accessibility of DNA in chromatin. Recent studies have implicated histone acetyltransferase (HAT) complexes and chromatin acetylation in DNA repair; however, the precise underlying mechanism remains poorly understood. Here, we show that the HAT cofactor Trrap and Tip60 HAT bind to the chromatin surrounding sites of DNA double-strand breaks (DSBs) in vivo. Trrap depletion impairs both DNA-damage-induced histone H4 hyperacetylation and accumulation of repair molecules at sites of DSBs, resulting in defective homologous recombination (HR) repair, albeit with the presence of a functional ATM-dependent DNA-damage signalling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in Trrap-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of Trrap is due to impeded chromatin accessibility at sites of DNA breaks. Thus, these data reveal that cells may use the same basic mechanism involving HAT complexes to regulate distinct cellular processes, such as transcription and DNA repair.