Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 79
Filtrar
1.
Nucleic Acids Res ; 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39287133

RESUMO

G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in guanine (G)-rich genomic regions. X-linked dystonia parkinsonism (XDP) is an inherited neurodegenerative disease in which a SINE-VNTR-Alu (SVA) retrotransposon, characterised by amplification of a G-rich repeat, is inserted into the coding sequence of TAF1, a key partner of RNA polymerase II. XDP SVA alters TAF1 expression, but the cause of this outcome in XDP remains unknown. To assess whether G4s form in XDP SVA and affect TAF1 expression, we first characterised bioinformatically predicted XDP SVA G4s in vitro. We next showed that highly stable G4s can form and stop polymerase amplification at the SVA region from patient-derived fibroblasts and neural progenitor cells. Using chromatin immunoprecipitazion (ChIP) with an anti-G4 antibody coupled to sequencing or quantitative PCR, we showed that XDP SVA G4s are folded even when embedded in a chromatin context in patient-derived cells. Using the G4 ligands BRACO-19 and quarfloxin and total RNA-sequencing analysis, we showed that stabilisation of the XDP SVA G4s reduces TAF1 transcripts downstream and around the SVA, and increases upstream transcripts, while destabilisation using the G4 unfolder PhpC increases TAF1 transcripts. Our data indicate that G4 formation in the XDP SVA is a major cause of aberrant TAF1 expression, opening the way for the development of strategies to unfold G4s and potentially target the disease.

2.
Nucleic Acids Res ; 51(10): 5144-5161, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-37021550

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.


Assuntos
Distrofia Muscular Facioescapuloumeral , Humanos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Fatores de Transcrição/metabolismo
3.
Genes Dev ; 30(19): 2199-2212, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798843

RESUMO

In order to understand whether early epigenetic mechanisms instruct the long-term behavior of neural stem cells (NSCs) and their progeny, we examined Uhrf1 (ubiquitin-like PHD ring finger-1; also known as Np95), as it is highly expressed in NSCs of the developing brain and rapidly down-regulated upon differentiation. Conditional deletion of Uhrf1 in the developing cerebral cortex resulted in rather normal proliferation and neurogenesis but severe postnatal neurodegeneration. During development, deletion of Uhrf1 lead to global DNA hypomethylation with a strong activation of the intracisternal A particle (IAP) family of endogenous retroviral elements, accompanied by an increase in 5-hydroxymethylcytosine. Down-regulation of Tet enzymes rescued the IAP activation in Uhrf1 conditional knockout (cKO) cells, suggesting an antagonistic interplay between Uhrf1 and Tet on IAP regulation. As IAP up-regulation persists into postnatal stages in the Uhrf1 cKO mice, our data show the lack of means to repress IAPs in differentiating neurons that normally never express Uhrf1 The high load of viral proteins and other transcriptional deregulation ultimately led to postnatal neurodegeneration. Taken together, these data show that early developmental NSC factors can have long-term effects in neuronal differentiation and survival. Moreover, they highlight how specific the consequences of widespread changes in DNA methylation are for certain classes of retroviral elements.


Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/fisiopatologia , Genes de Partícula A Intracisternal/genética , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Sobrevivência Celular/genética , Córtex Cerebral/embriologia , Metilação de DNA , Deleção de Genes , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/virologia , Ubiquitina-Proteína Ligases , Ativação Viral/genética
4.
Nucleic Acids Res ; 49(6): 3217-3241, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33675667

RESUMO

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.


Assuntos
Cromatina/virologia , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Transativadores/metabolismo , Transcriptoma , Sítios de Ligação , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos
5.
Nucleic Acids Res ; 49(2): 847-863, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33410915

RESUMO

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.


Assuntos
Quadruplex G , Regulação Neoplásica da Expressão Gênica/genética , Lipossarcoma/terapia , Terapia de Alvo Molecular , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Neoplasias de Tecidos Moles/terapia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Ligantes , Modelos Genéticos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteína Supressora de Tumor p53/metabolismo
6.
Mol Cell ; 54(4): 675-82, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24768537

RESUMO

A complex network of regulatory pathways links transcription to cell growth and proliferation. Here we show that cellular quiescence alters chromatin structure by promoting trimethylation of histone H4 at lysine 20 (H4K20me3). In contrast to pericentric or telomeric regions, recruitment of the H4K20 methyltransferase Suv4-20h2 to rRNA genes and IAP elements requires neither trimethylation of H3K9 nor interaction with HP1 proteins but depends on long noncoding RNAs (lncRNAs) that interact with Suv4-20h2. Growth factor deprivation and terminal differentiation lead to upregulation of these lncRNAs, increase in H4K20me3, and chromatin compaction. The results uncover a lncRNA-mediated mechanism that guides Suv4-20h2 to specific genomic loci to establish a more compact chromatin structure in growth-arrested cells.


Assuntos
Cromatina/metabolismo , Inativação Gênica , Genes de RNAr , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Proliferação de Células , Cromatina/genética , Regulação da Expressão Gênica , Genes de Partícula A Intracisternal , Loci Gênicos , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/genética , Metilação , Camundongos , Células NIH 3T3 , RNA Longo não Codificante/genética , Telômero/genética
7.
Nucleic Acids Res ; 48(19): 10890-10908, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33021676

RESUMO

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Retrovirus Endógenos/genética , Regulação da Expressão Gênica/imunologia , Infecções por HIV/genética , Regiões Promotoras Genéticas , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Células HEK293 , Infecções por HIV/imunologia , HIV-1 , Humanos , Macaca mulatta , Subpopulações de Linfócitos T/citologia
8.
Gut ; 70(3): 485-498, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503845

RESUMO

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/metabolismo , Animais , Diferenciação Celular , Células Epiteliais/metabolismo , Feminino , Inativação Gênica , Homeostase/genética , Humanos , Mutação com Perda de Função , Masculino , Camundongos
9.
EMBO J ; 36(18): 2726-2741, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28778956

RESUMO

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.


Assuntos
Replicação do DNA , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Metilação
10.
Nucleic Acids Res ; 47(17): 9069-9086, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350899

RESUMO

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.


Assuntos
Cromatina/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Endoderma/citologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 3-beta Nuclear de Hepatócito/genética , Código das Histonas , Histonas/metabolismo , Camundongos , Camundongos Knockout , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Transdução de Sinais
11.
J Am Soc Nephrol ; 31(2): 257-278, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932472

RESUMO

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion-induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCIInegF4/80hiCD11blow macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII+F4/80hi cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCIInegF4/80hi cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII+F4/80hi cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII+F4/80hi cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.


Assuntos
Células Dendríticas/imunologia , Rim/imunologia , Macrófagos/imunologia , Nefrite/imunologia , Injúria Renal Aguda/imunologia , Fatores Etários , Animais , Antígeno CD11b/análise , Receptor 1 de Quimiocina CX3C/análise , Proteínas de Ligação ao Cálcio/análise , Cisplatino/farmacologia , Antígenos de Histocompatibilidade Classe II/análise , Rim/efeitos dos fármacos , Rim/metabolismo , Lectinas Tipo C/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/análise , Receptores Imunológicos/análise
12.
Genes Dev ; 27(8): 859-72, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23599346

RESUMO

Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4-20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as "chromocenter scattering." We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4-20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Linhagem Celular , Segregação de Cromossomos/fisiologia , Histona-Lisina N-Metiltransferase/genética , Camundongos , Mutação , Estrutura Terciária de Proteína , Transporte Proteico , Coesinas
13.
Development ; 143(10): 1788-99, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27013243

RESUMO

The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1.


Assuntos
Apoptose/genética , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Retroelementos/genética , Resposta a Proteínas não Dobradas/genética , Animais , Perfilação da Expressão Gênica , Inativação Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Vírus da Leucemia Murina/genética , Lisina/metabolismo , Metilação , Camundongos , Sequências Repetitivas de Ácido Nucleico/genética , Transcrição Gênica
14.
Nat Chem Biol ; 13(3): 317-324, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28114273

RESUMO

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.


Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular
15.
Cell Mol Life Sci ; 74(11): 2055-2065, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28160052

RESUMO

Endogenous retroviruses (ERV) are an abundant class of repetitive elements in mammalian genomes. To ensure genomic stability, ERVs are largely transcriptionally silent. However, these elements also feature physiological roles in distinct developmental contexts, under which silencing needs to be partially relieved. ERV silencing is mediated through a heterochromatic structure, which is established by histone modification and DNA methylation machineries. This heterochromatic structure is largely refractory to transcriptional stimulation, however, challenges to the heterochromatic state, such as DNA replication, require re-establishment of the heterochromatic state in competition with transcriptional activators. In this review, we discuss the major pathways leading to efficient establishment of robust and inaccessible heterochromatin across ERVs.


Assuntos
Retrovirus Endógenos/genética , Inativação Gênica , Heterocromatina/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Humanos , Modelos Biológicos , Retroelementos/genética
16.
EMBO Rep ; 16(7): 836-50, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26012739

RESUMO

More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle (IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide shRNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Genes de Partícula A Intracisternal , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Montagem e Desmontagem da Cromatina , Instabilidade Genômica , Heterocromatina/genética , Interferência de RNA , RNA Interferente Pequeno , Proteína Nuclear Ligada ao X
17.
Development ; 140(15): 3128-38, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23824574

RESUMO

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse ß-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of ß-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for ß-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of ß-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/ß-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/ß-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.


Assuntos
Endoderma/embriologia , Endoderma/metabolismo , Proteínas HMGB/metabolismo , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Fatores de Transcrição SOXF/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/deficiência , Proteínas HMGB/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Organizadores Embrionários/citologia , Gravidez , Fatores de Transcrição SOXF/deficiência , Fatores de Transcrição SOXF/genética , Tetraploidia , Fator de Transcrição 4 , beta Catenina/deficiência , beta Catenina/genética
18.
PLoS Genet ; 9(1): e1003188, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23382689

RESUMO

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.


Assuntos
Diferenciação Celular , Histona-Lisina N-Metiltransferase/genética , Placa Neural , Fatores do Domínio POU , Proteínas de Xenopus/genética , Xenopus laevis , Animais , Técnicas de Cultura de Células , Linhagem da Célula , Cromatina/genética , Metilação de DNA , Embrião não Mamífero , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Placa Neural/crescimento & desenvolvimento , Placa Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
19.
Mol Syst Biol ; 10: 746, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25134515

RESUMO

The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.


Assuntos
Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Inativação Gênica , Marcadores Genéticos , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Mitose , Células NIH 3T3 , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade
20.
Nucleic Acids Res ; 41(5): 2797-806, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23345616

RESUMO

Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. Nuclear DNA is packaged into chromatin, and thus genome maintenance can be influenced by distinct chromatin environments. In particular, post-translational modifications of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic instability, demonstrating the important functions of H4K20 methylation in genome maintenance. In this review, we explain molecular mechanisms underlying these defects and discuss novel ideas for furthering our understanding of genome maintenance in higher eukaryotes.


Assuntos
Epigênese Genética , Instabilidade Genômica , Histonas/metabolismo , Lisina/metabolismo , Animais , Ciclo Celular , Cromatina/metabolismo , Dano ao DNA , Replicação do DNA , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa