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1.
EMBO Rep ; 25(2): 725-744, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38177923

RESUMO

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.


Assuntos
Herpesvirus Humano 6 , Proteínas Imediatamente Precoces , Infecções por Roseolovirus , Humanos , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Infecções por Roseolovirus/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Integração Viral , Instabilidade Genômica , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
2.
Mol Cell ; 72(2): 250-262.e6, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30270107

RESUMO

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.


Assuntos
Cromatina/genética , Reparo do DNA/genética , Histonas/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Humanos , Células K562 , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
3.
Mol Cell Proteomics ; 21(7): 100253, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35636729

RESUMO

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2-BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.


Assuntos
Fatores de Transcrição , Cromatina/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo
4.
Trends Biochem Sci ; 44(3): 226-240, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30638972

RESUMO

Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.


Assuntos
Proteína BRCA2/metabolismo , Anemia de Fanconi/metabolismo , Animais , Proteína BRCA2/genética , Dano ao DNA/genética , Anemia de Fanconi/genética , Feminino , Humanos , Mutação/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia
5.
Nucleic Acids Res ; 45(22): 12715-12722, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29036442

RESUMO

H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Histonas/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Alfa-Amanitina/farmacologia , Flavonoides/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histonas/metabolismo , Humanos , Modelos Genéticos , Nucleossomos/genética , Nucleossomos/metabolismo , Piperidinas/farmacologia
6.
Nat Commun ; 14(1): 6051, 2023 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-37770430

RESUMO

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.


Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Humanos , Linhagem Celular Tumoral , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Acetilação , Melanoma/genética , Melanoma/metabolismo , Melanócitos/metabolismo
7.
DNA Repair (Amst) ; 113: 103315, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35278769

RESUMO

In eukaryotic cells, DNA double-strand breaks (DSBs) can be repaired through two main pathways, non-homologous end-joining (NHEJ) or homologous recombination (HR). The selection of the repair pathway choice is governed by an antagonistic relationship between repair factors specific to each pathway, in a cell cycle-dependent manner. The molecular mechanisms of this decision implicate post-translational modifications of chromatin surrounding the break. Here, we discuss the recent advances regarding the function of the NuA4/TIP60 histone acetyltransferase/chromatin remodeling complex during DSBs repair. In particular, we emphasise the contribution of NuA4/TIP60 in repair pathway choice, in collaboration with the SAGA acetyltransferase complex, and how they regulate chromatin dynamics, modify non-histone substrates to allow DNA end resection and recombination.

8.
Methods Mol Biol ; 1983: 57-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31087293

RESUMO

The acetylation of the ε-amine of lysine residues has significant impacts on the cellular functions of proteins. Through the combination of unbiased and targeted analysis of acetylated proteins, biological insights on lysine acetylation are now routinely generated. To help in this endeavor, we describe detailed protocols for the identification of acetylated lysine residues and the preparation of multiple reagents for the characterization of these sites in order to obtain functional insights on this widespread modification.


Assuntos
Lisina Acetiltransferases/metabolismo , Lisina/metabolismo , Acetilação , Linhagem Celular , Ensaios Enzimáticos , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Humanos , Lisina/química , Lisina Acetiltransferases/química , Lisina Acetiltransferases/isolamento & purificação , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Leveduras/metabolismo
9.
Sci Rep ; 8(1): 14089, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237520

RESUMO

Regulation of the chromatin state is crucial for biological processes such as the regulation of transcription, DNA replication, and DNA damage repair. Here we show that knockdown of the BRD8 bromodomain protein - a subunit of the p400/Tip60 complex - leads to p21 induction, and concomitant cell cycle arrest in G1/S. We further demonstrate that the p53 transcriptional pathway is activated in BRD8-depleted cells, and this accounts for upregulation of not only p21 but also of pro-apoptotic genes, leading to subsequent apoptosis. Importantly, the DNA damage response (DDR) is induced upon BRD8 depletion, and DNA damage foci are detectable in BRD8-depleted cells under normal growth conditions. Consistently with an activated DDR, we find that in BRD8-depleted cells, the ATM-CHK2 DDR pathway is turned on but, CHK1 proteins levels are severely reduced and replication stress is detectable as enhanced replication protein A (RPA32) phosphorylation levels. Notably, acetylation of histone H4 at K16 (H4K16ac) is reduced in BRD8-depleted cells, suggesting that BRD8 may have a role in the recruitment and/or stabilization of the p400/Tip60 complex within chromatin, thereby facilitating DNA repair. Taken together, our results suggest that BRD8 is involved not only in p53-dependent gene suppression, but also in the maintenance of genome stability.


Assuntos
Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA/genética , Receptores dos Hormônios Tireóideos/genética , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Fosforilação , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição
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