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1.
Cell ; 163(1): 230-45, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26365490

RESUMO

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.


Assuntos
Células-Tronco Embrionárias/virologia , Retrovirus Endógenos/genética , Provírus/genética , Animais , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Células-Tronco de Carcinoma Embrionário/virologia , Epigênese Genética , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
2.
Cell ; 157(7): 1712-23, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24949978

RESUMO

In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.


Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Fúngico/genética , RNA não Traduzido/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA não Traduzido/metabolismo , Terminação da Transcrição Genética , Transcrição Gênica
3.
Genes Dev ; 33(11-12): 669-683, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30975723

RESUMO

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.


Assuntos
Antígenos CD4/genética , Fator 1 de Modelagem da Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos CD4/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Chaperonas de Histonas/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Domínios Proteicos
4.
Mol Cell ; 69(5): 879-892.e5, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29478807

RESUMO

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast. We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.


Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Chaperonas Moleculares/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Dano ao DNA/fisiologia , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Células K562 , Chaperonas Moleculares/genética , NF-kappa B/genética , Proteínas Nucleares/genética , Rad51 Recombinase/genética
5.
Mol Cell Proteomics ; 23(7): 100795, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38848995

RESUMO

At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Epigênese Genética , Chaperonas de Histonas , Histonas , Arabidopsis/genética , Arabidopsis/metabolismo , Histonas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Mutação , Processamento de Proteína Pós-Traducional , Regulação da Expressão Gênica de Plantas , Fator 1 de Modelagem da Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/genética
6.
Plant J ; 118(6): 1922-1936, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38493352

RESUMO

Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fator 1 de Modelagem da Cromatina , Proteínas de Ligação a DNA , Homeostase do Telômero , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator 1 de Modelagem da Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Mutação , Telômero/metabolismo , Telômero/genética , Cromossomos de Plantas/metabolismo
7.
EMBO J ; 40(10): e106632, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33739466

RESUMO

HIV-1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV-1-infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency-reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF-1 (chromatin assembly factor 1) is enriched on the HIV-1 long terminal repeat (LTR) and forms nuclear bodies with liquid-liquid phase separation (LLPS) properties. CAF-1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV-1 latency in different latency models and primary CD4+ T cells. Three disordered regions of the CHAF1A subunit are important for phase-separated CAF-1 nuclear body formation and play a key role in maintaining HIV-1 latency. Disruption of phase-separated CAF-1 bodies could be a potential strategy to reactivate latent HIV-1.


Assuntos
HIV-1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Células HEK293 , Humanos , Regiões Promotoras Genéticas/genética
8.
J Cell Sci ; 136(10)2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37129573

RESUMO

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.


Assuntos
Proteínas Cromossômicas não Histona , Histonas , Humanos , Histonas/genética , Proteína Centromérica A/genética , Células HeLa , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromatina , Centrômero/metabolismo , Chaperonas Moleculares/metabolismo , Instabilidade Cromossômica , Autoantígenos/genética , Fator 1 de Modelagem da Cromatina/genética
9.
Blood ; 142(8): 711-723, 2023 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-37216686

RESUMO

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.


Assuntos
Cromossomos Humanos Par 21 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Criança , Cromossomos Humanos Par 21/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberrações Cromossômicas , Citogenética , Genômica , Fator 1 de Modelagem da Cromatina/genética
10.
Nucleic Acids Res ; 51(8): 3770-3792, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36942484

RESUMO

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.


Assuntos
Cromatina , Histonas , Histonas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Cromatina/genética , Replicação do DNA , DNA/genética
11.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35074917

RESUMO

Early events of the retroviral life cycle are the targets of many host restriction factors that have evolved to prevent establishment of infection. Incoming retroviral DNAs are transcriptionally silenced before integration in most cell types, and efficient viral gene expression occurs only after formation of the provirus. The molecular machinery for silencing unintegrated retroviral DNAs of HIV-1 remains poorly characterized. Here, we identified the histone chaperones CHAF1A and CHAF1B as essential factors for silencing of unintegrated HIV-1 DNAs. Using RNAi-mediated knockdown (KD) of multiple histone chaperones, we found that KD of CHAF1A or CHAF1B resulted in a pronounced increase in expression of incoming viral DNAs. The function of these two proteins in silencing was independent of their interaction partner RBBP4. Viral DNA levels accumulated to significantly higher levels in CHAF1A KD cells over controls, suggesting enhanced stabilization of actively transcribed DNAs. Chromatin immunoprecipitation assays revealed no major changes in histone loading onto viral DNAs in the absence of CHAF1A, but levels of the H3K9 trimethylation silencing mark were reduced. KD of the H3K9me3-binding protein HP1γ accelerated the expression of unintegrated HIV-1 DNAs. While CHAF1A was critical for silencing HIV-1 DNAs, it showed no role in silencing of unintegrated retroviral DNAs of mouse leukemia virus. Our study identifies CHAF1A and CHAF1B as factors involved specifically in silencing of HIV-1 DNAs early in infection. The results suggest that these factors act by noncanonical pathways, distinct from their histone loading activities, to mediate silencing of newly synthesized HIV-1 DNAs.


Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , DNA Viral , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Provírus/genética , Integração Viral , Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Transcrição Gênica , Proteína 28 com Motivo Tripartido/metabolismo
12.
Mol Microbiol ; 119(5): 574-585, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36855815

RESUMO

The CUG-Ser1 clade-specific histone H3 variant (H3VCTG ) has been reported to be a negative regulator of planktonic to biofilm growth transition in Candida albicans. The preferential binding of H3VCTG at the biofilm gene promoters makes chromatin repressive for the biofilm mode of growth. The two evolutionarily conserved chaperone complexes involved in incorporating histone H3 are CAF-1 and HIRA. In this study, we sought to identify the chaperone complex(es) involved in loading H3VCTG . We demonstrate that C. albicans cells lacking either Cac1 or Cac2 subunit of the CAF-1 chaperone complex, exhibit a hyper-filamentation phenotype on solid surfaces and form more robust biofilms than wild-type cells, thereby mimicking the phenotype of the H3VCTG null mutant. None of the subunits of the HIRA chaperone complex shows any significant difference in biofilm growth as compared to the wild type. The occupancy of H3VCTG is found to be significantly reduced at the promoters of biofilm genes in the absence of CAF-1 subunits. Hence, we provide evidence that CAF-1, a chaperone known to load canonical histone H3 in mammalian cells, is involved in chaperoning of variant histone H3VCTG at the biofilm gene promoters in C. albicans. Our findings also illustrate the acquisition of an unconventional role of the CAF-1 chaperone complex in morphogenesis in C. albicans.


Assuntos
Candida albicans , Histonas , Animais , Histonas/genética , Histonas/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Cromatina , Fator 1 de Modelagem da Cromatina/química , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biofilmes , Mamíferos/genética , Mamíferos/metabolismo
13.
BJOG ; 131(2): 157-162, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37264725

RESUMO

OBJECTIVE: To examine the association of placental and fetal DNA copy number variants (CNVs) with fetal structural malformations (FSMs) in stillborn fetuses. DESIGN: A secondary analysis of stillbirth cases in the Stillbirth Collaborative Research Network (SCRN) study. SETTING: Multicenter, 59 hospitals in five geographic regions in the USA. POPULATION: 388 stillbirth cases of the SCRN study (2006-2008). METHODS: Fetal structural malformations were grouped by anatomic system and specific malformation type (e.g. central nervous system, thoracic, cardiac, gastrointestinal, skeletal, umbilical cord and craniofacial defects). Single-nucleotide polymorphism array detected CNVs of at least 500 kb. CNVs were classified into two groups: normal, defined as no CNVs >500 kb or benign CNVs, and abnormal, defined as pathogenic or variants of unknown clinical significance. MAIN OUTCOME MEASURES: The proportions of abnormal CNVs and normal CNVs were compared between stillbirth cases with and without FSMs using the Wald Chi-square test. RESULTS: The proportion of stillbirth cases with any FSMs was higher among those with abnormal CNVs than among those with normal CNVs (47.5 versus 19.1%; P-value <0.001). The most common organ system-specific FSMs associated with abnormal CNVs were cardiac defects, followed by hydrops, craniofacial defects and skeletal defects. A pathogenic deletion of 1q21.1 involving 46 genes (e.g. CHD1L) and a duplication of 21q22.13 involving four genes (SIM2, CLDN14, CHAF1B, HLCS) were associated with a skeletal and cardiac defect, respectively. CONCLUSION: Specific CNVs involving several genes were associated with FSMs in stillborn fetuses. The findings warrant further investigation and may inform counselling and care surrounding pregnancies affected by FSMs at risk for stillbirth.


Assuntos
Variações do Número de Cópias de DNA , Natimorto , Gravidez , Feminino , Humanos , Natimorto/epidemiologia , Natimorto/genética , Variações do Número de Cópias de DNA/genética , Aberrações Cromossômicas , Placenta , Feto/anormalidades , Diagnóstico Pré-Natal , Fator 1 de Modelagem da Cromatina/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética
14.
Mol Cell Proteomics ; 21(10): 100411, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36089195

RESUMO

Chromatin structure, transcription, DNA replication, and repair are regulated via locus-specific incorporation of histone variants and posttranslational modifications that guide effector chromatin-binding proteins. Here we report unbiased, quantitative interactomes for the replication-coupled (H3.1) and replication-independent (H3.3) histone H3 variants based on BioID proximity labeling, which allows interactions in intact, living cells to be detected. Along with a significant proportion of previously reported interactions detected by affinity purification followed by mass spectrometry, three quarters of the 608 histone-associated proteins that we identified are new, uncharacterized histone associations. The data reveal important biological nuances not captured by traditional biochemical means. For example, we found that the chromatin assembly factor-1 histone chaperone not only deposits the replication-coupled H3.1 histone variant during S-phase but also associates with H3.3 throughout the cell cycle in vivo. We also identified other variant-specific associations, such as with transcription factors, chromatin regulators, and with the mitotic machinery. Our proximity-based analysis is thus a rich resource that extends the H3 interactome and reveals new sets of variant-specific associations.


Assuntos
Chaperonas de Histonas , Histonas , Histonas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Cromatina , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Nucleossomos
15.
J Integr Plant Biol ; 65(1): 203-222, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36541721

RESUMO

Minichromosome Maintenance protein 10 (MCM10) is essential for DNA replication initiation and DNA elongation in yeasts and animals. Although the functions of MCM10 in DNA replication and repair have been well documented, the detailed mechanisms for MCM10 in these processes are not well known. Here, we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene. Although plant MCM10 possesses a similar crystal structure as animal MCM10, AtMCM10 is not essential for plant growth or development in Arabidopsis. AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro. The nucleosome density is decreased in Atmcm10, and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1 (CAF-1). Loss of both AtMCM10 and CAF-1 is embryo lethal, indicating that AtMCM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly. AtMCM10 interacts with both new and parental histones. Atmcm10 mutants have lower H3.1 abundance and reduced H3K27me1/3 levels with releasing some silenced transposons. We propose that AtMCM10 deposits new and parental histones during nucleosome assembly, maintaining proper epigenetic modifications and genome stability during DNA replication.


Assuntos
Arabidopsis , Histonas , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Replicação do DNA/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo
16.
Eur J Immunol ; 51(7): 1698-1714, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33949677

RESUMO

CD4+ CD8+ double-positive thymocytes give rise to both conventional TCRαß+ T cells and invariant natural killer T cells (iNKT cells), but these two kinds of cells display different characteristics. The molecular mechanism underlying iNKT cell lineage development and function acquisition remain to be elucidated. We show that the loss of chromatin assembly factor 1B (CHAF1b) maintains the normal development of conventional TCRαß+ T cells but severely impairs early development of iNKT cells. This dysregulation is accompanied by the impairment in chromatin activation and gene transcription at Vα14-Jα18 locus. Notably, ectopic expression of a Vα14-Jα18 TCR rescues Chaf1b-deficient iNKT cell developmental defects. Moreover, cytokine secretion and antitumor activity are substantially maintained in Vα14-Jα18 TCR transgene-rescued Chaf1b-deficient iNKT cells. Our study identifies CHAF1b as a critical factor that controls the early development but not function acquisition of iNKT cells via lineage- and stage-specific regulation.


Assuntos
Fator 1 de Modelagem da Cromatina/imunologia , Células T Matadoras Naturais/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Montagem e Desmontagem da Cromatina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/imunologia
17.
J Transl Med ; 20(1): 296, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35773729

RESUMO

PURPOSE: Aberrant epigenetic changes, like DNA methylation, histone modifications, or ubiquitination, could trigger metabolic disorders in human cancer cells. This study planed to uncover the biological roles of epigenetic SPOP/CHAF1A axis in modulating tumor autophagy during Diffuse large B-cell lymphoma (DLBCL) tumorigenesis. MATERIALS AND METHODS: The Immunohistochemistry (IHC) was performed to assess the CHAF1A expressions. The expression data of CHAF1A was derived from The Cancer Genome Atlas (TCGA), GSE32918 and GSE83632 datasets. Bioinformatic assays contain differential analysis, functional enrichment analysis and Kaplan-Meier survival curve analysis. The colony generation assay, Transwell assay and CCK-8 assays were conducted for the in vitro assays. The in vivo ubiquitination assays were used to assess regulations of SPOP on CHAF1A. The Chromatin immunoprecipitation (ChIP) assays were used to uncover epigenetic regulations of CHAF1A on TFEB. The relevant DLBCL cells were subcutaneously injected to SCID beige mice to establish the xenograft models. RESULTS: Bioinformatic results revealed that CHAF1A expressed highly in DLBCL that were validated in patients samples. Patients with high CHAF1A suffered from inferior prognosis with shorter survival months relative to those with low CHAF1A. High CHAF1A enhanced DLBCL aggressiveness, including cell proliferation, migration and in vivo growth. Mechanistically, E3 ubiquitin ligase SPOP binds to and induces the degradative ubiquitination of CHAF1A via recognizing a consensus SPOP-binding motif in CHAF1A. SPOP is down-regulated in DLBCL and habours two DLBCL-associated mutations. Deficient SPOP leads to accumulated CHAF1A proteins that promote malignant features of DLBCL. Subsequently, ChIP-qPCR assay revealed that CHAF1A directly binds to TFEB promoters to activate the expressions. High CHAF1A could enhance the transcriptional activity of TFEB and downstream genes. The SPOP/CHAF1A axis modulates TFEB-dependent transactivation to regulate the lysosomal biogenesis and autophagy. The in vivo models suggested that TFEB inhibition is effective to suppress growth of SPOP-deficient DLBCLs. CONCLUSIONS: CHAF1A is aberrantly elevated in SPOP-deficient DLBCL. The in-depth mechanism understanding of SPOP/CHAF1A/TFEB axis endows novel targets for DLBCL treatment.


Assuntos
Linfoma Difuso de Grandes Células B , Proteínas Nucleares , Proteínas Repressoras , Animais , Autofagia/fisiologia , Linhagem Celular Tumoral , Fator 1 de Modelagem da Cromatina/metabolismo , Xenoenxertos , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos SCID , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitinação
18.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232396

RESUMO

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.


Assuntos
Cromatina , Replicação do DNA , Aminoácidos/metabolismo , Arginina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/química , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , DNA/metabolismo , Humanos , Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
Int J Mol Sci ; 23(4)2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-35216276

RESUMO

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.


Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Cromatina/metabolismo , Domínios Proteicos/fisiologia , Proteínas Quinases/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
20.
Curr Genet ; 67(1): 129-139, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33025160

RESUMO

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells to maintain chromatin structure during DNA replication. The deposition of newly-synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. CAF-1 is an important histone chaperone complex and its main subunit, Cac1p, contains a PIP and WHD domain for interaction with PCNA and the DNA, respectively. While Cac1p subunit was extensively studied in different systems much less is known regarding the importance of the PIP and WHD domains in replication fork progression and genome stability. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in these Cac1p domains affect replication fork progression and post-replication characteristics. Our experiments revealed that mutations in the Cac1p WHD domain, which abolished the CAF-1-DNA interaction, slows down replication fork progression. In contrast, mutations in Cac1p PIP domain, abolishing Cac1p-PCNA interaction, lead to extended late-S/Anaphase duration, elevated number of RPA foci and increased spontaneous mutation rate. Our research shows that Cac1p WHD and PIP domains have distinct roles in high replisome progression and maintaining genome stability during cell cycle progression.


Assuntos
Fator 1 de Modelagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Replicação do DNA/genética , Humanos , Nucleossomos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Saccharomyces cerevisiae/genética
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