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1.
Nat Commun ; 12(1): 5224, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471130

RESUMO

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição
2.
Nat Commun ; 12(1): 5240, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475390

RESUMO

ß-actin is a crucial component of several chromatin remodeling complexes that control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes (PRCs). While previous work has shown that ß-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in ß-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of ß-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that ß-actin levels can induce changes in chromatin structure by affecting the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2. Our results show that changes in ß-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversible changes in 3D genome architecture. Our findings reveal that ß-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.


Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Actinas/genética , Animais , Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Fibroblastos , Dosagem de Genes , Técnicas de Inativação de Genes , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo
3.
Se Pu ; 39(10): 1094-1101, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505431

RESUMO

Histone post-translational modifications (HPTMs) have been believed to play crucial roles in the regulation of gene transcription. Thus, aberrant modification of histone can contribute to the occurrence and development of diseases such as tumors. To date, formalin fixed paraffin-embedded (FFPE) clinical tissues are believed to be one of the most valuable sample resources in the study of human diseases. Therefore, it is of great significance to reveal the molecular mechanism of cancer and discover the markers of tumor. Proteomics, based on high performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS), has become a powerful tool for HPTM identification. However, HPTM analysis of FFPE samples is yet to be explored; it has not been reported in China to our best knowledge. In this study, a new method based on HPLC-MS/MS was developed for the extraction and separation of histone proteins and analysis and quantification of HPTMs in FFPE tissues. First, the strategy for the extraction and separation of histone proteins from FFPE samples were optimized. After comparing the extraction efficiency of two different methods, which include the acid extraction of histone and extraction of total protein, a novel method was developed for histone extraction, separation, and HPTMs analysis by integrating dewaxed hydration treatment of FFPE tissues with protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Furthermore, the effects of operation parameters on histone extraction and HPTM identification were investigated, including number of paraffin embedded sections and chemical derivation of histone proteins. Thereafter, the identification and quantification of HPTMs were performed using reversed-phase HPLC-MS/MS in data independent acquisition (DIA) mode. Finally, the optimized methods were applied to quantitative analysis of HPTMs in FFPE tissues. A variety of HPTMs were identified; they included lysine methylation, acetylation, crotonylation, etc. Therefore, the spectrum of HPTMs on global level was set for human breast cancer and paracancerous tissues from two cases of FFPE clinical tissues. The sites holding differential HPTM level in cancer and paracancerous tissues were further obtained by quantifying the individual HPTMs. Thus, the relationship between HPTM level and tumor was discussed. Abnormal HPTM sites were characterized using cluster analysis, thus their similar tendency was found. For example, abnormal HPTM sites such as H3K9me3, H3K9ac, and H3K27me3 in cancers are associated with epigenetic regulation. It indicated that different epigenetic events might occur in cancer and paracancerous tissues. Interestingly, we found that the level of H4K20me3 were both significantly down-regulated in the two cancer tissues. HPTM had been thought to be a potential biomarker in breast cancer; therefore, these positive results indicated that our method is effective for HPTMs of clinical FFPE samples. Our study provides a useful tool for the isolation and analysis of HPTMs in clinical FFPE samples, showing the potential for the detection of epigenetic biomarker in cancer.


Assuntos
Histonas , Espectrometria de Massas em Tandem , Epigênese Genética , Formaldeído , Histonas/metabolismo , Humanos , Inclusão em Parafina , Processamento de Proteína Pós-Traducional
4.
J Med Food ; 24(9): 978-986, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34524028

RESUMO

Epigenetic regulation by histone acetyltransferase (HAT) is associated with various biological processes and the progression of diseases, including nonalcoholic fatty liver disease (NAFLD). The objective of this study was to investigate whether the hypolipidemic properties of black mulberry (Morus atropurpurea Roxb.) fruit extract (BME) contribute toward protection against NAFLD by HAT inhibition. HepG2 cells were treated with oleic and palmitic acids to induce lipid accumulation, which was significantly attenuated by the treatment with BME at 50 and 100 µg/mL. BME also markedly reduced the expression of proteins associated with lipogenesis, which was attributed to the BME-mediated downregulation of lipogenic genes in HepG2 cells. BME significantly inhibited in vitro total HAT and p300 activities. In addition, BME suppressed total acetylated lysine as well as specific histone acetylation of proteins H3K14 and H3K27 in HepG2 cells. Mice were then fed with either a chow diet or western diet (WD), with or without BME (1%, w/w) supplementation, for 12 weeks to confirm hypolipidemic activity of BME. BME attenuated serum nonesterified fatty acids and low-density lipoprotein (LDL) cholesterol levels, which was likely associated with the downregulation of hepatic lipogenic gene expression in WD-fed obese mice. Taken together, the hypolipidemic activity of BME was observed in HepG2 cells treated with fatty acids as well as in livers of obese mice, and the hepatoprotection of BME is likely associated with the inhibition of acetylation. Further investigation is warranted to determine whether BME can be developed into an efficacious dietary intervention to attenuate the progression of NAFLD by epigenetic regulation in clinical settings.


Assuntos
Morus , Hepatopatia Gordurosa não Alcoólica , Acetilação , Animais , Dieta Hiperlipídica/efeitos adversos , Epigênese Genética , Frutas/metabolismo , Células Hep G2 , Histonas/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia
5.
Nucleic Acids Res ; 49(15): 8961-8973, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365506

RESUMO

Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3(1-34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1-15) and H3-middle (e.g. 14-34) of H3(1-34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14PZP, in which a PHF14-unique insertion loop but not the core ß-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a 'ground' state (unmodified) H3(1-34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.


Assuntos
Histonas/química , Histonas/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Regulação Alostérica , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Proteínas de Peixe-Zebra/genética
6.
Nat Commun ; 12(1): 4800, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417450

RESUMO

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.


Assuntos
Cromatina/metabolismo , Genes Essenciais , Histonas/metabolismo , Lisina/metabolismo , Transcrição Genética , Sequência de Aminoácidos , Animais , Ciclo Celular/genética , Linhagem Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Modelos Biológicos , Nucleossomos/metabolismo , Conformação Proteica
7.
Nat Commun ; 12(1): 5056, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417458

RESUMO

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.


Assuntos
Epigênese Genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Micrometástase de Neoplasia , Ligação Proteica , Carga Tumoral
8.
Nat Commun ; 12(1): 4750, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362907

RESUMO

Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Mutação , Fatores de Transcrição
9.
Nat Cell Biol ; 23(8): 905-914, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354237

RESUMO

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Heterocromatina/fisiologia , Células Cultivadas , Sequência Conservada , Células Hep G2 , Histonas/metabolismo , Humanos
10.
Methods Mol Biol ; 2351: 3-22, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382181

RESUMO

Knowledge in gene transcription and chromatin regulation has been intensely studied for decades, but thanks to next-generation sequencing (NGS) techniques there has been a major leap forward in the last few years. Historically, identification of specific enhancer elements has led to the identification of master transcription factors (TFs) in the 1990s. Genetic and biochemical experiments have identified the key regulators controlling RNA polymerase II (RNAPII) transcription and structurally analyses have elucidated detailed mechanisms. NGS and the development of chromatin immunoprecipitation (ChIP) have accelerated the gain of knowledge in the recent years. By now, we have a dazzling wealth of techniques that are currently used to put gene expression into a genome-wide context. This book is an attempt to assemble useful protocols for many researchers within and nearby research areas. In general, these innovative techniques focus on enhancer and promoter studies. The techniques should also be of interest for related fields such as DNA repair and replication.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo
11.
Methods Mol Biol ; 2351: 147-161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382188

RESUMO

Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.


Assuntos
Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/metabolismo , Sítios de Ligação , Histonas/metabolismo , Ligação Proteica
12.
Methods Mol Biol ; 2351: 251-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382194

RESUMO

In this chapter, we describe the proteomic approach named "Native Chromatin Proteomics" (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Genômica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Cromatografia Líquida , Proteínas de Ligação a DNA/metabolismo , Análise de Dados , Genômica/métodos , Nucleossomos/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem
13.
Methods Mol Biol ; 2351: 275-288, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382195

RESUMO

Functionalization of the genome is carried out by proteins that bind to DNA to regulate gene expression. Since this process is highly dynamic, context-dependent, and rarely performed by single proteins alone, we here describe ChIP-SICAP to identify proteins that co-localize with a protein of interest on the genome. Benefiting from its nature as a dual purification approach via ChIP and DNA biotinylation, ChIP-SICAP distinguishes genuine chromatin-binders and is uniquely placed to identify novel players in genome regulation.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Biotinilação , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Peptídeo Hidrolases , Ligação Proteica , Proteômica/métodos
14.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360892

RESUMO

The explosive development of next-generation sequencing-based technologies has allowed us to take an unprecedented look at many molecular signatures of the non-coding genome. In particular, the ChIP-seq (Chromatin ImmunoPrecipitation followed by sequencing) technique is now very commonly used to assess the proteins associated with different non-coding DNA regions genome-wide. While the analysis of such data related to transcription factor binding is relatively straightforward, many modified histone variants, such as H3K27me3, are very important for the process of gene regulation but are very difficult to interpret. We propose a novel method, called HERON (HiddEn MaRkov mOdel based peak calliNg), for genome-wide data analysis that is able to detect DNA regions enriched for a certain feature, even in difficult settings of weakly enriched long DNA domains. We demonstrate the performance of our method both on simulated and experimental data.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/genética , DNA/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Adulto , Algoritmos , Expressão Gênica , Regulação da Expressão Gênica , Hipocampo/embriologia , Hipocampo/metabolismo , Código das Histonas/genética , Humanos , Fígado/metabolismo , Metilação , Distribuição Normal , Ligação Proteica
15.
Nat Commun ; 12(1): 5016, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408138

RESUMO

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
16.
Nat Commun ; 12(1): 5015, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408139

RESUMO

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/química , Proteômica
17.
Sci Rep ; 11(1): 17351, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34456333

RESUMO

Coronavirus disease 2019 (COVID-19) is raging worldwide. This potentially fatal infectious disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the complete mechanism of COVID-19 is not well understood. Therefore, we analyzed gene expression profiles of COVID-19 patients to identify disease-related genes through an innovative machine learning method that enables a data-driven strategy for gene selection from a data set with a small number of samples and many candidates. Principal-component-analysis-based unsupervised feature extraction (PCAUFE) was applied to the RNA expression profiles of 16 COVID-19 patients and 18 healthy control subjects. The results identified 123 genes as critical for COVID-19 progression from 60,683 candidate probes, including immune-related genes. The 123 genes were enriched in binding sites for transcription factors NFKB1 and RELA, which are involved in various biological phenomena such as immune response and cell survival: the primary mediator of canonical nuclear factor-kappa B (NF-κB) activity is the heterodimer RelA-p50. The genes were also enriched in histone modification H3K36me3, and they largely overlapped the target genes of NFKB1 and RELA. We found that the overlapping genes were downregulated in COVID-19 patients. These results suggest that canonical NF-κB activity was suppressed by H3K36me3 in COVID-19 patient blood.


Assuntos
COVID-19/genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Histonas/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Sítios de Ligação , COVID-19/metabolismo , Estudos de Casos e Controles , Epigênese Genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Aprendizado de Máquina , Transdução de Sinais
18.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445278

RESUMO

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Epigênese Genética , Histonas/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Caracteres Sexuais , Injeções de Esperma Intracitoplásmicas , Adulto , Feminino , Humanos , Recém-Nascido , Masculino , Metilação , Gravidez
19.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445348

RESUMO

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.


Assuntos
Fibroblastos/fisiologia , Histonas/metabolismo , PPAR gama/fisiologia , Ligamento Periodontal/fisiologia , Acetilação , Diferenciação Celular/genética , Células Cultivadas , Cementogênese/genética , Cementogênese/fisiologia , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/química , Humanos , Osteogênese/genética , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Processamento de Proteína Pós-Traducional/genética
20.
Science ; 373(6552): 306-315, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437148

RESUMO

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fatores de Transcrição/química
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