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1.
Mol Cell ; 84(5): 967-980.e10, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38242130

RESUMO

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.


Assuntos
Histonas , Óxido Nítrico , Histonas/genética , Histonas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Óxido Nítrico/metabolismo , Complexo Mediador/metabolismo , Oxirredutases/metabolismo
2.
EMBO J ; 43(7): 1187-1213, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38383863

RESUMO

Histone modifications commonly integrate environmental cues with cellular metabolic outputs by affecting gene expression. However, chromatin modifications such as acetylation do not always correlate with transcription, pointing towards an alternative role of histone modifications in cellular metabolism. Using an approach that integrates mass spectrometry-based histone modification mapping and metabolomics with stable isotope tracers, we demonstrate that elevated lipids in acetyltransferase-depleted hepatocytes result from carbon atoms derived from deacetylation of hyperacetylated histone H4 flowing towards fatty acids. Consistently, enhanced lipid synthesis in acetyltransferase-depleted hepatocytes is dependent on histone deacetylases and acetyl-CoA synthetase ACSS2, but not on the substrate specificity of the acetyltransferases. Furthermore, we show that during diet-induced lipid synthesis the levels of hyperacetylated histone H4 decrease in hepatocytes and in mouse liver. In addition, overexpression of acetyltransferases can reverse diet-induced lipogenesis by blocking lipid droplet accumulation and maintaining the levels of hyperacetylated histone H4. Overall, these findings highlight hyperacetylated histones as a metabolite reservoir that can directly contribute carbon to lipid synthesis, constituting a novel function of chromatin in cellular metabolism.


Assuntos
Carbono , Histonas , Animais , Camundongos , Histonas/metabolismo , Carbono/metabolismo , Lipogênese , Cromatina , Acetiltransferases/metabolismo , Lipídeos , Acetilação , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo
3.
Mol Cell ; 74(5): 1037-1052.e7, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31029542

RESUMO

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.


Assuntos
Complexo Repressor Polycomb 1/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cromatina/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição E2F6/genética , Heterocromatina/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Proteínas Repressoras/genética , Fator de Transcrição DP1/genética , Fatores de Transcrição/genética , Fatores Estimuladores Upstream/genética
4.
Mol Cell Proteomics ; 23(7): 100799, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38866077

RESUMO

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry-based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.


Assuntos
Neoplasias da Mama , Histonas , Processamento de Proteína Pós-Traducional , Histonas/metabolismo , Humanos , Animais , Camundongos , Neoplasias da Mama/metabolismo , Feminino , Espectrometria de Massas/métodos , Acilação , Especificidade de Órgãos , Acetilação , Proteômica/métodos
5.
Blood ; 142(9): 812-826, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37294917

RESUMO

Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.


Assuntos
Edição de Genes , Proteína Supressora de Tumor p53 , Humanos , Edição de Genes/métodos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células-Tronco Hematopoéticas/metabolismo , RNA/metabolismo , Sistemas CRISPR-Cas
6.
Genes Dev ; 31(4): 399-412, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28275002

RESUMO

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of cis-regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the cis-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping cis-regulatory elements with the ability to efficiently promote transcription.


Assuntos
Regulação da Expressão Gênica/genética , Genes Essenciais/genética , Macrófagos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Fatores de Transcrição/genética
7.
Mol Cell Proteomics ; 21(7): 100243, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35577067

RESUMO

Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning-based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein-RNA interactions and suggest a role in rewiring protein-protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain-containing octamer-binding protein]-paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.


Assuntos
Processamento de Proteína Pós-Traducional , Proteoma , Aminoácidos/metabolismo , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas , Metilação , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo
8.
Proteomics ; 23(23-24): e2200435, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37727062

RESUMO

The combined activity of epigenetic features, which include histone post-translational modifications, DNA methylation, and nucleosome positioning, regulates gene expression independently from changes in the DNA sequence, defining how the shared genetic information of an organism is used to generate different cell phenotypes. Alterations in epigenetic processes have been linked with a multitude of diseases, including cancer, fueling interest in the discovery of drugs targeting the proteins responsible for writing, erasing, or reading histone and DNA modifications. Mass spectrometry (MS)-based proteomics has emerged as a versatile tool that can assist drug discovery pipelines from target validation, through target deconvolution, to monitoring drug efficacy in vivo. Here, we provide an overview of the contributions of MS-based proteomics to epigenetic drug discovery, describing the main approaches that can be used to support different drug discovery pipelines and highlighting how they contributed to the development and characterization of epigenetic drugs.


Assuntos
Histonas , Proteômica , Histonas/metabolismo , Proteômica/métodos , Metilação de DNA , Epigênese Genética , Processamento de Proteína Pós-Traducional
9.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30389668

RESUMO

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Organismos Geneticamente Modificados , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
10.
Fungal Genet Biol ; 167: 103800, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37146898

RESUMO

In eukaryotes, the combination of different histone post-translational modifications (PTMs) - the histone code - impacts the chromatin organization as compact and transcriptionally silent heterochromatin or accessible and transcriptionally active euchromatin. Although specific histone PTMs have been studied in fungi, an overview of histone PTMs and their relative abundance is still lacking. Here, we used mass spectrometry to detect and quantify histone PTMs in three fungal species belonging to three distinct taxonomic sections of the genus Aspergillus (Aspergillus niger, Aspergillus nidulans (two strains), and Aspergillus fumigatus). We overall detected 23 different histone PTMs, including a majority of lysine methylations and acetylations, and 23 co-occurrence patterns of multiple histone PTMs. Among those, we report for the first time the detection of H3K79me1, H3K79me2, and H4K31ac in Aspergilli. Although all three species harbour the same PTMs, we found significant differences in the relative abundance of H3K9me1/2/3, H3K14ac, H3K36me1 and H3K79me1, as well as the co-occurrence of acetylation on both K18 and K23 of histone H3 in a strain-specific manner. Our results provide novel insights about the underexplored complexity of the histone code in filamentous fungi, and its functional implications on genome architecture and gene regulation.


Assuntos
Aspergillus nidulans , Histonas , Histonas/genética , Histonas/metabolismo , Código das Histonas/genética , Processamento de Proteína Pós-Traducional , Heterocromatina , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo
11.
BMC Cancer ; 23(1): 1236, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38102575

RESUMO

BACKGROUND: Currently, main treatment strategies for early-stage non-small cell lung cancer (ES-NSCLC) disease are surgery or stereotactic body radiation therapy (SBRT), with successful local control rates for both approaches. However, regional and distant failure remain critical in SBRT, and it is paramount to identify predictive factors of response to identify high-risk patients who may benefit from more aggressive approaches. The main endpoint of the MONDRIAN trial is to identify multi-omic biomarkers of SBRT response integrating information from the individual fields of radiomics, genomics and proteomics. METHODS: MONDRIAN is a prospective observational explorative cohort clinical study, with a data-driven, bottom-up approach. It is expected to enroll 100 ES-NSCLC SBRT candidates treated at an Italian tertiary cancer center with well-recognized expertise in SBRT and thoracic surgery. To identify predictors specific to SBRT, MONDRIAN will include data from 200 patients treated with surgery, in a 1:2 ratio, with comparable clinical characteristics. The project will have an overall expected duration of 60 months, and will be structured into five main tasks: (i) Clinical Study; (ii) Imaging/ Radiomic Study, (iii) Gene Expression Study, (iv) Proteomic Study, (v) Integrative Model Building. DISCUSSION: Thanks to its multi-disciplinary nature, MONDRIAN is expected to provide the opportunity to characterize ES-NSCLC from a multi-omic perspective, with a Radiation Oncology-oriented focus. Other than contributing to a mechanistic understanding of the disease, the study will assist the identification of high-risk patients in a largely unexplored clinical setting. Ultimately, this would orient further clinical research efforts on the combination of SBRT and systemic treatments, such as immunotherapy, with the perspective of improving oncological outcomes in this subset of patients. TRIAL REGISTRATION: The study was prospectively registered at clinicaltrials.gov (NCT05974475).


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Radiocirurgia , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/patologia , Multiômica , Estadiamento de Neoplasias , Estudos Observacionais como Assunto , Proteômica , Radiocirurgia/métodos
12.
EMBO Rep ; 22(3): e50852, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33586907

RESUMO

Transition from proliferative-to-invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down-regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR-depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re-expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor-initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient-rich conditions by repressing translation of selected ISR RNAs.


Assuntos
Melanoma , Preparações Farmacêuticas , RNA Longo não Codificante , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Fosforilação , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo
13.
Nucleic Acids Res ; 49(2): 791-804, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398338

RESUMO

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.


Assuntos
Enterócitos/metabolismo , Histonas/metabolismo , Intestino Delgado/citologia , Celulas de Paneth/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Animais , Catepsina L/metabolismo , Diferenciação Celular , Homeostase , Mucosa Intestinal/citologia , Camundongos , Microvilosidades/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Organoides , Domínios Proteicos , Tripsina/metabolismo
14.
Mol Cell ; 53(1): 49-62, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24289921

RESUMO

H3K27me3 is deposited at promoters by the preferential association of Polycomb repressive complex 2 (PRC2) with CpG-rich DNA elements regulating development by repressing gene transcription. H3K27 is also present in mono- and dimethylated states; however, the functional roles of H3K27me1 and H3K27me2 deposition remain poorly characterized. Here, we show that PRC2 activity is not only associated with H3K27me3 but also regulates all forms of H3K27 methylation in a spatially defined manner, contributing to different genomic functions in mouse embryonic stem cells. H3K27me1 accumulates within transcribed genes, promotes transcription, and is regulated by Setd2-dependent H3K36me3 deposition. Contrarily, H3K27me2 is present on approximately 70% of total histone H3 and is distributed in large chromatin domains, exerting protective functions by preventing firing of non-cell-type-specific enhancers. Considering that only 5%-10% of deregulated genes in PRC2-deficient cells are direct H3K27me3 targets, our data support an active role for all H3K27 methylated forms in regulating transcription and determining cell identity.


Assuntos
Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/imunologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Histonas/genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo
15.
Nucleic Acids Res ; 48(1): 96-115, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777917

RESUMO

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.


Assuntos
Epigênese Genética , MicroRNAs/genética , Proteínas do Fator Nuclear 90/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Animais , Arginina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Marcação por Isótopo , Lisina/metabolismo , Metilação , MicroRNAs/biossíntese , MicroRNAs/classificação , Proteínas do Fator Nuclear 90/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo
16.
Methods ; 184: 19-28, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31605746

RESUMO

Aberrations in histone post-translational modifications (PTMs) have been implicated with the development of numerous pathologies, including cancer. Therefore, profiling histone PTMs in patient samples could provide information useful for the identification of epigenetic biomarkers, as well as for the discovery of potential novel targets. While antibody-based methods have been traditionally employed to analyze histone PTM in clinical samples, mass spectrometry (MS) can provide a more comprehensive, unbiased and quantitative view on histones and their PTMs. To combine the power of MS-based methods and the potential offered by histone PTM profiling of clinical samples, we have recently developed a series of methods for the extraction and enrichment of histones from different types of patient samples, including formalin-fixed paraffin-embedded tissues, fresh- and optimal cutting temperature-frozen tissues, and primary cells. Here, we provide a detailed description of these protocols, together with indications on the expected results and the most suitable workflow to be used downstream of each procedure.


Assuntos
Técnicas de Preparação Histocitológica/métodos , Histonas/análise , Manejo de Espécimes/métodos , Células Cultivadas , Feminino , Histonas/metabolismo , Humanos , Masculino , Metilação , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
17.
Mol Cell ; 49(4): 645-56, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23352454

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina , Ilhas de CpG , Proteínas de Ligação a DNA/isolamento & purificação , Dioxigenases , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Imunoprecipitação , Redes e Vias Metabólicas/genética , Camundongos , N-Acetilglucosaminiltransferases/isolamento & purificação , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas/isolamento & purificação , Transdução de Sinais/genética , Sítio de Iniciação de Transcrição
18.
EMBO Rep ; 19(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29507079

RESUMO

Mitochondria are the energy-generating hubs of the cell. In spite of considerable advances, our understanding of the factors that regulate the molecular circuits that govern mitochondrial function remains incomplete. Using a genome-wide functional screen, we identify the poorly characterized protein Zinc finger CCCH-type containing 10 (Zc3h10) as regulator of mitochondrial physiology. We show that Zc3h10 is upregulated during physiological mitochondriogenesis as it occurs during the differentiation of myoblasts into myotubes. Zc3h10 overexpression boosts mitochondrial function and promotes myoblast differentiation, while the depletion of Zc3h10 results in impaired myoblast differentiation, mitochondrial dysfunction, reduced expression of electron transport chain (ETC) subunits, and blunted TCA cycle flux. Notably, we have identified a loss-of-function mutation of Zc3h10 in humans (Tyr105 to Cys105) that is associated with increased body mass index, fat mass, fasting glucose, and triglycerides. Isolated peripheral blood mononuclear cells from individuals homozygotic for Cys105 display reduced oxygen consumption rate, diminished expression of some ETC subunits, and decreased levels of some TCA cycle metabolites, which all together derive in mitochondrial dysfunction. Taken together, our study identifies Zc3h10 as a novel mitochondrial regulator.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Idoso , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Ciclo do Ácido Cítrico , Biologia Computacional/métodos , Metabolismo Energético , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Proteoma , Proteômica/métodos
19.
J Immunol ; 200(7): 2439-2454, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29500242

RESUMO

The enzymes of the poly-ADP-ribose polymerase (PARP) superfamily control many relevant cellular processes, but a precise understanding of their activities in different physiological or disease contexts is largely incomplete. We found that transcription of several Parp genes was dynamically regulated upon murine macrophage activation by endotoxin. PARP14 was strongly induced by several inflammatory stimuli and translocated into the nucleus of stimulated cells. Quantitative mass spectrometry analysis showed that PARP14 bound to a group of IFN-stimulated gene (ISG)-encoded proteins, most with an unknown function, and it was required for their nuclear accumulation. Moreover, PARP14 depletion attenuated transcription of primary antiviral response genes regulated by the IFN regulatory transcription factor 3, including Ifnb1, thus reducing IFN-ß production and activation of ISGs involved in the secondary antiviral response. In agreement with the above-mentioned data, PARP14 hindered Salmonella typhimurium proliferation in murine macrophages. Overall, these data hint at a role of PARP14 in the control of antimicrobial responses and specifically in nuclear activities of a subgroup of ISG-encoded proteins.


Assuntos
Interferon beta/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Poli(ADP-Ribose) Polimerases/genética , Salmonella typhimurium/imunologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Endotoxinas/imunologia , Edição de Genes , Ativação de Macrófagos/genética , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , Salmonella typhimurium/crescimento & desenvolvimento
20.
Nucleic Acids Res ; 46(8): 3817-3832, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618087

RESUMO

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.


Assuntos
Código das Histonas , Histonas/metabolismo , Neoplasias/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Xenoenxertos , Código das Histonas/genética , Histonas/genética , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Células Tumorais Cultivadas
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