RESUMO
AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.
Assuntos
Cromatina , Reparo do DNA , Proteínas de Ligação a DNA , Imunidade , Fatores de Transcrição , Humanos , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Recombinação Homóloga/genética , Imunidade/genética , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/imunologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transativadores , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via the concerted interplay among different layers, including DNA, histone post-translational modifications (hPTMs) and chromatin-associated proteins. Here, we employ multi-layered proteomics to characterize chromatin-mediated functional interactions of repair proteins, signatures of hPTMs and the DNA-bound proteome during DNA double-strand break (DSB) repair at high temporal resolution. Our data illuminate the dynamics of known and novel DDR-associated factors both at chromatin and at DSBs. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining (NHEJ), homologous recombination (HR) and DSB repair pathway choice. We reveal histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and involved in poly-ADP-ribose polymerase-inhibitor sensitivity. Furthermore, we distinguish hPTMs that are globally induced by DNA damage from those specifically acquired at sites flanking DSBs (γH2AX foci-specific) and profiled their dynamics during the DDR. Integration of complementary chromatin layers implicates G9A-mediated monomethylation of H3K56 in DSBs repair via HR. Our data provide a dynamic chromatin-centered view of the DDR that can be further mined to identify novel mechanistic links and cell vulnerabilities in DSB repair.
Assuntos
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Proteômica , Reparo do DNA , Reparo do DNA por Junção de Extremidades , DNA , Reparo de DNA por RecombinaçãoRESUMO
Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.
Assuntos
Distúrbios no Reparo do DNA , Histonas , Criança , Humanos , Histonas/genética , Nucleotidiltransferases/genética , Imunidade , DNARESUMO
Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mecanotransdução Celular , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Via de Sinalização Hippo , Humanos , Masculino , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Mecânico , Fatores de Transcrição de Domínio TEA , Transativadores , Fatores de Transcrição/metabolismo , Via de Sinalização WntRESUMO
Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state. Trim24, a previously unrecognized protein in the network, converges with OSN on multiple enhancers and suppresses the expression of developmental genes while activating cell cycle genes. Consistently, Trim24 significantly improved efficiency of cellular reprogramming, demonstrating its direct functionality in establishing pluripotency. Collectively, ChIP-SICAP provides a powerful tool to decode chromatin protein composition, further enhanced by its integrative capacity to perform ChIP-seq.
Assuntos
Cromatina/química , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/genética , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Diferenciação Celular , Reprogramação Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Marcação por Isótopo , Espectrometria de Massas/métodos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
Assuntos
Cromatina/metabolismo , Receptores de Glucocorticoides/metabolismo , Sumoilação , Sítios de Ligação , Regulação da Expressão Gênica , Células HEK293 , Humanos , Correpressor 1 de Receptor Nuclear/metabolismo , Coativador 1 de Receptor Nuclear , Mapeamento de Interação de Proteínas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/efeitos dos fármacosRESUMO
The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.
Assuntos
Recombinação Homóloga , Proteínas Inibidoras de Diferenciação/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição E2F1/metabolismo , Células HEK293 , Humanos , Proteínas Inibidoras de Diferenciação/química , Masculino , Proteínas de Neoplasias/química , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismoRESUMO
Chromatin is the assembly of genomic DNA and proteins packaged in the nucleus of eukaryotic cells, which together are crucial in regulating a plethora of cellular processes. Histones may be the best known class of protein constituents in chromatin, which are decorated by a range of post-translational modifications to recruit accessory proteins and protein complexes to execute specific functions, ranging from DNA compaction, repair, transcription, and duplication, all in a dynamic fashion and depending on the cellular state. The key role of chromatin in cellular fitness is emphasized by the deregulation of chromatin determinants predisposing to different diseases, including cancer. For this reason, deep investigation of chromatin composition is fundamental to better understand cellular physiology. Proteomic approaches have played a crucial role to understand critical aspects of this complex interplay, benefiting from the ability to identify and quantify proteins and their modifications in an unbiased manner. This review gives an overview of the proteomic approaches that have been developed by combining mass spectrometry-based with tailored biochemical and genetic methods to examine overall protein make-up of chromatin, to characterize chromatin domains, to determine protein interactions, and to decipher the broad spectrum of histone modifications that represent the quintessence of chromatin function.
Assuntos
Cromatina , Código das Histonas , DNA/genética , Epigênese Genética , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.
Assuntos
Espectrometria de Massas/métodos , Metaloendopeptidases/química , Proteínas/análise , Proteômica/métodos , Estreptavidina/química , Tripsina/química , Arginina/análogos & derivados , Arginina/química , Biotinilação/métodos , Imunoprecipitação da Cromatina/métodos , Células HeLa , Humanos , Lisina/análogos & derivados , Lisina/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteólise , Fatores de Transcrição/metabolismoRESUMO
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 & desenvolvimentoRESUMO
The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type-specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K4me1/K36me2 marks transcribed enhancers, while H3K4me1/K36me3 and H3K4me1/K79me2 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics.
Assuntos
Cromatina/metabolismo , Código das Histonas , Macrófagos/metabolismo , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Genoma , Histonas/metabolismo , Íntrons , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Proteômica , Transcrição GênicaRESUMO
Eukaryotic Initiation Translation Factor 2A (EIF2A) is considered to be primarily responsible for the initiation of translation when a cell is subjected to stressful conditions. However, information regarding this protein is still incomplete. Using a combination of proteomic approaches, we demonstrated that EIF2A is the molecular target of the naturally occurring bioactive compound cannabidiolic acid (CBDA) within human glioblastoma cells. This finding allowed us to undertake a study aimed at obtaining further information on the functions that EIF2A plays in tumor cells. Indeed, our data showed that CBDA is able to activate EIF2A when the cells are in no-stress conditions. It induces conformational changes in the protein structure, thus increasing EIF2A affinity towards the proteins participating in the Eukaryotic Translation Machinery. Consequently, following glioblastoma cells incubation with CBDA we observed an enhanced neosynthesis of proteins involved in the stress response, nucleic acid translation and organization, and protein catabolism. These changes in gene expression resulted in increased levels of ubiquitinated proteins and accumulation of the autophagosome. Our results, in addition to shedding light on the molecular mechanism underlying the biological effect of a phytocannabinoid in cancer cells, demonstrated that EIF2A plays a critical role in regulation of protein homeostasis.
Assuntos
Fator de Iniciação 2 em Eucariotos , Glioblastoma , Humanos , Glioblastoma/metabolismo , Glioblastoma/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Linhagem Celular Tumoral , Proteostase/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteômica/métodosRESUMO
The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.
Assuntos
Aminoácidos de Cadeia Ramificada , Cisteína , Animais , Humanos , Camundongos , Aurora Quinase B , Modelos Animais de Doenças , Oxirredução , TransaminasesRESUMO
The Schlafen gene family was first described in mice as a regulator of thymocyte development. Further studies showed involvement of human orthologs in different processes related with viral replication, cellular proliferation, and differentiation. In recent years, a new role for human Slfn11 in DNA replication and chromatin remodeling was described. As commonly observed in many gene families, Slfn paralogs show a tissue-specific expression. This made it difficult to reach conclusions which can be valid in different biological models regarding the function of the different Schlafen proteins. In the present study, we investigate the involvement of SLFN5 in cell-cycle regulation and cell proliferation. A careful analysis of SLFN5 expression revealed that SLFN5 is highly expressed in proliferating tissues and that the protein is ubiquitously present in all the tissues and cell line models we analyzed. Very interestingly, SLFN5 expression oscillates during cell cycle, peaking during S phase. The fact that SLFN5 interacts with protein phosphatase 2A and that SLFN5 depletion causes cell cycle arrest and cellular apoptosis, suggests a direct involvement of this human paralog in cell cycle progression and cellular proliferation. We substantiated our in vitro and in cellulo results using Xenopus laevis oocytes to show that mRNA depletion of the unique Slfn gene present in Xenopus, whose protein sequence shares 80% of homology with SLFN5, recapitulates the phenotype observed in human cells preventing the resumption of meiosis during oocyte development.
RESUMO
Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1-G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Fator de Transcrição GATA2/metabolismo , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinogênese , Aberrações Cromossômicas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Fator de Transcrição GATA2/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Translocação Genética , Células Tumorais CultivadasRESUMO
Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.
Assuntos
Receptores Androgênicos , Animais , Embrião de Galinha , Masculino , Próstata , ProteômicaRESUMO
BACKGROUND: The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. METHODS: Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. RESULTS: HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. CONCLUSIONS: Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC's transactivating and transrepressing functions.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Linhagem Celular Tumoral , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Cromatina , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genéticaRESUMO
Transcription factors (TFs) control cell fates by precisely orchestrating gene expression. However, how individual TFs promote transcriptional diversity remains unclear. Here, we use the Hox TF Ultrabithorax (Ubx) as a model to explore how a single TF specifies multiple cell types. Using proximity-dependent Biotin IDentification in Drosophila, we identify Ubx interactomes in three embryonic tissues. We find that Ubx interacts with largely non-overlapping sets of proteins with few having tissue-specific RNA expression. Instead most interactors are active in many cell types, controlling gene expression from chromatin regulation to the initiation of translation. Genetic interaction assays in vivo confirm that they act strictly lineage- and process-specific. Thus, functional specificity of Ubx seems to play out at several regulatory levels and to result from the controlled restriction of the interaction potential by the cellular environment. Thereby, it challenges long-standing assumptions such as differential RNA expression as determinant for protein complexes.
Assuntos
Linhagem da Célula/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Homeodomínio/genética , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , RNA/metabolismo , Fatores de Transcrição/genéticaRESUMO
The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.