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2.
Nucleic Acids Res ; 48(19): 10940-10952, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010150

RESUMO

ATR functions as a master regulator of the DNA-damage response. ATR activation requires the ATR activator, topoisomerase IIß-binding protein 1 (TopBP1). However, the underlying mechanism of TopBP1 regulation and how its regulation affects DNA replication remain unknown. Here, we report a specific interaction between TopBP1 and the histone demethylase PHF8. The TopBP1/PHF8 interaction is mediated by the BRCT 7+8 domain of TopBP1 and phosphorylation of PHF8 at Ser854. This interaction is cell-cycle regulated and phosphorylation-dependent. PHF8 is phosphorylated by CK2, which regulates binding of PHF8 to TopBP1. Importantly, PHF8 regulates TopBP1 protein level by preventing its ubiquitination and degradation mediated by the E3 ligase UBR5. Interestingly, PHF8pS854 is likely to contribute to regulation of TopBP1 stability and DNA replication checkpoint. Further, both TopBP1 and PHF8 are required for efficient replication fork restart. Together, these data identify PHF8 as a TopBP1-binding protein and provide mechanistic insight into how PHF8 regulates TopBP1 stability to maintain DNA replication.


Assuntos
Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Fosforilação , Ligação Proteica , Domínios Proteicos
3.
Zhonghua Zhong Liu Za Zhi ; 42(8): 648-652, 2020 Aug 23.
Artigo em Chinês | MEDLINE | ID: mdl-32867456

RESUMO

Objective: To investigate the relationship between KDM6A mutation or expression and clinicopathological characteristics of gastric cancer. Methods: Fifty-seven cases of gastric cancer tissues were analyzed by second-generation sequencing, and bioinformation database such as Cbioportal, Kaplan Meier-Plotter, and the Human Protein Atlas were used to analyze the relationship between KDM6A mutation and clinicopathological characteristics of gastric cancer. Results: Among 57 gastric cancer samples, 14 were KDM6A mutation, and the mutation proportion was 24.6%. Compared with the non-mutation group, the Borrmann classification, T stage, TNM stage and tumor diameter of KDM6A mutant group were significantly different (all P<0.05). The median survival time of the KDM6A mutant patients was 53.5 months, significantly shorter than 72.0 months of the KDM6A non-mutation patients (P=0.007). The analysis result of Kaplan Meier-Plotter database showed that, among all of the 875 patients, 655 patients had low KDM6A expression and 220 patients had high expression. The median survival time of patients with low expression was 23.5 months, significantly shorter than 30.8 months of patients with high expression (P=0.002). In male, gastric cancer patients with stage Ⅲ, intestinal type, diffuse type, simple surgical treatment and fluorouracil chemotherapy, the expression of KDM6A is related to the patient's overall survival time (all P<0.05). The analysis result of Cbioportal database showed that, among all of the 1 172 gastric cancer patients, 70 patients with KDM6A mutation, 1100 patients with non-mutation. The median overall survival time of mutant patients was 28.9 months, significantly shorter than 35.9 months of non-mutation patients (P<0.001). The analysis result of Human Protein Atlas database showed that, among all of the 355 gastric cancer patients, 97 patients had high KDM6A expression and 258 patients had low KDM6A expression. The median survival time of patients with low expression was 13.7 months, significantly shorter than 19.8 months of patients with high expression (P=0.022). Conclusions: The survival time of gastric cancer patients with KDM6A mutation or low expression is shorter. The mutation and expression of KDM6A are related to clinical pathological factors, which may become a potential target for the diagnosis and treatment of gastric cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Histona Desmetilases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Histona Desmetilases/genética , Humanos , Metástase Linfática , Masculino , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida
4.
Nat Commun ; 11(1): 4673, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938917

RESUMO

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.


Assuntos
Proteínas de Ligação a DNA/genética , Haploinsuficiência , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/etiologia , Fatores de Transcrição/genética , Proteínas ras/metabolismo , Anormalidades Múltiplas/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/genética
5.
Mol Cell Biol ; 40(20)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32817139

RESUMO

Lysine demethylase 6A (KDM6A), also known as UTX, belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases, which also includes UTY and KDM6B (JMJD3). The KDM6A protein contains six tetratricopeptide repeat (TPR) domains and an enzymatic Jumonji C (JmjC) domain that catalyzes the removal of di- and trimethylation on H3K27. KDM6A physically associates with histone H3 lysine 4 monomethyltransferases MLL3 (KMT2C) and MLL4 (KMT2D). Since its identification as an H3K27 demethylase in 2007, studies have reported KDM6A's critical roles in cell differentiation, development, and cancer. KDM6A is important for differentiation of embryonic stem cells and development of various tissues. Mutations of KDM6A cause Kabuki syndrome. KDM6A is frequently mutated in cancers and functions as a tumor suppressor. KDM6A is redundant with UTY and functions largely independently of its demethylase activity. It regulates gene expression, likely through the associated transcription factors and MLL3/4 on enhancers. However, KDM6A enzymatic activity is required in certain cellular contexts. Functional redundancy between H3K27 demethylase activities of KDM6A and KDM6B in vivo has yet to be determined. Further understanding of KDM6A functions and working mechanisms will provide more insights into enhancer regulation and may help generate novel therapeutic approaches to treat KDM6A-related diseases.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Histona Desmetilases/genética , Neoplasias/genética , Domínio Catalítico/genética , Montagem e Desmontagem da Cromatina/genética , Células-Tronco Embrionárias/citologia , Genes Supressores de Tumor , Histona Desmetilases/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(33): 19888-19895, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747552

RESUMO

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Histona Desmetilases/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Histona Desmetilases/química , Histona Desmetilases/genética , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , RNA Polimerase II/genética
7.
J Chromatogr A ; 1625: 461294, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709337

RESUMO

It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 µM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.


Assuntos
Corydalis/química , Distribuição Contracorrente/métodos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Histona Desmetilases/antagonistas & inibidores , Interface Usuário-Computador , Bioensaio , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Histona Desmetilases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Solventes , Espectrometria de Massas em Tandem
8.
Life Sci ; 258: 118095, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32679142

RESUMO

AIMS: This study focused on investigating the potential role of long non-coding RNA (lncRNA) lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in the progression of osteoarthritis (OA). MATERIALS AND METHODS: qRT-PCR assay was applied to detect gene expression, while western blot was performed to measure levels of apoptosis-related proteins. CCK-8, colony formation and TUNEL assays were conducted to explore the functional role of LOXL1-AS1 in OA. ChIP assay was utilized to assess the affinity between JunD proto-oncogene, AP-1 transcription factor subunit (JUND) and LOXL1-AS1 promoter. Mechanism experiments were implemented to investigate the underlying molecular mechanism of LOXL1-AS1. KEY FINDINGS: LOXL1-AS1 was up-regulated in OA cartilage tissues. Silencing LOXL1-AS1 hampered proliferation and inflammation, yet promoting apoptosis in chondrocytes. LOXL1-AS1 was transcriptionally activated by JUND1. LOXL1-AS1 sequestered miR-423-5p and abolished miR-423-5p-mediated repression on lysine demethylase 5C (KDM5C), thus promoted the development of OA. SIGNIFICANCE: LncRNA LOXL1-AS1 is transcriptionally activated by JUND and facilitates the proliferation and inflammation of chondrocytes via elevating miR-423-5p-mediated KDM5C in OA, which may provide potential therapeutic target for OA.


Assuntos
Histona Desmetilases/metabolismo , MicroRNAs/metabolismo , Osteoartrite/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Transcrição Genética , Apoptose/genética , Sequência de Bases , Proliferação de Células/genética , Condrócitos/metabolismo , Condrócitos/patologia , Progressão da Doença , Inativação Gênica , Histona Desmetilases/genética , Humanos , Inflamação/genética , Inflamação/patologia , MicroRNAs/genética , Osteoartrite/patologia , RNA Longo não Codificante/genética
9.
PLoS One ; 15(7): e0236101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678829

RESUMO

Dysregulation of histone demethylase Jumonji-C domain-containing protein 5 (JMJD5) has been identified as a great effect on tumorigenesis. Silibinin is a commonly used anti-hepatotoxic drug and exhibits anticancer effect in various cancers. However, the antitumor mechanism between silibinin and JMJD5 in oral squamous cell carcinoma (OSCC) remains unclear. In this study, the clinical significance of JMJD5 on OSCC patients was assessed through tissue microarray. Furthermore, mice bearing patient-derived tumor xenografts (PDTXs) and tongue cancer cell lines were treated with silibinin and evaluated for tumor growth and JMJD5 expression. High expression of JMJD5 in oral cancer was significantly associated with tumor size (P = 0.0241), cervical node metastasis (P = 0.0001) and clinical stage (P = 0.0002), was associated with worse survival rate compared with that of the total cohort (P = 0.0002). Collectively the data indicate that JMJD5 expression may be suitable for detection of unfavorable prognosis in OSCC patients, based in part on its apparent role as a marker of metastasis. In addition, silibinin inhibits cancer growth in vitro and in PDTX models. Furthermore, metastasis-associated protein 1 (MTA1) could regulate the expression for JMJD5 and had a positive correlation with JMJD5. Moreover, silibinin could downregulate JMJD5 and MTA1 in oral cancer. Present study thus identifies that JMJD5 might be an essential prognostic indicator and therapeutic target against OSCC progression. In addition, silibinin is a potential candidate among novel chemotherapeutic agents or adjuvants for modulating JMJD5 in OSCC, through a mechanism likely involving MTA1/JMJD5 axis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Histona Desmetilases/metabolismo , Neoplasias Bucais/patologia , Proteínas Repressoras/metabolismo , Silibina/farmacologia , Transativadores/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Prognóstico , Proteínas Repressoras/genética , Taxa de Sobrevida , Transativadores/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Cancer Res Clin Oncol ; 146(11): 2843-2850, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32725356

RESUMO

PURPOSE: Demethylation of DNA through enzymes like LSD1 showed a crucial impact on different kind of cancers. Epigenetic modifications in cervical cancer are still not fully investigated nevertheless of high interest for a therapeutic use. METHODS: Tumor samples of 250 cervical cancer patients were immunochemically stained and evaluated based on Immunoreactive Score. Results were statistically analyzed for clinical and pathological parameters. RESULTS: Our patient collective showed a disadvantage for 10-year survival for patients with a strong expression of LSD1 in the cytoplasm of cervical cancer cells. The results of the correlational analysis further revealed a negative correlation of LSD1 to G-protein coupled estrogen receptor (GPER). CONCLUSIONS: Epigenetic changes through enzymes like LSD1 may also be of interest for patients with cervical cancer. A combined therapy with other proteins relayed to cervical cancer like GPER might be of interest for future investigations.


Assuntos
Adenocarcinoma/enzimologia , Carcinoma de Células Escamosas/enzimologia , Histona Desmetilases/metabolismo , Neoplasias do Colo do Útero/enzimologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Feminino , Histona Desmetilases/análise , Humanos , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/patologia
11.
Nat Commun ; 11(1): 2082, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350257

RESUMO

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.


Assuntos
Células Endócrinas/citologia , Células Endócrinas/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Histona Desmetilases/metabolismo , Ilhotas Pancreáticas/citologia , Transdução de Sinais , Tretinoína/metabolismo , Adulto , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Células Endócrinas/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/embriologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Adulto Jovem
12.
Proc Natl Acad Sci U S A ; 117(24): 13670-13679, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32471953

RESUMO

Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Inibidores Enzimáticos/administração & dosagem , Histona Desmetilases/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Fator Estimulador de Colônias/genética , Fatores de Transcrição STAT/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fator Estimulador de Colônias/metabolismo , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Transdução de Sinais/efeitos dos fármacos
13.
Gene ; 752: 144758, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32422235

RESUMO

Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). While initial studies of LSD1 inhibitors suggested these compounds may be used to induce differentiation of acute myeloid leukemia (AML), the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we used precision nuclear run-on sequencing (PRO-seq) and ChIP-seq in AML cell lines to probe for the earliest regulatory events associated with INCB059872 treatment. The changes in nascent transcription could be traced back to a loss of CoREST activity and activation of GFI1-regulated genes. INCB059872 is in phase I clinical trials, and we evaluated a pre-treatment bone marrow sample of a patient who showed a clinical response to INCB059872 while being treated with azacitidine. We used single-cell RNA-sequencing (scRNA-seq) to show that INCB059872 caused a shift in gene expression that was again associated with GFI1/GFI1B regulation. Finally, we treated mice with INCB059872 and performed scRNA-seq of lineage-negative bone marrow cells, which showed that INCB059872 triggered accumulation of megakaryocyte early progenitor cells with gene expression hallmarks of stem cells. Accumulation of these stem/progenitor cells may contribute to the thrombocytopenia observed in patients treated with LSD1 inhibitors.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Exoma/métodos
14.
Mol Cell ; 78(5): 903-914.e4, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32396821

RESUMO

LSD1 (lysine specific demethylase; also known as KDM1A), the first histone demethylase discovered, regulates cell-fate determination and is overexpressed in multiple cancers. LSD1 demethylates histone H3 Lys4, an epigenetic mark for active genes, but requires the CoREST repressor to act on nucleosome substrates. To understand how an accessory subunit (CoREST) enables a chromatin enzyme (LSD1) to function on a nucleosome and not just histones, we have determined the crystal structure of the LSD1/CoREST complex bound to a 191-bp nucleosome. We find that the LSD1 catalytic domain binds extranucleosomal DNA and is unexpectedly positioned 100 Å away from the nucleosome core. CoREST makes critical contacts with both histone and DNA components of the nucleosome, explaining its essential function in demethylating nucleosome substrates. Our studies also show that the LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.


Assuntos
Histona Desmetilases/metabolismo , Histona Desmetilases/ultraestrutura , Sequência de Aminoácidos , Domínio Catalítico , Cromatina/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Cristalografia por Raios X/métodos , DNA/genética , DNA/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica
15.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269126

RESUMO

Human papillomavirus 16 (HPV16) E7 has long been known to stabilize the tumor suppressor TP53. However, the molecular mechanism of TP53 stabilization by HPV16 E7 has remained obscure, and this stabilization can occur independently of the E2F-regulated MDM2 inhibitor p14ARF Here, we report that the damage-induced noncoding (DINO) lncRNA (DINOL) is the "missing link" between HPV16 E7 and increased TP53 levels. DINO levels are decreased in cells where TP53 is inactivated, either by HPV16 E6, by expression of a dominant negative TP53 minigene, or by TP53 depletion. DINO levels are increased in HPV16 E7-expressing cells. HPV16 E7 causes increased DINO expression independently of RB1 degradation and E2F1 activation. Similar to what is seen with the adjacent CDKN1A locus, DINO expression is regulated by the histone demethylase KDM6A. DINO stabilizes TP53 in HPV16 E7-expressing cells, and as it is a TP53 transcriptional target, DINO levels further increase. As with expression of other oncogenes, such as adenovirus E1A or MYC, HPV16 E7-expressing cells are sensitized to cell death under conditions of metabolic stress, which in the case of E7 has been linked to TP53 activation. Consistent with earlier studies, we show that HPV16 E7-expressing keratinocytes are highly sensitive to metabolic stress induced by starvation or the antidiabetic drug metformin. Sensitivity of HPV16 E7-expressing cells to metabolic stress is rescued by DINO depletion. Moreover, DINO depletion decreases sensitivity to the DNA damage-inducing chemotherapy agent doxorubicin. This work identifies DINO as a critical mediator of TP53 stabilization and activation in HPV16 E7-expressing cells.IMPORTANCE Viral oncoproteins, including HPV16 E6 and E7, have been instrumental in elucidating the activities of cellular signaling networks, including those governed by the TP53 tumor suppressor. Our study demonstrates that the long noncoding RNA DINO is the long-sought missing link between HPV16 E7 and elevated TP53 levels. Importantly, the TP53-stabilizing DINO plays a critical role in the cell death response of HPV16 E7-expressing cells to metabolic stress or DNA damage.


Assuntos
Histona Desmetilases/genética , Interações Hospedeiro-Patógeno/genética , Proteínas E7 de Papillomavirus/genética , RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/genética , Antibióticos Antineoplásicos/farmacologia , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doxorrubicina/farmacologia , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Papillomavirus Humano 16 , Humanos , Hipoglicemiantes/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/virologia , Metformina/farmacologia , Proteínas E7 de Papillomavirus/metabolismo , Cultura Primária de Células , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
16.
Nucleic Acids Res ; 48(9): 4915-4927, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232336

RESUMO

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , Histona Desmetilases/fisiologia , Proteínas de Homeodomínio/fisiologia , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Células HeLa , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos
17.
Cancer Res ; 80(12): 2599-2611, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32312832

RESUMO

Zinc finger protein 143 (ZNF143) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence-specific DNA. The exact biological role of ZNF143 in hepatocellular carcinoma (HCC) has not been investigated. Here we report that ZNF143 is overexpressed in HCC tissues and its overexpression correlates with poor prognosis. Gain- and loss-of-function experiments showed that ZNF143 promoted HCC cell proliferation, colony formation, and tumor growth in vitro and in vivo. ZNF143 accelerated HCC cell-cycle progression by activating cell division cycle 6 (CDC6). Mechanistically, ZNF143 promoted expression of CDC6 by directly activating transcription of histone demethylase mineral dust-induced gene (MDIG), which in turn reduced H3K9me3 enrichment in the CDC6 promoter region. Consistently, ZNF143 expression correlated significantly with MDIG and CDC6 expression in HCC. Collectively, we propose a model for a ZNF143-MDIG-CDC6 oncoprotein axis that provides novel insight into ZNF143, which may serve as a therapeutic target in HCC. SIGNIFICANCE: These findings describe the mechanism by which ZNF143 promotes HCC proliferation and provide important clues for exploring new targets and strategies for clinical treatment of human liver cancer.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Dioxigenases/genética , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Transativadores/metabolismo , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Estudos de Coortes , Metilação de DNA , Conjuntos de Dados como Assunto , Dioxigenases/metabolismo , Células HEK293 , Hepatectomia , Histona Desmetilases/metabolismo , Histonas/genética , Humanos , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Análise Serial de Tecidos , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Cell Biol ; 22(4): 380-388, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231309

RESUMO

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.


Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Processamento de Proteína Pós-Traducional , Zigoto/metabolismo , Animais , Implantação do Embrião , Embrião de Mamíferos , Feminino , Fertilização/genética , Heterocromatina/química , Heterocromatina/metabolismo , Histona Desmetilases/genética , Histonas/genética , Masculino , Metáfase , Metilação , Camundongos , Camundongos Knockout , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transcrição Genética , Zigoto/citologia , Zigoto/crescimento & desenvolvimento
19.
Cell Mol Life Sci ; 77(17): 3341-3350, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32193608

RESUMO

It is well-established that Lysine-specific demethylase 1 (LSD1, also known as KDM1A) roles as a lysine demethylase canonically acting on H3K4me1/2 and H3K9me1/2 for regulating gene expression. Though the discovery of non-histone substrates methylated by LSD1 has largely expanded the functions of LSD1 as a typical demethylase, recent groundbreaking studies unveiled its non-catalytic functions as a second life for this demethylase. We and others found that LSD1 is implicated in the interaction with a line of proteins to exhibit additional non-canonical functions in a demethylase-independent manner. Here, we present an integrated overview of these recent literatures charging LSD1 with unforeseen functions to re-evaluate and summarize its non-catalytic biological roles beyond the current understanding of its demethylase activity. Given LSD1 is reported to be ubiquitously overexpressed in a variety of tumors, it has been generally considered as an innovative target for cancer therapy. We anticipate that these non-canonical functions of LSD1 will arouse the consideration that extending the LSD1-based drug discovery to targeting LSD1 protein interactions non-catalytically, not only its demethylase activity, may be a novel strategy for cancer prevention.


Assuntos
Histona Desmetilases/metabolismo , Autofagia , Desmetilação , Proteína 7 com Repetições F-Box-WD/metabolismo , Histona Desmetilases/química , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitinação
20.
Comp Biochem Physiol B Biochem Mol Biol ; 243-244: 110434, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32201355

RESUMO

Histone demethylation modification is an important means of gene expression regulation and is widely involved in biological processes such as animal reproduction and development. Histone lysine demethylases (Kdm) plays an important role in the demethylation of histones. To understand the role of histone demethylation in scallops, we identified the Kdm gene family of the Yesso scallop Patinopecten yessoensis, and analyzed its expression during the gonad and early development. The results showed that the P. yessoensis has a complete Kdm family including seventeen members that belong to sixteen subfamilies (Hif1an, Hspbap1, Jarid2, Jmjd4, Jmjd6, Jmjd7, Jmjd8, Kdm1, Kdm2, Kdm3, Kdm4, Kdm5, Kdm6, Kdm7, Kdm8 and Kdm9). The Kdm genes showed five different expression patterns in the early development of scallop, with some of them (e.g. Jmjd7, Jmjd8 and Kdm8) being highly expressed in only one or two stage and the others (e.g. Kdm1A, Kdm9, Jmjd4 and Jmjd6) in several consecutive stages. During gonadal development, the Kdm genes also display various expression patterns. Some genes (e.g. Kdm2, Kdm4 and Jmjd7) display preferential expression in the testis, and the others have no obvious sex bias but show stage preference (resting, proliferative, growing or maturation stage). These results suggest that various histone demethylation modifications (e.g. H3K4, H3K9 and H3K27) may participate in the regulation of gametogenesis and early development of Yesso scallop. It will facilitate a better understanding of the epigenetic contributions to mollusk development.


Assuntos
Gametogênese/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Pectinidae/metabolismo , Sequência de Aminoácidos , Animais , Desmetilação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Histona Desmetilases/genética , Masculino , Pectinidae/embriologia , Pectinidae/genética , Pectinidae/crescimento & desenvolvimento , Filogenia , Testículo/metabolismo
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