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1.
Int J Cancer ; 146(2): 400-412, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31271662

RESUMO

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.


Assuntos
Antineoplásicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Adulto , Idoso , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desmetilação do DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Aguda Bifenotípica/patologia , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Tretinoína/farmacologia , Tretinoína/uso terapêutico
2.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 660-665, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762234

RESUMO

OBJECTIVE: To investigate the ability of osteogenic differentiation and the expression of histone demethylases KDM6B in bone marrow mesenchymal stem cells (BMSCs) in diabetic environment. METHODS: Diabetic model rats was successfully established, and BMSCs from diabetic model rats and normal rats were isolated and cultured for further study. When cultured cells, we added high concentration of glucose and advanced glycosylation products (AGE) in the medium to imitating the diabetic environment. BMSCs were divided into 6 groups: diabetes group (derived from diabets SD rats), normal group (derived from normal SD rats), high glucose group (30 mmol/L D-glucose), normal glucose group (5.5 mmol/L D-glucose), AGE group (AGE 300 µg/mL) and BSA group (BSA 300 µg/mL). BMSCs in diabetes group were derived from diabetes SD rats, while others were derived from normal SD rats. After 7 d of osteogenic induction, the cells were examined the ability of osteogenic differentiation by alkaline phosphatase (ALP) staining, the transcription levels of Runt-related transcription factor 2 (Runx2) and KDM6B were determined by RT-PCR, and the expression levels of H3K27Me3 protein were examined by Western bolt. RESULTS: Compared with the control groups, the numbers of ALP stained cells and the mRNA levels of Runx2 and KDM6B in diabetes group, high glucose group and AGE group were all decreased (P < 0.05), while H3K27Me3 protein expression levels were all increased (P < 0.05). CONCLUSION: The ability of osteogenic differentiation of BMSCs in diabetic environment was weakened, and the expression of Runx2 mRNA was inhibited, which may be related to the increased expression of H3K27Me3 after the inhibition of KDM6B expression.


Assuntos
Diabetes Mellitus , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Animais , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ratos , Ratos Sprague-Dawley
3.
Genome Biol ; 20(1): 170, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429787

RESUMO

BACKGROUND: Circadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood. RESULTS: Here we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20-30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression. CONCLUSIONS: Circadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Arabidopsis/genética , Modelos Biológicos , Processamento de Proteína Pós-Traducional
4.
Gastroenterology ; 157(6): 1646-1659.e11, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31442435

RESUMO

BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.


Assuntos
Carcinogênese/genética , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metilação de DNA , Conjuntos de Dados como Assunto , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sobrevida , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cell ; 75(5): 905-920.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422875

RESUMO

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.


Assuntos
Diferenciação Celular/fisiologia , Metilação de DNA/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Transcrição Genética/fisiologia , Animais , Linhagem Celular , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
6.
Life Sci ; 234: 116788, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445935

RESUMO

Livin is an important member of the human inhibitor of apoptosis proteins (IAPs) family. IAPs are proteins with antiapoptotic abilities, and their functions are different from the Bcl-2 (B-cell lymphoma-2) family proteins. However, the precise role of Livin in colon cancer progression remains unclear. The purpose of this study is to assess the effect of overexpression Livin in colon cancer cells and to examine its molecular mechanism. We demonstrated that Livin induced a colon cancer phenotype, including proliferation and migration, by regulating H2A.XY39ph (histone family 2A variant (H2AX) phosphorylated on the 39th serine site). We elucidated that Livin degraded Jumonji-C domain-containing 6 protein (JMJD6), which was mediated by the proteasome murine double minute 2 (MDM2), thereby regulating H2A.XY39ph. Above all, the overexpression of JMJD6 recovered H2A.XY39ph in colon cancer cells with a high level of Livin, thus inhibiting colon cancer malignancy progression. These results reveal a previously unrecognized role for Livin in regulating the tumor-initiating capacity in colon cancer and provide a novel treatment strategy in cancer via the interruption of H2A.XY39ph function and the interaction between H2A.XY39ph and JMJD6.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo/patologia , Histonas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interação de Proteínas , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Neoplasias/genética , Proteólise
7.
Nucleic Acids Res ; 47(17): 9005-9023, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31363749

RESUMO

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.


Assuntos
Ilhas de CpG/genética , Proteínas F-Box/fisiologia , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/fisiologia , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Cromatina/enzimologia , Cromatina/metabolismo , Metilação de DNA , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Camundongos , Modelos Genéticos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/metabolismo , RNA Polimerase II/metabolismo
8.
Oncol Rep ; 42(4): 1431-1440, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364745

RESUMO

Jumonji domain­containing protein 2A (JMJD2A) has been identified to promote cell proliferation in bladder cancer; however, it remains undetermined whether JMJD2A regulates cell migration and invasion in bladder cancer. The aim of the present study was to further investigate the roles of JMJD2A in bladder cancer. The expression levels of JMJD2A in bladder cancer tissues and cell lines were established by RT­qPCR assays and western blot analysis. Moreover, by gain­ and loss­of­function assays, the effects of JMJD2A on migration and invasion as well as proliferation were investigated in bladder cancer cells. The results revealed that the expression level of JMJD2A was significantly upregulated in bladder cancer tissues and cell lines compared to adjacent non­tumor tissues and a human immortalized bladder urothelial cell line. Kaplan­Meier survival analysis indicated that patients with high JMJD2A expression level had shorter overall survival. Moreover, JMJD2A could promote cell migration and invasion by facilitating epithelial­mesenchymal transition (EMT) in bladder cancer. In addition, it was determined that JMJD2A promoted EMT through regulation of SLUG expression. Collectively, our findings revealed that JMJD2A may act as an oncogene and participate in bladder cancer progression, which provides a promising therapeutic strategy for patients with bladder cancer.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transição Epitelial-Mesenquimal , Humanos , Histona Desmetilases com o Domínio Jumonji/biossíntese , Histona Desmetilases com o Domínio Jumonji/genética , Gradação de Tumores , Invasividade Neoplásica , Fatores de Transcrição da Família Snail/genética , Transcrição Genética , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
9.
Nat Commun ; 10(1): 3319, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346162

RESUMO

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Neuroblastoma/tratamento farmacológico , Receptores de Superfície Celular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinogênese , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/fisiopatologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética
10.
Biosci Biotechnol Biochem ; 83(11): 2090-2096, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31334687

RESUMO

Myostatin (Mstn) is an important growth/differentiation factor, and knockdown of Mstn reduces fat content. Here, we knocked down Mstn expression in C2C12 myoblasts and then induced adipogenic trans-differentiation in the cells. The effects of Mstn knockdown on lipid droplet contents and H3K27me3 marker expression on adipocyte-specific genes were detected. The results showed that Mstn knockdown reduced the formation of lipid droplets, downregulated the expression of adipocyte-specific genes, and increased H3K27me3 marker expression on adipocyte-specific genes. Chromatin immunoprecipitation analysis showed that the SMAD2/SMAD3 complex could combine with the Jumonji D3 (Jmjd3) promoter and that Mstn regulated Jmjd3 expression through this process. Jmjd3 overexpression removed the H3K27me3 marker and increased the expression of adipocyte-specific genes. Overall, our results showed that Mstn regulated Jmjd3 expression through SMAD2/SMAD3, thus affecting the H3K27me3 marker on adipocyte-specific genes and the trans-differentiation from myocytes to adipocytes.


Assuntos
Adipócitos/citologia , Transdiferenciação Celular/genética , Técnicas de Silenciamento de Genes , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células Musculares/citologia , Miostatina/genética , Proteínas Smad Reguladas por Receptor/metabolismo , Adipócitos/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/genética , Histonas/química , Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Células Musculares/metabolismo , Miostatina/deficiência , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
11.
Science ; 365(6454)2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31320558

RESUMO

DNA viruses typically eject genomic DNA into the nuclei of host cells after entry. It is unclear, however, how nuclear pathogen-derived DNA triggers innate immune responses. We report that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) recognizes pathogenic DNA and amplifies interferon-α/ß (IFN-α/ß) production. Upon DNA virus infection, nuclear-localized hnRNPA2B1 senses viral DNA, homodimerizes, and is then demethylated at arginine-226 by the arginine demethylase JMJD6. This results in hnRNPA2B1 translocation to the cytoplasm where it activates the TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway, leading to IFN-α/ß production. Additionally, hnRNPA2B1 facilitates N 6-methyladenosine (m6A) modification and nucleocytoplasmic trafficking of CGAS, IFI16, and STING messenger RNAs. This, in turn, amplifies the activation of cytoplasmic TBK1-IRF3 mediated by these factors. Thus, hnRNPA2B1 plays important roles in initiating IFN-α/ß production and enhancing stimulator of interferon genes (STING)-dependent cytoplasmic antiviral signaling.


Assuntos
Núcleo Celular/imunologia , Núcleo Celular/virologia , Infecções por Vírus de DNA/imunologia , DNA Viral/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Imunidade Inata , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Células HEK293 , Herpesvirus Humano 1/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Fator Regulador 3 de Interferon , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7
12.
Artif Cells Nanomed Biotechnol ; 47(1): 2891-2899, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31307234

RESUMO

JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.


Assuntos
Carcinogênese , Proteína p300 Associada a E1A/metabolismo , Proteínas F-Box/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Osteossarcoma/patologia , Acetilação , Animais , Proteínas Reguladoras de Apoptose/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas F-Box/química , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Lisina/metabolismo , Metilação , Camundongos , Nucleossomos/metabolismo , Proteínas Proto-Oncogênicas/genética , Transcrição Genética
13.
Eur J Med Chem ; 177: 316-337, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158747

RESUMO

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 µM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinonas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Piridinas/química , Piridinas/metabolismo , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Relação Estrutura-Atividade
14.
Cell Prolif ; 52(5): e12638, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31152465

RESUMO

OBJECTIVES: Terminally differentiated stratified squamous epithelial cells play an important role in barrier protection of the skin. The integrity of epidermal cells is maintained by tight regulation of proliferation and differentiation. The aim of this study was to investigate the role of epigenetic regulator H3K4me3 and its demethylase Jarid1b in the control of epithelial cell differentiation. MATERIALS AND METHODS: RT-qPCR, Western blotting and IHC were used to detect mRNA and protein levels. We analysed cell proliferation by CCK8 assay and cell migration by wound healing assay. ChIP was used to measure H3K4me3 enrichment. A chamber graft model was established for epidermal development. RESULTS: Our studies showed that H3K4me3 was decreased during epidermal differentiation. The H3K4me3 demethylase Jarid1b positively controlled epidermal cell differentiation in vitro and in vivo. Mechanistically, we found that Jarid1b substantially increased the expression of mesenchymal-epithelial transition (MET)-related genes, among which Ovol1 positively regulated differentiation gene expression. In addition, Ovol1 expression was repressed by PI3K-AKT pathway inhibitors and overexpression (O/E) of the PI3K-AKT pathway suppressor Ship1. Knockdown (KD) of Ship1 activated downstream PI3K-AKT pathway and enhanced Ovol1 expression in HaCaT. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not that of Pten, by directly binding to its promoter to modulate H3K4me3 enrichment. CONCLUSION: Our results identify an essential role of Jarid1b in the regulation of the Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Transição Epitelial-Mesenquimal , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genética
15.
Nucleic Acids Res ; 47(16): 8424-8438, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31251802

RESUMO

ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3-DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3-DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.


Assuntos
RNA Helicases DEAD-box/genética , DNA/genética , Histona Desmetilases com o Domínio Jumonji/genética , Diester Fosfórico Hidrolases/genética , RNA Mensageiro/genética , Transcrição Genética/efeitos dos fármacos , Sistemas CRISPR-Cas , RNA Helicases DEAD-box/metabolismo , DNA/química , DNA/metabolismo , Elementos Facilitadores Genéticos , Edição de Genes/métodos , Regulação da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Inflamação , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lipopolissacarídeos/farmacologia , Modelos Biológicos , NF-kappa B/genética , NF-kappa B/metabolismo , Conformação de Ácido Nucleico , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Transdução de Sinais , Sítio de Iniciação de Transcrição
16.
Nat Commun ; 10(1): 2669, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209209

RESUMO

The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density defines super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here, we purify Mediator from neural stem cells (NSCs) and identify 75 protein-protein interaction partners. We identify super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalize with Mediator at enhancers and super enhancers. Transcription factor families with high affinity for Mediator dominate enhancers and super enhancers and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator of NSCs. Tcf4 interacts with Mediator, colocalizes with Mediator at super enhancers and regulates neurogenic transcription factor genes with super enhancers and broad H3K4me3 domains. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Complexo Mediador/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Fator de Transcrição 4/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transcrição Genética/fisiologia
17.
Eur J Haematol ; 103(3): 215-224, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31206203

RESUMO

AIM: This study aimed to investigate the possible functions of interaction between JARID1B and miR-137 in ALL. METHODS: The levels of H3K4me3 and H3K4me2 and the expression of JARID1B and miR-137 were analyzed in six ALL cell lines and 30 ALL patients. The effects of miR-137 and JARID1B on cell proliferation and apoptosis were investigated by silencing or promoting the respective genes. The interaction between miR-137 and JARID1B was confirmed by double-luciferase report assay. RESULTS: The histone H3K4 expressions and miR-137 expression were lower in 30 ALL patients and in six ALL cell lines, while the expression of JARID1B was elevated. A negative correlation was observed between JARID1B and miR-137. Over-expression of miR-137 led to decreasing cell proliferation and increasing apoptosis in MOLT-4 and BALL-1 cells. MiR-137 inhibitor up-regulated JARID1B in these two cell lines, while promoted proliferation in BALL-1 cells only. Dual-luciferase report assay suggested that JARID1B was a direct target of miR-137 in ALL cell lines. CONCLUSIONS: The expression of miR-137 was declined in ALL, and JARID1B was directly repressed by miR-137. Aberrant JARID1B expression could result in abnormal histone methylation, which might be one cause of ALL.


Assuntos
Regulação Leucêmica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferência de RNA , Proteínas Repressoras/genética , Regiões 3' não Traduzidas , Adolescente , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Genes Reporter , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Repressoras/metabolismo , Adulto Jovem
18.
Genetics ; 212(3): 631-654, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31123043

RESUMO

Fumarase is a well-characterized TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate. In mammals, fumarase acts as a tumor suppressor, and loss-of-function mutations in the FH gene in hereditary leiomyomatosis and renal cell cancer result in the accumulation of intracellular fumarate-an inhibitor of α-ketoglutarate-dependent dioxygenases. Fumarase promotes DNA repair by nonhomologous end joining in mammalian cells through interaction with the histone variant H2A.Z, and inhibition of KDM2B, a H3 K36-specific histone demethylase. Here, we report that Saccharomyces cerevisiae fumarase, Fum1p, acts as a response factor during DNA replication stress, and fumarate enhances survival of yeast lacking Htz1p (H2A.Z in mammals). We observed that exposure to DNA replication stress led to upregulation as well as nuclear enrichment of Fum1p, and raising levels of fumarate in cells via deletion of FUM1 or addition of exogenous fumarate suppressed the sensitivity to DNA replication stress of htz1Δ mutants. This suppression was independent of modulating nucleotide pool levels. Rather, our results are consistent with fumarate conferring resistance to DNA replication stress in htz1Δ mutants by inhibiting the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation. Although the timing of checkpoint activation and deactivation remained largely unaffected by fumarate, sensors and mediators of the DNA replication checkpoint were required for fumarate-dependent resistance to replication stress in the htz1Δ mutants. Together, our findings imply metabolic enzymes and metabolites aid in processing replicative intermediates by affecting chromatin modification states, thereby promoting genome integrity.


Assuntos
Replicação do DNA , Fumaratos/metabolismo , Código das Histonas , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Histonas/genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
19.
Nat Commun ; 10(1): 2063, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048693

RESUMO

RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me3 histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 tends to bind motifs located in active chromatin states that are depleted for heterochromatic modifications. However, the underlying mechanism remains unknown. Here, we show that REF6 preferentially bind to hypo-methylated CTCTGYTY motifs in vivo, and that CHG methylation decreases REF6 DNA binding affinity in vitro. In addition, crystal structures of ZnF-clusters in complex with DNA oligonucleotides reveal that 5-methylcytosine is unfavorable for REF6 binding. In drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, in which non-CG methylation is significantly reduced, REF6 can ectopically bind a small number of new target loci, most of which are located in or neighbored with short TEs in euchromatic regions. Collectively, our findings reveal that DNA methylation, likely acting in combination with other epigenetic modifications, may partially explain why REF6 binding is depleted in heterochromatic loci.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Metilação de DNA/fisiologia , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição/metabolismo , 5-Metilcitosina/metabolismo , Epigênese Genética/fisiologia , Eucromatina/metabolismo , Heterocromatina/metabolismo , Mutação , Plantas Geneticamente Modificadas , Dedos de Zinco/fisiologia
20.
Molecules ; 24(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060229

RESUMO

Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.


Assuntos
Neoplasias da Mama/enzimologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas Nucleares/genética , Radiossensibilizantes/farmacologia , Proteínas Repressoras/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/química , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/química , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação
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