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1.
Mol Cell ; 82(20): 3901-3918.e7, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36206767

RESUMO

How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Histona Metiltransferases , Evasão Tumoral , Animais , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cromatina , Metilação de DNA , Neoplasias de Cabeça e Pescoço/genética , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Interferons/genética , Proteínas Nucleares/metabolismo , Receptores de Interferon/genética , Retroelementos , Evasão Tumoral/genética
2.
Nature ; 573(7773): 281-286, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31485078

RESUMO

Enzymes that catalyse CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B), are indispensable for mammalian tissue development and homeostasis1-4. They are also implicated in human developmental disorders and cancers5-8, supporting the critical role of DNA methylation in the specification and maintenance of cell fate. Previous studies have suggested that post-translational modifications of histones are involved in specifying patterns of DNA methyltransferase localization and DNA methylation at promoters and actively transcribed gene bodies9-11. However, the mechanisms that control the establishment and maintenance of intergenic DNA methylation remain poorly understood. Tatton-Brown-Rahman syndrome (TBRS) is a childhood overgrowth disorder that is defined by germline mutations in DNMT3A. TBRS shares clinical features with Sotos syndrome (which is caused by haploinsufficiency of NSD1, a histone methyltransferase that catalyses the dimethylation of histone H3 at K36 (H3K36me2)8,12,13), which suggests that there is a mechanistic link between these two diseases. Here we report that NSD1-mediated H3K36me2 is required for the recruitment of DNMT3A and maintenance of DNA methylation at intergenic regions. Genome-wide analysis shows that the binding and activity of DNMT3A colocalize with H3K36me2 at non-coding regions of euchromatin. Genetic ablation of Nsd1 and its paralogue Nsd2 in mouse cells results in a redistribution of DNMT3A to H3K36me3-modified gene bodies and a reduction in the methylation of intergenic DNA. Blood samples from patients with Sotos syndrome and NSD1-mutant tumours also exhibit hypomethylation of intergenic DNA. The PWWP domain of DNMT3A shows dual recognition of H3K36me2 and H3K36me3 in vitro, with a higher binding affinity towards H3K36me2 that is abrogated by TBRS-derived missense mutations. Together, our study reveals a trans-chromatin regulatory pathway that connects aberrant intergenic CpG methylation to human neoplastic and developmental overgrowth.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Intergênico/metabolismo , Histonas/metabolismo , Animais , Linhagem Celular , DNA Metiltransferase 3A , Estudo de Associação Genômica Ampla , Transtornos do Crescimento/genética , Transtornos do Crescimento/fisiopatologia , Humanos , Camundongos , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Síndrome de Sotos/genética , Síndrome de Sotos/fisiopatologia
3.
BMC Microbiol ; 23(1): 278, 2023 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-37775764

RESUMO

BACKGROUND: Allyl isothiocyanate (AITC) is a natural product with high volatility that is used as a biofumigant to alleviate soil-borne plant diseases, and problems such as root knot nematodes (RKNs) that necessitate continuous cropping. However, little research has assessed the effects of AITC fumigation on medicinal plants. RESULTS: AITC significantly reduced the population of RKNs in soil (p < 0.0001) and showed an excellent RKN disease control effect within 6 months after sowing Panax notoginseng (p < 0.0001). The seedling survival rate of 2-year-old P. notoginseng was approximately 1.7-fold higher after soil treatment with AITC (p = 0.1008). 16S rRNA sequencing indicated that the AITC treatment affected bacterial richness rather than diversity in consecutively cultivated (CC) soil. Furthermore, biomarkers with statistical differences between AITC-treated and untreated CC soil showed that Pirellulales (order), Pirellulaceae (family), Pseudomonadaceae (family), and Pseudomonas (genus) played important roles in the AITC-treated group. In addition, the microbiome functional phenotypes predicted using the BugBase tool suggested that AITC treatment is more conducive to improving CC soil through changes in the bacterial community structure. Crucially, our research also suggested that AITC soil treatment significantly increases soil organic matter (p = 0.0055), total nitrogen (p = 0.0054), and available potassium (p = 0.0373), which promotes the survival of a succeeding medicinal plant (Polygonatum kingianum). CONCLUSION: AITC is an ecologically friendly soil treatment that affects the top 10 bacterial richness but not diversity. It could also provide a basis for a useful agricultural soil management measure to alleviate soil sickness.


Assuntos
Plantas Medicinais , Solo , Solo/química , Fumigação , RNA Ribossômico 16S/genética , Microbiologia do Solo , Bactérias/genética
4.
FASEB J ; 32(5): 2574-2586, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29279353

RESUMO

Preeclampsia (PE), a hypertensive disorder of pregnancy, is a leading cause of maternal and fetal morbidity and mortality. Although the etiology is unknown, PE is thought to be caused by defective implantation and decidualization in pregnancy. Pregnant blood pressure high (BPH)/5 mice spontaneously develop placentopathies and maternal features of human PE. We hypothesized that BPH/5 implantation sites have transcriptomic alterations. Next-generation RNA sequencing of implantation sites at peak decidualization, embryonic day (E)7.5, revealed complement gene up-regulation in BPH/5 vs. controls. In BPH/5, expression of complement factor 3 was increased around the decidual vasculature of E7.5 implantation sites and in the trophoblast giant cell layer of E10.5 placentae. Altered expression of VEGF pathway genes in E5.5 BPH/5 implantation sites preceded complement dysregulation, which correlated with abnormal vasculature and increased placental growth factor mRNA and VEGF164 expression at E7.5. By E10.5, proangiogenic genes were down-regulated, whereas antiangiogenic sFlt-1 was up-regulated in BPH/5 placentae. We found that early local misexpression of VEGF genes and abnormal decidual vasculature preceded sFlt-1 overexpression and increased complement deposition in BPH/5 placentae. Our findings suggest that abnormal decidual angiogenesis precedes complement activation, which in turn contributes to the aberrant trophoblast invasion and poor placentation that underlie PE.-Sones, J. L., Merriam, A. A., Seffens, A., Brown-Grant, D.-A., Butler, S. D., Zhao, A. M., Xu, X., Shawber, C. J., Grenier, J. K., Douglas, N. C. Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.


Assuntos
Decídua , Regulação da Expressão Gênica , Neovascularização Patológica/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Decídua/irrigação sanguínea , Decídua/metabolismo , Decídua/patologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Physiol Genomics ; 50(5): 385-392, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29521599

RESUMO

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.


Assuntos
Indutores da Angiogênese/metabolismo , Celecoxib/farmacologia , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Placenta/metabolismo , Pré-Eclâmpsia/genética , Animais , Inibidores de Ciclo-Oxigenase 2/farmacologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Pré-Eclâmpsia/metabolismo , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Mol Cell Proteomics ; 14(1): 251-62, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25385071

RESUMO

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3ß and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteômica/métodos , Proteínas tau/metabolismo , Quinase 5 Dependente de Ciclina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas do Tecido Nervoso/genética , Fosforilação , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Leveduras , Proteínas tau/genética
7.
Proc Natl Acad Sci U S A ; 109(22): 8652-7, 2012 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-22589305

RESUMO

The SNF1/AMP-activated protein kinases are αßγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the ß and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking ß- and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.


Assuntos
Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glucose/farmacologia , Immunoblotting , Modelos Biológicos , Mutação , Fosforilação/efeitos dos fármacos , Multimerização Proteica , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Treonina/genética , Treonina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
J Biol Chem ; 288(43): 31052-8, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24019512

RESUMO

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αßγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Domínio Catalítico , Deleção de Genes , Glucose/genética , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
Proc Natl Acad Sci U S A ; 108(16): 6349-54, 2011 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-21464305

RESUMO

The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 ß-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the ß-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.


Assuntos
Glicogênio/biossíntese , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Glucose/genética , Glucose/metabolismo , Glicogênio/genética , Mutação , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
10.
Cell Rep ; 43(1): 113629, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38165806

RESUMO

The interplay between metabolism and chromatin signaling is implicated in cancer progression. However, whether and how metabolic reprogramming in tumors generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway, which drives aggressive and chemo-resistant disease. Using a chromatin-focused CRISPR screen, we report that NRF2 activation sensitizes LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDACs). This association is observed across cultured cells, mouse models, and patient-derived xenografts. Integrative epigenomic, transcriptomic, and metabolomic analysis demonstrates that HDAC inhibition causes widespread redistribution of H4ac and its reader protein, which transcriptionally downregulates metabolic enzymes. This results in reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest NRF2 activation as a potential biomarker for effective repurposing of HDAC inhibitors to treat solid tumors.


Assuntos
Fator 2 Relacionado a NF-E2 , Neoplasias , Animais , Humanos , Camundongos , Cromatina , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Reprogramação Metabólica , Fator 2 Relacionado a NF-E2/metabolismo
11.
Sci Adv ; 10(35): eadp0975, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39196936

RESUMO

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.


Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , DNA Metiltransferase 3A , Histonas , Neoplasias , Nucleossomos , Ligação Proteica , Ubiquitinação , Nucleossomos/metabolismo , Histonas/metabolismo , Humanos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Regiões Promotoras Genéticas , Microscopia Crioeletrônica , Linhagem Celular Tumoral
12.
bioRxiv ; 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38562823

RESUMO

During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

13.
bioRxiv ; 2023 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-37162970

RESUMO

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

14.
Nat Commun ; 14(1): 4259, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-37460547

RESUMO

Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.


Assuntos
Linfoma , Neoplasias , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , Cromatina
15.
bioRxiv ; 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38116029

RESUMO

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights: The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions.The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs.Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture.The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

16.
Eukaryot Cell ; 10(3): 313-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21216941

RESUMO

The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , Ativação Enzimática , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
Mol Cancer Ther ; 20(12): 2362-2371, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34552007

RESUMO

Chondrosarcomas are inherently resistant to chemotherapy and radiotherapy, pointing to an unmet need for new treatment options. Immune checkpoint inhibitors, which have shown remarkable promise in multiple solid cancer types, have limited efficacy in chondrosarcomas. Mutations in IDH1/2 genes, which result in progressive increases in DNA and histone methylation, are observed in 50% of conventional chondrosarcomas, suggesting that epigenetic dysregulation represents a potential barrier for tumor progression and target for therapeutic intervention. Here, we demonstrated that combined treatment of FDA-approved inhibitors of DNA methyltransferases (DNMTs) 5-aza-2'-deoxycytidine (5-aza), and histone deacetylases (HDACs) suberanilohydroxamic acid (SAHA) impaired the proliferation of chondrosarcoma cell lines in vitro and in xenograft studies. Transcriptomic analysis reveals that chondrosarcoma cells treated with 5-aza and SAHA markedly elevated the expression of IFN-stimulated genes including PD-L1, indicating that these epigenetic drugs induced a potent innate immune response. We demonstrated that 5-aza and SAHA resulted in both genomic and epigenomic instability, as shown by elevated DNA damage response and derepression of retrotransposons, respectively, which in turn activated pattern recognition receptors (PRRs) and the downstream IFN signaling pathways. Importantly, the cytotoxic effects of 5-aza and SAHA can be rescued by depletion of PRRs such as cGAS and MAVS, and potentiated by depletion of the RNA-editing enzyme ADAR1. Together, our results demonstrate preclinical activity of combined DNMT and HDAC inhibition against chondrosarcomas and suggest that targeted epigenetic therapies could represent a new therapeutic approach in the treatment of chondrosarcomas, and this is being tested in an ongoing clinical trial (NCT04340843).


Assuntos
Condrossarcoma/tratamento farmacológico , Epigênese Genética/genética , Inibidores de Histona Desacetilases/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos Nus
18.
Biochim Biophys Acta ; 1789(5): 395-402, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19358899

RESUMO

Histone acetylation and methylation are linked to a variety of nuclear activities, most notably transcriptional regulation. Both synergistic and antagonistic relationships between these two modifications have been reported in different systems. Here we show that the budding yeast histone H4 arginine 3 (R3) methyltransferase Hmt1p binds acetylated histones H3 and H4, and importantly, that acetylated H4 is a significantly better methylation substrate for Hmt1p. Kinetic studies show that acetylation at any of the four acetylatable lysine residues of histone H4 results in more efficient methylation. Among the four, K8 acetylation imposes the strongest effect on reducing K(M), consistent with the observed acetylation-stimulated interaction. In vivo, hmt1Delta cells rescue the transcriptional defect caused by GCN5 deletion, indicating that one of the functions of Gcn5p is to neutralize the negative effect of Hmt1p. Mutating either K8 or R3 to alanine causes similar growth defects in selective histone and gcn5 mutant background, suggesting that these two residues function in the same pathway for optimal vegetative growth. Together, these results reveal a functional connection between histone acetylation, methylation, and two of the responsible enzymes, Gcn5p and Hmt1p.


Assuntos
Histona Acetiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Acetilação , Arginina/metabolismo , Redes Reguladoras de Genes , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Mutação , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
19.
PLoS One ; 15(6): e0233710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530963

RESUMO

BACKGROUND: Phlebopus portentosus and mealy bugs form a fungus-insect gall on the roots of host plants. The fungus and mealy bugs benefit mutually through the gall, which is the key link in the nutritional mechanism of P. portentosus. The cavity of the fungus-insect gall provides an ideal shelter for mealy bugs survival and reproduction, but how does P. portentosus benefit from this symbiotic relationship? METHODOLOGY AND RESULTS: Anatomical examination of fungus-insect galls revealed that one or more mealy bugs of different generations were living inside the galls. The mealy bug's mouthpart could penetrate through the mycelium layer of the inside of the gall and suck plant juice from the host plant root. Mealy bugs excreted honeydew inside or outside the galls. The results of both honeydew agar medium and quartz tests showed that the honeydew can attract and promote the mycelial growth of P. portentosus. A test of the relationship between the honeydew and the formation of the fungus-insect gall showed that honeydew promoted gall formation. CONCLUSIONS: All experimental results in this study show that the honeydew secreted by mealy bugs can attract and promote the mycelial growth of P. portentosus, forming a fungus-insect gall, because mealy bugs' honeydew is rich in amino acids and sugars.


Assuntos
Basidiomycota/fisiologia , Hemípteros/fisiologia , Tumores de Planta/microbiologia , Animais , Basidiomycota/crescimento & desenvolvimento , Basidiomycota/patogenicidade , Fabaceae/microbiologia , Fabaceae/parasitologia , Hemípteros/patogenicidade , Tumores de Planta/parasitologia
20.
Nat Med ; 25(11): 1691-1698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31700187

RESUMO

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.


Assuntos
Pneumopatias/terapia , Pulmão/crescimento & desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Regeneração/genética , Acilação/genética , Animais , Blastocisto/metabolismo , Diferenciação Celular/genética , Metilação de DNA/genética , Modelos Animais de Doenças , Histonas/genética , Humanos , Pulmão/patologia , Pneumopatias/patologia , Camundongos , Organogênese/genética , Células-Tronco Pluripotentes/transplante , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , beta Catenina/genética
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