RESUMO
DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.
Assuntos
Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Bactérias/metabolismo , Instabilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Instabilidade Genômica , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Mutagênese , Mutação , Fatores de Transcrição/metabolismoRESUMO
Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
Assuntos
Oócitos , Zigoto , Animais , Criança , Feminino , Humanos , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citoplasma/genética , Citoplasma/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Impressão Genômica/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.
Assuntos
Senescência Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Genoma Humano , Oncogenes , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Fibroblastos , Heterocromatina/genética , Humanos , Hibridização in Situ FluorescenteRESUMO
In vertebrates, DNA methyltransferase 1 (DNMT1) contributes to preserving DNA methylation patterns, ensuring the stability and heritability of epigenetic marks important for gene expression regulation and the maintenance of cellular identity. Previous structural studies have elucidated the catalytic mechanism of DNMT1 and its specific recognition of hemimethylated DNA. Here, using solution nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, we demonstrate that the N-terminal region of human DNMT1, while flexible, encompasses a conserved globular domain with a novel α-helical bundle-like fold. This work expands our understanding of the structure and dynamics of DNMT1 and provides a structural framework for future functional studies in relation with this new domain.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Animais , Humanos , Domínio Catalítico , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Estrutura Terciária de Proteína , Conformação Proteica em alfa-HéliceRESUMO
The earliest hematopoietic stem and progenitor cells (HSPCs) are generated from the ventral wall of the dorsal aorta, through endothelial-to-hematopoietic transition during vertebrate embryogenesis. Notch signaling is crucial for HSPC generation across vertebrates; however, the precise control of Notch during this process remains unclear. In the present study, we used multi-omics approaches together with functional assays to assess global DNA methylome dynamics during the endothelial cells to HSPCs transition in zebrafish, and determined that DNA methyltransferase 1 (Dnmt1) is essential for HSPC generation via repression of Notch signaling. Depletion of dnmt1 resulted in decreased DNA methylation levels and impaired HSPC production. Mechanistically, we found that loss of dnmt1 induced hypomethylation of Notch genes and consequently elevated Notch activity in hemogenic endothelial cells, thereby repressing the generation of HSPCs. This finding deepens our understanding of HSPC specification in vivo, which will provide helpful insights for designing new strategies for HSPC generation in vitro.
Assuntos
Hemangioblastos , Peixe-Zebra , Animais , Metilação de DNA/genética , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.
Assuntos
Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Camundongos , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , DNA Metiltransferase 3B , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , DNA Metiltransferase 3A/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genéticaRESUMO
Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Conformação de Ácido Nucleico , RNA , Humanos , Cromatina/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , RNA/genética , RNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismoRESUMO
Recently, extracellular vesicles (EVs) have been emphasized in regulating the hypoxic tumor microenvironment of breast cancer (BC), where tumor-associated fibroblasts (TAFs) play a significant role. In this study, we describe possible molecular mechanisms behind the pro-tumoral effects of EVs, secreted by hypoxia (HP)-induced TAFs, on BC cell growth, metastasis, and chemoresistance. These mechanisms are based on long noncoding RNA H19 (H19) identified by microarray analysis. We employed an in silico approach to identify differentially expressed lncRNAs that were associated with BC. Subsequently, we explored possible downstream regulatory mechanisms. We isolated EVs from TAFs that were exposed to HP, and these EVs were denoted as HP-TAF-EVs henceforth. MTT, transwell, flow cytometry, and TUNEL assays were performed to assess the malignant phenotypes of BC cells. A paclitaxel (TAX)-resistant BC cell line was constructed, and xenograft tumor and lung metastasis models were established in nude mice for in vivo verification. Our observation revealed that lncRNA H19 was significantly overexpressed, whereas miR-497 was notably downregulated in BC. HP induced activation of TAFs and stimulated the secretion of EVs. Coculture of HP-TAF-EVs and BC cells led to an increase in TAX resistance of the latter. HP-TAF-EVs upregulated methylation of miR-497 by delivering lncRNA H19, which recruited DNMT1, thus lowering the expression of miR-497. In addition, lncRNA H19-containing HP-TAF-EVs hindered miR-497 expression, enhancing tumorigenesis and TAX resistance of BC cells in vivo. Our study presents evidence for the contribution of lncRNA H19-containing HP-TAF-EVs in the reduction of miR-497 expression through the recruitment of DNMT1, which in turn promotes the growth, metastasis, and chemoresistance of BC cells.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Vesículas Extracelulares , MicroRNAs , RNA Longo não Codificante , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Transformação Celular Neoplásica , Resistencia a Medicamentos Antineoplásicos/genética , Vesículas Extracelulares/genética , Hipóxia , Camundongos Nus , MicroRNAs/genética , RNA Longo não Codificante/genética , Microambiente Tumoral/genéticaRESUMO
In recent years, accumulating evidence has demonstrated the role of long noncoding RNAs (lncRNAs) in colon cancer. We aim to investigate the role of MIR143HG, also known as CARMN (Cardiac mesoderm enhancer-associated noncoding RNA) in colon cancer and explore the related mechanisms. An RNAseq data analysis was performed to screen differentially expressed lncRNAs associated with colon cancer. Next, MIR143HG expression was quantified in colon cancer cells. Moreover, the contributory roles of MIR143HG in the progression of colon cancer with the involvement of DNMT1 and HOXB7 (Homeobox B7) were evaluated after restored MIR143HG or depleted HOXB7. Finally, the effects of MIR143HG were investigated in vivo by measuring tumor formation in nude mice. High-throughput transcriptome sequencing was employed to validate the specific mechanisms by which MIR143HG and HOXB7 affect tumor growth in vivo. MIR143HG was found to be poorly expressed, while HOXB7 was highly expressed in colon cancer. MIR143HG could promote HOXB7 methylation by recruiting DNMT1 to reduce HOXB7 expression. Upregulation of MIR143HG or downregulation of HOXB7 inhibited cell proliferation, invasion and migration and facilitated apoptosis in colon cancer cells so as to delay the progression of colon cancer. The same trend was identified in vivo. Our study provides evidence that restoration of MIR143HG suppressed the progression of colon cancer via downregulation of HOXB7 through DNMT1-mediated HOXB7 promoter methylation. Thus, MIR143HG may be a potential candidate for the treatment of colon cancer.
Assuntos
Neoplasias do Colo , DNA (Citosina-5-)-Metiltransferase 1 , Proteínas de Homeodomínio , RNA Longo não Codificante , Animais , Camundongos , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Homeodomínio/genética , Metilação , Metiltransferases , Camundongos Nus , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Fatores de Transcrição , HumanosRESUMO
Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and embryonic stem cells. Mechanistically, Naa10p facilitates binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates, including the ICRs of the imprinted allele during S phase. Moreover, the lethal Ogden syndrome-associated mutation of human Naa10p disrupts its binding to the ICR of H19 and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated Ogden syndrome to defective DNA methylation and genomic imprinting.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Deficiências do Desenvolvimento/genética , Epigênese Genética , Impressão Genômica , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , RNA Longo não Codificante/genética , Animais , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Deleção de Genes , Genes Letais , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/patologia , Acetiltransferase N-Terminal A/deficiência , Acetiltransferase N-Terminal E/deficiência , Ligação Proteica , RNA Longo não Codificante/metabolismo , Fase S/genéticaRESUMO
Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.
Assuntos
Transdução de Sinais , Neoplasias de Mama Triplo Negativas , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteômica , Proliferação de Células , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Receptores ErbB/metabolismoRESUMO
DNA methylation can be considered the most prominent in controlling the gene expression responsible for the balance between cell proliferation and cell death. In this study, we aimed to analyze the distinct contributions of Dnmt1 and Dnmt3a enzymes in oocyte maturation, survival, autophagy, reactive oxygen species (ROS) production, and compensation capacity of Dnmt3b and Dnmt3l enzymes in mouse oocytes. Following confirming the suppression of Dnmt1or Dnmt3a through siRNA application, the assessment involved immunofluorescence staining for Dnmts, 5mC, p62, and ROS levels. Cell death rates showed a noticeable increase while oocyte maturation rates exhibited significant reduction. Global DNA methylation showed a decline, concomitant with elevated p62 and ROS levels upon Dnmt1 or Dnmt3a knockdown. Remarkably, silencing of Dnmt1 led to an upsurge in Dnmt3a expression, whereas Dnmt3a knockdown triggered an increase in Dnmt1 levels. Furthermore, Dnmt3l expression exhibited a notable decrease after silencing of either Dnmt1 or Dnmt3a, while Dnmt3b levels remained comparable between control and siRNA-treated groups. Collectively, this study underscores the pivotal roles of Dnmt1 and Dnmt3a in orchestrating various facets of oocyte development, encompassing maturation, survival, autophagy, and ROS production. These findings offer valuable insights into the intricate regulatory network governed by DNA methylation machinery within the context of oocyte physiology.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Camundongos , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Espécies Reativas de Oxigênio/metabolismo , Oócitos/metabolismo , Fatores de Transcrição/genética , RNA Interferente Pequeno , HomeostaseRESUMO
BACKGROUND: The faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear. METHODS: The shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation. RESULTS: We investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3ß collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback. CONCLUSIONS: Targeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais , Humanos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Animais , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Camundongos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Transdução de Sinais , Proliferação de CélulasRESUMO
Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Neoplasias da Glândula Tireoide , Humanos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Ativação Transcricional , Metilação de DNA , Neoplasias da Glândula Tireoide/genética , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismoRESUMO
ßIV-spectrin is a membrane-associated cytoskeletal protein that maintains the structural stability of cell membranes and integral proteins such as ion channels and transporters. Its biological functions are best characterized in the brain and heart, although recently we discovered a fundamental new role in the vascular system. Using cellular and genetic mouse models, we reported that ßIV-spectrin acts as a critical regulator of developmental and tumor-associated angiogenesis. ßIV-spectrin was shown to selectively express in proliferating endothelial cells (EC) and suppress VEGF/VEGFR2 signaling by enhancing receptor internalization and degradation. Here we examined how these events impact the downstream kinase signaling cascades and target substrates. Based on quantitative phosphoproteomics, we found that ßIV-spectrin significantly affects the phosphorylation of epigenetic regulatory enzymes in the nucleus, among which DNA methyltransferase 1 (DNMT1) was determined as a top substrate. Biochemical and immunofluorescence results showed that ßIV-spectrin inhibits DNMT1 function by activating ERK/MAPK, which in turn phosphorylates DNMT1 at S717 to impede its nuclear localization. Given that DNMT1 controls the DNA methylation patterns genome-wide, and is crucial for vascular development, our findings suggest that epigenetic regulation is a key mechanism by which ßIV-spectrin suppresses angiogenesis.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Sistema de Sinalização das MAP Quinases , Proteômica , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Animais , Proteômica/métodos , Camundongos , Fosforilação , Humanos , Neovascularização Fisiológica , Espectrina/metabolismo , Espectrina/genética , Fosfoproteínas/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais/metabolismo , AngiogêneseRESUMO
Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Pareamento Cromossômico/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Espermatócitos/crescimento & desenvolvimento , Espermatogênese/genética , Ubiquitina-Proteína Ligases/genética , Células-Tronco Germinativas Adultas/citologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Epigênese Genética/genética , Heterocromatina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Espermatócitos/fisiologia , Espermatogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.
RESUMO
BACKGROUND: Human liver cancer stem-like cells (HLCSLCs) are widely acknowledged as significant factors in the recurrence and eradication of hepatocellular carcinoma (HCC). The sustenance of HLCSLCs' stemness is hypothesized to be intricately linked to the epigenetic process of DNA methylation modification of genes associated with anticancer properties. The present study aimed to elucidate the stemness-maintaining mechanism of HLCSLCs and provide a novel idea for the clearance of HLCSLCs. METHODS: The clinical relevance of DNMT1 and SOCS1 in hepatocellular carcinoma (HCC) patients was evaluated through the GEO and TCGA databases. Cellular immunofluorescence assay, methylation-specific PCR, chromatin immunoprecipitation were conducted to explore the expression of DNMT1 and SOCS1 and the regulatory relationship between them in HLCSLCs. Spheroid formation, soft agar colony formation, expression of stemness-associated molecules, and tumorigenicity of xenograft in nude mice were used to evaluate the stemness of HLCSLCs. RESULTS: The current analysis revealed a significant upregulation of DNMT1 and downregulation of SOCS1 in HCC tumor tissues compared to adjacent normal liver tissues. Furthermore, patients exhibiting an elevated DNMT1 expression or a reduced SOCS1 expression had low survival. This study illustrated the pronounced expression and activity of DNMT1 in HLCSLCs, which effectively targeted the promoter region of SOCS1 and induced hypermethylation, consequently suppressing the expression of SOCS1. Notably, the stemness of HLCSLCs was reduced upon treatment with DNMT1 inhibitors in a concentration-dependent manner. Additionally, the overexpression of SOCS1 in HLCSLCs significantly mitigated their stemness. The knockdown of SOCS1 expression reversed the effect of DNMT1 inhibitor on the stemness of HLCSLCs. DNMT1 directly binds to the SOCS1 promoter. In vivo, DNMT1 inhibitors suppressed SOCS1 expression and inhibited the growth of xenograft. CONCLUSION: DNMT1 targets the promoter region of SOCS1, induces hypermethylation of its CpG islands, and silences its expression, thereby promoting the stemness of HLCSLCs.
RESUMO
Cellular apoptosis is a central mechanism leveraged by chemotherapy to treat human cancers. 5-Methylcytosine (m5C) modifications installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Here, we reported that promoter methylation by DNMT1 coordinated with mRNA methylation by NSun2 to regulate osteosarcoma cell apoptosis. DNMT1 was induced during osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was suppressed. DNMT1 was found to repress NSun2 expression by methylating the NSun2 promoter. Moreover, DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced the expression of these anti-apoptotic genes via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma cells to apoptosis. Collectively, our findings establish crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and adding additional layers to the control of gene expression at different epigenetic levels.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Humanos , Metilação , RNA Mensageiro/genética , Citosina , DNA , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Apoptose/genéticaRESUMO
DNA methylation can deactivate tumor suppressor genes thus causing cancers. Two DNA methylation inhibitors have been approved by the Food and Drug Administration (FDA) and have entered clinical use. However, these inhibitors are nucleoside analogues that can be incorporated into DNA or RNA and induce significant side effects. DNMT1 and DNMT3 are key enzymes involved in DNA methylation. In the acute myeloid leukemia model, a non-nucleoside DNMT1-specific inhibitor has shown lower toxicity and improved pharmacokinetics compared to traditional nucleoside drugs. DNMT3 is also implicated in certain specific cancers. Thus, developing non-nucleoside inhibitors for DNMT1 or DNMT3 can help in understanding their roles in carcinogenesis and provide targeted treatment options in certain cancers. Although no non-nucleoside inhibitors have yet entered clinical trials, in this review, we focus on DNMT1 or DNMT3 selective inhibitors. For DNMT1 selective inhibitors, we have compiled information on the repurposed drugs, derivative compounds and selective inhibitors identified through virtual screening. Additionally, we have outlined potential targets for DNMT1, including protein-protein complex, RNA mimics and aptamers. Compared to DNMT1, research on DNMT3-specific inhibitors has been less extensive. In this context, our exploration has identified a limited number of molecular inhibitors, and we have proposed specific long non-coding RNAs (lncRNAs) as potential contributors to the selective inhibition of DNMT3. This collective effort aims to offer valuable insights into the development of non-nucleoside inhibitors that selectively target DNMT1 or DNMT3.