RESUMO
DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
Assuntos
Metilação de DNA , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Timina DNA Glicosilase/metabolismo , 5-Metilcitosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citidina Desaminase/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Timina DNA Glicosilase/genética , Transcrição GênicaRESUMO
BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.
Assuntos
Adenocarcinoma , Adenoma , Neoplasias do Colo , Neoplasias Colorretais , Animais , Humanos , Camundongos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Oxigenases de Função Mista/genética , Fenótipo , Proteínas Proto-Oncogênicas/genéticaRESUMO
How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation.
Assuntos
Reparo do DNA , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Timina DNA Glicosilase/metabolismo , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Camundongos Knockout , Timina DNA Glicosilase/genéticaRESUMO
Numerous parallelisms exist between development and cancer. In this article, I review some of the founding ideas linking development and cancer, and highlight clinical conditions exhibiting features of both developmental derangement and cancer predisposition, including cohesinopathies, rasopathies, phakomatoses, Proteus syndrome and other overgrowth disorders, recessive chromosome breakage syndromes, and dominant hereditary cancer syndromes. I suggest that these disorders encompass a continuous spectrum spanning clinical genetics and clinical oncology, and derive some general implications that might be useful in the future for the treatment of these diseases.
Assuntos
Deficiências do Desenvolvimento/genética , Neoplasias/genética , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/diagnóstico , Epigênese Genética , Predisposição Genética para Doença , Humanos , Neoplasias/complicações , Neoplasias/diagnóstico , FenótipoRESUMO
BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.
Assuntos
Reparo do DNA , Retroelementos , Humanos , Reparo de Erro de Pareamento de DNA , Proteínas Recombinantes/genética , Metilação de DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismoRESUMO
Beta-catenin is involved in intercellular adhesion and participates in the Wnt signaling pathway. This study evaluated the expression pattern and prognostic value of ß-catenin in a series of endometrial carcinoma patients. Immunohistochemical analyses were used to assess the expression and subcellular localization of ß-catenin from tissue sections of 74 patients with endometrial carcinoma. No correlation was found between beta-catenin expression and clinicopathological parameters. Patients expressing nuclear ß-catenin (n = 13; 16%) showed a more favorable prognosis than patients expressing membranous ß-catenin; the 5-year disease-related survival rate was 100% for cases expressing nuclear ß-catenin, compared with 73.8% (SE 0.08) of cases expressing membranous ß-catenin (p = 0.04). Although statistical significance was not reached (p = 0.15), cases expressing nuclear ß-catenin showed a 5-year disease-free survival rate of 90.9% (SE 0.08) compared with 67.4% (SE 0.08) of cases expressing membranous ß-catenin. Univariate Cox analysis revealed that membranous ß-catenin expression was found to be associated with a relative risk of death of 33.9 (p = 0.04). The stage of disease (p = 0.0006), histology (p = 0.003), and grading (p = 0.008) were also significantly correlated with disease-free survival according to univariate Cox analyses. Determining ß-catenin expression and localization patterns may predict survival in patients with endometrial cancer and, therefore, should be considered a potential prognostic marker of disease.
RESUMO
Proteolysis-targeting chimeras (PROTACs) are a promising new class of drugs that selectively degrade cellular proteins of interest. PROTACs that target oncogene products are avidly being explored for cancer therapies, and several are currently in clinical trials. Drug resistance is a substantial challenge in clinical oncology, and resistance to PROTACs has been reported in several cancer cell models. Here, using proteomic analysis, we found intrinsic and acquired resistance mechanisms to PROTACs in cancer cell lines mediated by greater abundance or production of the drug efflux pump MDR1. PROTAC-resistant cells were resensitized to PROTACs by genetic ablation of ABCB1 (which encodes MDR1) or by coadministration of MDR1 inhibitors. In MDR1-overexpressing colorectal cancer cells, degraders targeting either the kinases MEK1/2 or the oncogenic mutant GTPase KRASG12C synergized with the dual epidermal growth factor receptor (EGFR/ErbB)/MDR1 inhibitor lapatinib. Moreover, compared with single-agent therapies, combining MEK1/2 degraders with lapatinib improved growth inhibition of MDR1-overexpressing KRAS-mutant colorectal cancer xenografts in mice. Together, our findings suggest that concurrent blockade of MDR1 will likely be required with PROTACs to achieve durable protein degradation and therapeutic response in cancer.
Assuntos
Neoplasias Colorretais , Animais , Humanos , Camundongos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistência a Medicamentos , Lapatinib , Inibidores de Proteínas Quinases , Proteólise , Proteômica , Proteínas Proto-Oncogênicas p21(ras)RESUMO
The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Inibidores Enzimáticos/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Treonina/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
DNA methylation is a critical process in the regulation of gene expression with dramatic effects in development and continually expanding roles in oncogenesis. 5-Methylcytosine was once considered to be an inherited and stably repressive epigenetic mark, which can be only removed by passive dilution during multiple rounds of DNA replication. However, in the past two decades, physiologically controlled DNA demethylation and deamination processes have been identified, thereby revealing the function of cytosine methylation as a highly regulated and complex state-not simply a static, inherited signature or binary on-off switch. Alongside these fundamental discoveries, clinical studies over the past decade have revealed the dramatic consequences of aberrant DNA demethylation. In this review we discuss DNA demethylation and deamination in the context of 5-methylcytosine as critical processes for physiological and physiopathological transitions within three states-development, immune maturation, and oncogenic transformation; and we describe the expanding role of DNA demethylating drugs as therapeutic agents in cancer.
RESUMO
BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Macrófagos/enzimologia , Linfócitos T/enzimologia , Timina DNA Glicosilase/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Citosina/análogos & derivados , Citosina/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Dioxigenases/deficiência , Elementos Facilitadores Genéticos , Expressão Gênica , Loci Gênicos , Hematopoese/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolissacarídeos/farmacologia , Longevidade/genética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/imunologia , Timina DNA Glicosilase/deficiênciaRESUMO
While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.
Assuntos
Carcinogênese/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Melanoma/metabolismo , Fatores do Domínio POU/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Estudos de Coortes , Variações do Número de Cópias de DNA , Progressão da Doença , Técnicas de Silenciamento de Genes , Haploinsuficiência , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/mortalidade , Melanoma/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Fatores do Domínio POU/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , RNA Interferente Pequeno , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/secundário , Melanoma Maligno CutâneoRESUMO
Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.
Assuntos
Cílios/metabolismo , Reparo do DNA , Perda do Embrião/genética , Endodesoxirribonucleases/genética , Inativação Gênica , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Padronização Corporal , Cílios/ultraestrutura , Proteínas do Citoesqueleto , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário , Endodesoxirribonucleases/metabolismo , Extremidades/embriologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca(2+) handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca(2+) handling. To this end, SR Ca(2+) uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr(17), the Ca(2+)-calmodulin-dependent kinase IIdelta site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr(17). Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca(2+) handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retículo Sarcoplasmático/metabolismo , Treonina/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas Hedgehog/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Receptor Patched-1/metabolismo , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (Ë66â¯kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.
Assuntos
Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dano ao DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Células HEK293 , Humanos , Células MCF-7 , Mutação , Neoplasias/genética , SumoilaçãoRESUMO
Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
Assuntos
Inibidores Enzimáticos/farmacologia , Melanoma/patologia , Timina DNA Glicosilase/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Timina DNA Glicosilase/antagonistas & inibidores , Timina DNA Glicosilase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Laser capture microdissection (LCM) permits isolation of pure cell populations from which RNA can be extracted, amplified, and subjected to microarray analysis, allowing information to be obtained on the gene expression profile of defined cell types. To avoid amplification artifacts and detect genes expressed at different levels, it is important to optimize the choice of both RNA amplification step and microarray platform. We captured by LCM the same colon cancer biopsy and conducted a cross comparison of distinct RNA amplification methods and different chip platforms. We tested two RNA amplification methods with different chemistry: the one-cycle Ovation system (NuGEN) and the two-cycle Ribo OA method (Arcturus). We also compared two different whole genome platforms, based on Affymetrix technology: the U133 plus 2.0 and the X3P array, with probe sets closer to the 3' end of transcripts. After RNA amplification, microarray analysis, and data normalization, we investigated reproducibility and correlation of different methods and arrays. Our results indicate that the Arcturus Ribo OA method is superior for both array choices, especially in combination with X3P arrays, showing the lowest variance and Spearman correlation of 0.986. The quicker NuGEN procedure, when coupled with X3P arrays, also yielded excellent results (correlation of 0.951). These observations will be useful for planning large-scale analyses of LCM-dissected clinical samples.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Microdissecção/métodos , Microscopia Confocal/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Neoplásico/genética , Adenocarcinoma/patologia , Biotinilação , Neoplasias do Colo/patologia , Secções Congeladas , Humanos , Mucosa Intestinal/química , Mucosa Intestinal/ultraestrutura , Microdissecção/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Estabilidade de RNA , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/isolamento & purificação , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k(st) values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20- to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity.
Assuntos
Endodesoxirribonucleases/metabolismo , Idoxuridina/metabolismo , Idoxuridina/farmacologia , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Animais , Pareamento Incorreto de Bases , Linhagem Celular , Ilhas de CpG , Embrião de Mamíferos , Endodesoxirribonucleases/genética , Inativação Gênica , Humanos , Camundongos , Especificidade por SubstratoRESUMO
BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brca1ΔC/ΔC mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1ΔC/ΔC;53bp1-/- mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance.