RESUMO
Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.
Assuntos
Carcinoma Hepatocelular , Endonucleases , Neoplasias Hepáticas , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Endonucleases/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Predisposição Genética para DoençaRESUMO
Members of the E2F transcription factor family regulate the expression of genes important for DNA replication and mitotic cell division in most eukaryotes. Homologs of the retinoblastoma (RB) tumor suppressor inhibit the activity of E2F factors, thus controlling cell cycle progression. Organisms such as budding and fission yeast have lost genes encoding E2F and RB, but have gained genes encoding other proteins that take on E2F and RB cell cycle-related functions. In addition to regulating cell proliferation, E2F and RB homologs have non-canonical functions outside the mitotic cell cycle in a variety of eukaryotes. For example, in both mammals and plants, E2F and RB homologs localize to DNA double-strand breaks (DSBs) and directly promote repair by homologous recombination (HR). Here, we discuss the parallels between mammalian E2F1 and RB and their Arabidopsis homologs, E2FA and RB-related (RBR), with respect to their recruitment to sites of DNA damage and how they help recruit repair factors important for DNA end resection. We also explore the question of whether this role in DNA repair is a conserved ancient function of the E2F and RB homologs in the last eukaryotic common ancestor or whether this function evolved independently in mammals and plants.
RESUMO
The E2F1 transcription factor and RB tumor suppressor are best known for their roles in regulating the expression of genes important for cell cycle progression but, they also have transcription-independent functions that facilitate DNA repair at sites of damage. Depending on the type of DNA damage, E2F1 can recruit either the GCN5 or p300/CBP histone acetyltransferases to deposit different histone acetylation marks in flanking chromatin. At DNA double-strand breaks, E2F1 also recruits RB and the BRG1 ATPase to remodel chromatin and promote loading of the MRE11-RAD50-NBS1 complex. Knock-in mouse models demonstrate important roles for E2F1 post-translational modifications in regulating DNA repair and physiological responses to DNA damage. This review highlights how E2F1 moonlights in DNA repair, thus revealing E2F1 as a versatile protein that recruits many of the same chromatin-modifying enzymes to sites of DNA damage to promote repair that it recruits to gene promoters to regulate transcription.
Assuntos
Dano ao DNA , Reparo do DNA , Fator de Transcrição E2F1/metabolismo , Proteína do Retinoblastoma/metabolismo , Acetilação , Animais , Montagem e Desmontagem da Cromatina , Fator de Transcrição E2F1/genética , Histonas/metabolismo , Humanos , Proteína do Retinoblastoma/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.
Assuntos
Proteína de Ligação a CREB/metabolismo , Quebras de DNA de Cadeia Dupla , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição E2F1/metabolismo , Histonas/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Técnicas de Introdução de Genes , Histona Acetiltransferases , Lisina Acetiltransferase 5/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Radiação Ionizante , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The retinoblastoma (RB) tumor suppressor is known as a master regulator of the cell cycle. RB is mutated or functionally inactivated in the majority of human cancers. This transcriptional regulator exerts its function in cell cycle control through its interaction with the E2F family of transcription factors and with chromatin remodelers and modifiers that contribute to the repression of genes important for cell cycle progression. Over the years, studies have shown that RB participates in multiple processes in addition to cell cycle control. Indeed, RB is known to interact with over 200 different proteins and likely exists in multiple complexes. RB, in some cases, acts through its interaction with E2F1, other members of the pocket protein family (p107 and p130), and/or chromatin remodelers and modifiers. RB is a tumor suppressor with important chromatin regulatory functions that affect genomic stability. These functions include the role of RB in DNA repair, telomere maintenance, chromosome condensation and cohesion, and silencing of repetitive regions. In this review we will discuss recent advances in RB biology related to RB, partner proteins, and their non-transcriptional functions fighting back against genomic instability.
Assuntos
Reparo do DNA , Inativação Gênica , Instabilidade Genômica , Proteína do Retinoblastoma/metabolismo , Homeostase do Telômero , Animais , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Humanos , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismoRESUMO
Tissue culture and mouse model studies show that the presence of the arginine (R) or proline (P) coding single nucleotide polymorphism (SNP) of the tumor suppressor gene p53 at codon 72 (p53 R72P) differentially affects the responses to genotoxic insult. Compared to the P variant, the R variant shows increased apoptosis in most cell cultures and mouse model tissues in response to genotoxins, and epidemiological studies suggest that the R variant may enhance cancer survival and reduce the risks of adverse reactions to genotoxic cancer treatment. As ionizing radiation (IR) treatment is often used in cancer therapy, we sought to test the physiological effects of IR in mouse models of the p53 R72P polymorphism. By performing blood counts, immunohistochemical (IHC) staining and survival studies in mouse populations rigorously controlled for strain background, sex and age, we discovered that p53 R72P polymorphism did not differentially affect the physiological response to IR. Our findings suggest that genotyping for this polymorphism to personalize IR therapy may have little clinical utility.
Assuntos
Lesões Experimentais por Radiação/genética , Proteína Supressora de Tumor p53/genética , Substituição de Aminoácidos , Animais , Apoptose , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Feminino , Intestinos/patologia , Intestinos/efeitos da radiação , Masculino , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Lesões Experimentais por Radiação/patologia , Baço/patologia , Baço/efeitos da radiação , Timo/patologia , Timo/efeitos da radiaçãoRESUMO
The retinoblastoma (RB) tumor suppressor is recognized as a master regulator that controls entry into the S phase of the cell cycle. Its loss leads to uncontrolled cell proliferation and is a hallmark of cancer. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double-strand breaks (DSBs) dependent on E2F1 and ATM kinase activity and promotes DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1, and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability, and renders mice hypersensitive to IR. Importantly, depletion of RB in osteosarcoma and breast cancer cell lines results in sensitivity to DNA-damaging drugs, which is further exacerbated by poly-ADP ribose polymerase (PARP) inhibitors. We uncovered a novel, nontranscriptional function for RB in HR, which could contribute to genome instability associated with RB loss.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Recombinação Homóloga/genética , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Helicases/genética , Reparo do DNA/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Raios gama , Técnicas de Introdução de Genes , Instabilidade Genômica/genética , Humanos , Masculino , Camundongos , Mutagênicos/farmacologia , Mutação , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transporte Proteico/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Irradiação Corporal Total/mortalidadeRESUMO
DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.
Assuntos
Dano ao DNA , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptor TIE-2/metabolismo , Tirosina/metabolismo , Angiotensina I/metabolismo , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , Humanos , Ligação Proteica , Transporte Proteico , Proto-Oncogene Mas , Tolerância a Radiação/genética , Radiação Ionizante , Receptor TIE-2/genéticaRESUMO
A hydrogen bond of the type C-Hâ â â X (X=O or N) is known to influence the structure and function of chemical and biological systems in solution. C-Hâ â â O hydrogen bonding in solution has been extensively studied, both experimentally and computationally, whereas the equivalent thermodynamic parameters have not been enumerated experimentally for C-Hâ â â N hydrogen bonds. This is, in part, due to the lack of systems that exhibit persistent C-Hâ â â N hydrogen bonds in solution. Herein, a class of molecule based on a biologically active norharman motif that exhibits unsupported intermolecular C-Hâ â â N hydrogen bonds in solution has been described. A pairwise interaction leads to dimerisation to give bond strengths of about 7â kJ mol-1 per hydrogen bond, which is similar to chemically and biologically relevant C-Hâ â â O hydrogen bonding. The experimental data is supported by computational work, which provides additional insight into the hydrogen bonding by consideration of electrostatic and orbital interactions and allowed a comparison between calculated and extrapolated NMR chemical shifts.
RESUMO
Gold(I)-catalysed direct allylic etherifications have been successfully carried out with chirality transfer to yield enantioenriched, γ-substituted secondary allylic ethers. Our investigations include a full substrate-scope screen to ascertain substituent effects on the regioselectivity, stereoselectivity and efficiency of chirality transfer, as well as control experiments to elucidate the mechanistic subtleties of the chirality-transfer process. Crucially, addition of molecular sieves was found to be necessary to ensure efficient and general chirality transfer. Computational studies suggest that the efficiency of chirality transfer is linked to the aggregation of the alcohol nucleophile around the reactive π-bound Au-allylic ether complex. With a single alcohol nucleophile, a high degree of chirality transfer is predicted. However, if three alcohols are present, alternative proton transfer chain mechanisms that erode the efficiency of chirality transfer become competitive.
RESUMO
A mild gold-catalyzed protodeboronation reaction, which does not require acid or base additives and can be carried out in "green" solvents, is described. As a result, the reaction is very functional-group-tolerant, even to acid- and base-sensitive functional groups, and should allow for the boronic acid group to be used as an effective traceless directing or blocking group. The reaction has also been extended to deuterodeboronations for regiospecific ipso-deuterations of aryls and heteroaryls from the corresponding organoboronic acid. Based on density functional theory calculations, a mechanism is proposed that involves nucleophilic attack of water at boron followed by rate-limiting B-C bond cleavage and facile protonolysis of a Au-σ-phenyl intermediate.
RESUMO
A direct Pd-catalyzed C-H functionalization of benzoquinone (BQ) can be controlled to give either mono- or disubstituted BQ, including the installation of two different groups in a one-pot procedure. BQ can now be directly functionalized with aryl, heteroaryl, cycloalkyl, and cycloalkene groups and, moreover, the reaction is conducted in environmentally benign water or acetone as solvents.
RESUMO
The far-infrared spectroscopy of the troposphere (FIRST) instrument is a Fourier-transform spectrometer developed to measure the Earth's thermal emission spectrum with a particular emphasis on the far-infrared. FIRST has observed the atmosphere from both the ground looking up and from a high-altitude balloon looking down. A recent absolute laboratory calibration of FIRST under ground-like operating conditions showed accuracy to better than 0.3 K at near-ambient temperatures (270-325 K) but reduced accuracy at lower temperatures. This paper presents calibration results for balloon-flight conditions using a cold blackbody to simulate the space view used for on-board calibration. An unusual detector nonlinearity was discovered and corrected, and stray light was measured and removed. Over most of the range of Earth scene temperatures (205-300 K), the accuracy of FIRST is 0.35-0.15 K (one sigma).
RESUMO
A gold(I)-catalysed direct thioetherification reaction between allylic alcohols and thiols is presented. The reaction is generally highly regioselective (S(N)2'). This dehydrative allylation procedure is very mild and atom economical, producing only water as the by-product and avoiding any unnecessary waste/steps associated with installing a leaving or activating group on the substrate. Computational studies are presented to gain insight into the mechanism of the reaction. Calculations indicate that the regioselectivity is under equilibrium control and is ultimately dictated by the thermodynamic stability of the products.
Assuntos
Compostos Alílicos/química , Ouro/química , Propanóis/química , Sulfetos/química , Catálise , Modelos Moleculares , EstereoisomerismoRESUMO
In response to DNA damage, the E2F1 transcription factor is phosphorylated at serine 31 (serine 29 in mouse) by the ATM or ATR kinases, which promotes E2F1 protein stabilization. Phosphorylation of E2F1 also leads to the recruitment of E2F1 to sites of DNA damage, where it functions to enhance DNA repair. To study the role of this E2F1 phosphorylation event in vivo, a knock-in mouse model was generated, in which serine 29 was mutated to alanine. The S29A mutation impairs E2F1 stabilization in response to ultraviolet (UV) radiation and doxorubicin treatment, but has little effect on the expression of E2F target genes. The apoptotic and proliferative responses to acute UV radiation exposure are also similar between wild-type and E2f1(S29A/) (S29A) mice. As expected, the S29A mutation prevents E2F1 association with damaged DNA and reduces DNA repair efficiency. Moreover, E2f1(S29A/) (S29A) mice display increased sensitivity to UV-induced skin carcinogenesis. This knock-in mouse model thus links the ability of E2F1 to directly promote DNA repair with the suppression of tumor development.
Assuntos
Carcinogênese/genética , Reparo do DNA , Fator de Transcrição E2F1/fisiologia , Neoplasias Cutâneas/genética , Raios Ultravioleta , Substituição de Aminoácidos , Animais , Células Cultivadas , Dano ao DNA , Técnicas de Introdução de Genes , Genes Supressores de Tumor , Camundongos , Camundongos Transgênicos , Fosforilação , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transporte ProteicoRESUMO
The promoter of p53 induced gene 3 (PIG3) contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n that is known as a p53 binding site. In this study, we investigated whether other potential molecules could bind to this PIG3 promoter (TGYCC)n motif. Ligand-chromatography combined with liquid chromatography-tandem mass spectrometry analyses indicated direct interactions of prohibitin and/or prohibiton with the (TGYCC)15 motif, which was confirmed by electrophoretic mobility shift assay and super-gel shift analysis with anti-prohibitin and anti-prohibiton antibodies. Using the chromatin immunopercipipation assay, we further demonstrated that prohibitin and prohibiton associated with the (TGYCC)15 motif in vivo regardless of the p53 status and apoptotic stress. We also found that prohibitin and prohibiton up-regulated PIG3 transcription independent of p53, although p53 obviously enhanced this process, and that the knock-down of prohibitin and prohibiton inhibited camptothecin-induced apoptosis. Taken together, our findings suggest that prohibitin and prohibiton contribute to PIG3-mediated apoptosis by binding to the PIG3 promoter (TGYCC)15 motif.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo , Apoptose/genética , Apoptose/fisiologia , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Repetições de Microssatélites , Proibitinas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A large number of epidemiological studies have linked a common single-nucleotide polymorphism (SNP) in the human p53 gene to risk for developing a variety of cancers. This SNP encodes either an arginine or proline at position 72 (R72P) of the p53 protein, which can alter the apoptotic activity of p53 via transcriptional and non-transcriptional mechanisms. This SNP has also been reported to modulate the development of human papilloma virus (HPV)-driven cancers through differential targeting of the p53 variant proteins by the E6 viral oncoprotein. Mouse models for the p53 R72P polymorphism have recently been developed but a role for this SNP in modifying cancer risk in response to viral and chemical carcinogens has yet to be established experimentally. Here, we demonstrate that the p53 R72P polymorphism modulates the hyperprolferative, apoptotic and inflammatory phenotypes caused by expression of the HPV16 E6 and E7 oncoproteins. Moreover, the R72P SNP also modifies the carcinogenic response to the chemical carcinogen 4NQO, in the presence and absence of the HPV16 transgene. Our findings confirm several human epidemiological studies associating the codon 72 proline variant with increased risk for certain cancers but also suggest that there are tissue-specific differences in how the R72P polymorphism influences the response to environmental carcinogens.
Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Neoplasias Esofágicas/etiologia , Interação Gene-Ambiente , Inflamação/etiologia , Neoplasias Bucais/etiologia , Infecções por Papillomavirus/etiologia , Polimorfismo de Nucleotídeo Único/genética , Proteína Supressora de Tumor p53/fisiologia , Animais , Western Blotting , Carcinógenos/toxicidade , Células Cultivadas , DNA Viral/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas Oncogênicas Virais/fisiologia , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/fisiologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Queratinócitos/fisiologia , Neoplasias Cutâneas/genética , Pele/patologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Animais , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Pele/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoAssuntos
Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Arteriopatias Oclusivas/diagnóstico , Trombose/diagnóstico por imagem , Constipação Intestinal/etiologia , Feminino , Humanos , Imageamento Tridimensional , Dor Lombar/etiologia , Pessoa de Meia-Idade , Debilidade Muscular/etiologia , Transtornos de Sensação/etiologia , Tomografia Computadorizada por Raios X , Retenção Urinária/etiologiaRESUMO
Signal transduction pathways converge upon sequence-specific DNA binding factors to reprogram gene expression. Transcription factors, in turn, team up with chromatin modifying activities. However, chromatin is not simply an endpoint for signaling pathways. Histone modifications relay signals to other proteins to trigger more immediate responses than can be achieved through altered gene transcription, which might be especially important to time-urgent processes such as the execution of cell-cycle check points, chromosome segregation, or exit from mitosis. In addition, histone-modifying enzymes often have multiple nonhistone substrates, and coordination of activity toward different targets might direct signals both to and from chromatin.