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1.
J Coll Physicians Surg Pak ; 31(8): 937-940, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34320711

RESUMO

OBJECTIVE: To investigate the expression of miR-22-3p in breast cancer and the mechanism of targeting PLAGL2 to inhibit the invasion and migration in human breast cancer. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Oncology and Department of General Surgery, The People's Hospital of China Three Gorges University, China, from March 2019 to December 2020. METHODOLOGY: The miR-22-3p expression level in 41 paired human primary breast invasive ductal carcinoma tissues and para-cancer tissues was obtained by real-time fluorescence quantitative reverse transcriptase PCR (qRT-PCR). The effect of miR-22-3p on the proliferation of breast cancer cells was detected by growth curve method. Online software TargetScan was used to predict the target genes of miR-22-3p. The prediction results were verified by luciferase reporter gene assay and qRT⁃PCR. RESULTS: MiR-22-3p expression was significantly decreased in the breast cancer tissues than in para⁃carcinoma normal breast tissues (p<0.05). Over-expression of miR-22-3p can inhibit the proliferation of MCF-7 cells significantly. Pleomorphic adenoma gene-like protein 2(PLAGL2) is the predicted target gene of miR-22-3p. MiR-22-3p binds to its predicted target gene PLAGL2-3'UTR. The expression of miR-22-3p was negatively correlated with PLAGL2 in MCF-7 cells. CONCLUSION: MiR-22-3p could suppress the proliferation of breast cancer by targeting PLAGL2. This suggests that miR-22-3p may be a strategy of choice for targeted therapy of breast cancer. Key Words: Breast cancer, MiR-22-3p, PLAGL2, Cell proliferation.


Assuntos
Neoplasias da Mama , MicroRNAs , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , China , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
2.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205342

RESUMO

Little is known about the impairments and pathological changes in the visual system in mild brain trauma, especially repetitive mild traumatic brain injury (mTBI). The goal of this study was to examine and compare the effects of repeated head impacts on the neurodegeneration, axonal integrity, and glial activity in the optic tract (OT), as well as on neuronal preservation, glial responses, and synaptic organization in the lateral geniculate nucleus (LGN) and superior colliculus (SC), in wild-type mice and transgenic animals with overexpression of human TDP-43 mutant protein (TDP-43G348C) at 6 months after repeated closed head traumas. Animals were also assessed in the Barnes maze (BM) task. Neurodegeneration, axonal injury, and gliosis were detected in the OT of the injured animals of both genotypes. In the traumatized mice, myelination of surviving axons was mostly preserved, and the expression of neurofilament light chain was unaffected. Repetitive mTBI did not induce changes in the LGN and the SC, nor did it affect the performance of the BM task in the traumatized wild-type and TDP-43 transgenic mice. Differences in neuropathological and behavioral assessments between the injured wild-type and TDP-43G348C mice were not revealed. Results of the current study suggest that repetitive mTBI was associated with chronic damage and inflammation in the OT in wild-type and TDP-43G348C mice, which were not accompanied with behavioral problems and were not affected by the TDP-43 genotype, while the LGN and the SC remained preserved in the used experimental conditions.


Assuntos
Lesões Encefálicas Traumáticas/patologia , Trato Óptico/patologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gliose , Masculino , Aprendizagem em Labirinto , Camundongos Transgênicos , Sinapses/patologia
3.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205418

RESUMO

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Humanos , Estruturas R-Loop
4.
Nat Commun ; 12(1): 4255, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253720

RESUMO

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Complexos Multiproteicos/metabolismo , Animais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Proliferação de Células , Células HEK293 , Humanos , Meiose , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/metabolismo , Mutação/genética , Fenótipo , Rad51 Recombinase/metabolismo , Troca de Cromátide Irmã , Análise de Sobrevida
5.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206257

RESUMO

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunidade Inata , Miócitos Cardíacos/metabolismo , Receptores Toll-Like/genética , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Citocinas/metabolismo , Regulação da Expressão Gênica , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
6.
Int J Mol Sci ; 22(12)2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-34203082

RESUMO

Ideal Plant Architecture 1 (IPA1) encodes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14 (SPL14) with a pleiotropic effect on regulating rice development and biotic stress responses. To investigate the role of IPA1 in early seedling development, we developed a pair of IPA1/ipal-NILs and found that seed germination and early seedling growth were retarded in the ipa1-NIL. Analysis of the soluble sugar content, activity of amylase, and expression of the α-amylase genes revealed that the starch metabolism was weakened in the ipa1-NIL germinating seeds. Additionally, the content of bioactive gibberellin (GA) was significantly lower than that in the IPA1-NIL seeds at 48 h of imbibition. Meanwhile, the expression of GA synthesis-related gene OsGA20ox1 was downregulated, whereas the expression of GA inactivation-related genes was upregulated in ipa1-NIL seeds. In addition, the expression of OsWRKY51 and OsWRKY71 was significantly upregulated in ipa1-NIL seeds. Using transient dual-luciferase and yeast one-hybrid assays, IPA1 was found to directly activate the expression of OsWRKY51 and OsWRKY71, which would interfere with the binding affinity of GA-induced transcription factor OsGAMYB to inhibit the expression of α-amylase genes. In summary, our results suggest that IPA1 negatively regulates seed germination and early seedling growth by interfering with starch metabolism via the GA and WRKY pathways.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Giberelinas/metabolismo , Oryza/fisiologia , Desenvolvimento Vegetal , Plântula/crescimento & desenvolvimento , Transdução de Sinais , Amido/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Fenótipo , Ligação Proteica , alfa-Amilases/metabolismo
7.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202641

RESUMO

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Predisposição Genética para Doença , Mutação , Neoplasias/genética , Biomarcadores Tumorais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gerenciamento Clínico , Regulação da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/terapia , Especificidade de Órgãos , Ligação Proteica , Relação Estrutura-Atividade
8.
Nat Commun ; 12(1): 4126, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226554

RESUMO

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.


Assuntos
Proteína BRCA1/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA , Recombinação Homóloga , Proteínas de Neoplasias/metabolismo , RNA , Proteína BRCA1/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , DNA Helicases , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Ftalazinas , Piperazinas , RNA Helicases , RNA Mensageiro , Rad51 Recombinase , Reparo de DNA por Recombinação
9.
Nucleic Acids Res ; 49(12): 6832-6848, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34157114

RESUMO

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/fisiologia
10.
Nat Commun ; 12(1): 3621, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131149

RESUMO

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.


Assuntos
Cromatina , Glioma/genética , Sequências Reguladoras de Ácido Nucleico , Sítios de Ligação , Neoplasias Encefálicas/genética , Imunoprecipitação da Cromatina , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Epigenômica , Proteína Forkhead Box M1 , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Código das Histonas , Histonas , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
11.
Nucleic Acids Res ; 49(11): 6213-6237, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086943

RESUMO

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.


Assuntos
Processamento Alternativo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Éxons , Feminino , Células HeLa , Código das Histonas , Humanos , Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
12.
Nucleic Acids Res ; 49(11): 6196-6212, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086947

RESUMO

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.


Assuntos
Chaperonas de Histonas/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Evolução Biológica , Sequência Conservada , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Expressão Gênica , Células HEK293 , Chaperonas de Histonas/química , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidade , Oncogenes , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/crescimento & desenvolvimento
13.
Nucleic Acids Res ; 49(11): 6569-6586, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107018

RESUMO

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DnaB Helicases/química , Vibrio cholerae/enzimologia , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DnaB Helicases/metabolismo , Modelos Moleculares , Conformação Proteica , Serina/química
14.
Nucleic Acids Res ; 49(11): 6296-6314, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107019

RESUMO

Metal-induced genes are usually transcribed at relatively low levels under normal conditions and are rapidly activated by heavy metal stress. Many of these genes respond preferentially to specific metal-stressed conditions. However, the mechanism by which the general transcription machinery discriminates metal stress from normal conditions and the regulation of MTF-1-meditated metal discrimination are poorly characterized. Using a focused RNAi screening in Drosophila Schneider 2 (S2) cells, we identified a novel activator, the Drosophila gawky, of metal-responsive genes. Depletion of gawky has almost no effect on the basal transcription of the metallothionein (MT) genes, but impairs the metal-induced transcription by inducing the dissociation of MTF-1 from the MT promoters and the deficient nuclear import of MTF-1 under metal-stressed conditions. This suggests that gawky serves as a 'checkpoint' for metal stress and metal-induced transcription. In fact, regular mRNAs are converted into gawky-controlled transcripts if expressed under the control of a metal-responsive promoter, suggesting that whether transcription undergoes gawky-mediated regulation is encrypted therein. Additionally, lack of gawky eliminates the DNA binding bias of MTF-1 and the transcription preference of metal-specific genes. This suggests a combinatorial control of metal discrimination by gawky, MTF-1, and MTF-1 binding sites.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Metais/toxicidade , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cobre/toxicidade , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Metalotioneína/genética , Regiões Promotoras Genéticas , Interferência de RNA , Splicing de RNA , Estresse Fisiológico/genética
15.
Nucleic Acids Res ; 49(11): 6596-6603, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34110422

RESUMO

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.


Assuntos
Bacteriófago M13/fisiologia , DNA de Cadeia Simples/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas Virais/genética , Regiões 5' não Traduzidas , Bacteriófago M13/genética , Bacteriófago M13/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Mutação , Proteínas Virais/metabolismo , Replicação Viral
17.
Nat Commun ; 12(1): 3636, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140467

RESUMO

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ratos , Mutações Sintéticas Letais/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
18.
Nat Commun ; 12(1): 3705, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140493

RESUMO

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Linfoma de Células T Periférico/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas de Ligação a RNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transdução de Sinais/genética , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
19.
Nat Commun ; 12(1): 3428, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103526

RESUMO

Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.


Assuntos
Aborto Habitual/metabolismo , Vilosidades Coriônicas/metabolismo , Ácido Succínico/metabolismo , Aborto Habitual/enzimologia , Aborto Habitual/genética , Animais , Estudos de Casos e Controles , Hipóxia Celular , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaboloma , Camundongos Endogâmicos C57BL , Gravidez , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Risco , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Trofoblastos/metabolismo , Trofoblastos/patologia
20.
Rev Assoc Med Bras (1992) ; 67(1): 64-70, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34161481

RESUMO

OBJECTIVE: Bladder cancer under the age of 40 is extremely rare. Bladder cancer development involves complex and multi-stage processes, one of which is the DNA damage repair mechanism. In this retrospective study, we aimed to evaluate the histopathological features of bladder urothelial carcinoma seen in patients under 40 years of age and tumor microsatellite instability status using immunohistochemistry. METHODS: A total of 50 patients under the age of 40 with urothelial bladder carcinoma from two different centers in the same country were included. Expression of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 was analyzed by immunohistochemistry. RESULTS: Age at the time of diagnosis ranged from 17 to 40 years old. Most tumors were non-invasive papillary urothelial carcinoma. Two cases had nuclear loss of MSH-6 and PMS-2. We observed that tumor grade, tumor stage, presence of tumor differentiation, and infiltrative growth pattern of the tumor have significant impact on prognosis, but microsatellite instability does not have an effective role in bladder carcinogenesis in young patients. CONCLUSIONS: Our results indicate that the presence of microsatellite instability is not related to the low tumor grade and stage in urothelial neoplasms in young patients, suggesting that urothelial carcinoma of the bladder in young patients may represent a genetically stable form of neoplasia.


Assuntos
Carcinoma de Células de Transição , Instabilidade de Microssatélites , Adolescente , Adulto , Carcinoma de Células de Transição/genética , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Estudos Retrospectivos , Bexiga Urinária/metabolismo , Adulto Jovem
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