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1.
Mol Cell ; 81(15): 3043-3045, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358457

RESUMO

Neguembor et al. (2021) use super-resolution microscopy to illuminate genome packaging inside the cell nucleus. They discover that transcription and topoisomerases protect chromatin from collapsing in a crumpled state refractory to DNA loop extrusion by cohesin proteins.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , DNA/genética , Humanos
2.
Pestic Biochem Physiol ; 178: 104923, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34446199

RESUMO

Cell division cycle protein 37 (Cdc37) is a molecular chaperone that actively participates in many intracellular physiological and biochemical processes as well as pathogen infection. However, the function of Cdc37 in silkworm cells under Bombyx mori nucleopolyhedrovirus (BmNPV) infection is unknown. We cloned and identified BmCdc37, a Cdc37 gene from B. mori, which is highly conserved among other species. After BmNPV infection, the expression level of the BmCdc37 gene was up-regulated and showed an expression pattern similar to the BmHsp90 gene, which relies on Cdc37 to stabilize and activate specific protein kinases. The immunofluorescence, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays all indicated that BmCdc37 interacts with BmHsp90 in silkworm cells. Both BmCdc37 and BmHsp90 promote the reproduction of BmNPV. Co-expression of BmCdc37 and BmHsp90 was better at promoting virus proliferation than overexpression alone. These findings all indicate that BmCdc37 plays an active role in the proliferation of BmNPV.


Assuntos
Bombyx , Animais , Bombyx/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , Nucleopoliedrovírus
3.
Mol Cell ; 81(16): 3368-3385.e9, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34375583

RESUMO

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , RNA/genética , Fatores de Transcrição/genética , Adenosina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Elementos Reguladores de Transcrição/genética , Ativação Transcricional/genética
4.
Medicine (Baltimore) ; 100(32): e26474, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34397869

RESUMO

ABSTRACT: This study is to identify potential biomarkers and therapeutic targets for lung adenocarcinoma (LUAD).GSE6044 and GSE118370 raw data from the Gene Expression Omnibus database were normalized with Robust Multichip Average. After merging these two datasets, the combat function of sva packages was used to eliminate batch effects. Then, limma packages were used to filtrate differentially expressed genes. We constructed protein-protein interaction relationships using STRING database and hub genes were identified based on connectivity degrees. The cBioportal database was used to explore the alterations of the hub genes. The promoter methylation of cyclin dependent kinase 1 (CDK1) and polo-like Kinase 1 (PLK1) and their association with tumor immune infiltration in patients with LUAD were investigated using DiseaseMeth version 2.0 and TIMER databases. The Cancer Genome Atlas-LUAD dataset was used to perform gene set enrichment analysis.We identified 10 hub genes, which were upregulated in LUAD, among which 8 were successfully verified in the Cancer Genome Atlas and Oncomine databases. Kaplan-Meier analysis indicated that the expressions of CDK1 and PLK1 in LUAD patients were associated with overall survival and disease-free survival. The methylation levels in the promoter regions of these 2 genes in LUAD patients were lower than those in normal lung tissues. Their expressions in LUAD were associated with tumor stages and relative abundance of tumor infiltrating immune cells, such as B cells, CD4+ T cells, and macrophages. Moreover, cell cycle, DNA replication, homologous recombination, mismatch repair, P53 signaling pathway, and small cell lung cancer signaling were significantly enriched in CDK1 and PLK1 high expression phenotype.CDK1 and PLK1 may be used as potential biomarkers and therapeutic targets for LUAD.


Assuntos
Adenocarcinoma de Pulmão/genética , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Proteína Quinase CDC2/biossíntese , Proteínas de Ciclo Celular/biossíntese , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Prognóstico , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , RNA Neoplásico/genética , Transdução de Sinais
5.
J Biol Regul Homeost Agents ; 35(Special Issue on Internal Medicine n.1)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34348833

RESUMO

Acute lymphoblastic leukemia (ALL) is a malignant disease of the hematopoietic system. At present, the mechanism and pathogenesis of ALL have not been fully clarified. This study aimed to illustrate the roles of Cdc10 protein-dependent transcript 1 (CDT1) in ALL. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was performed to examine serum levels of CDT1 in childhood ALL patients and healthy volunteers. The interaction between CDT1 expression and prognosis of childhood ALL was analyzed. Meanwhile, expressions of CDT1 in ALL cell lines were determined. Furthermore, CDT1 knockdown model was constructed in ALL cells, and Cell Counting Kit-8 (CCK-8), and Transwell assays were conducted to analyze the effect of CDT1 on the biological functions of ALL cells. Potential mechanism was further explored through detecting the expressions of Epithelial-to-mesenchymal transition (EMT)-related genes. RT-qPCR results indicated that serum level of CDT1 in childhood ALL patients was remarkably higher than that of healthy volunteers. Childhood ALL patients with high expression of CDT1 had lower overall survival rate compared with those expressing low expression of CDT1. CDT1 knockdown remarkably decreased the proliferation and metastasis abilities of pediatric ALL cells. Results of western blot showed that CDT1 might contribute to the malignant progression of childhood ALL via activating EMT. The findings showed that elevated CDT1 facilitated ALL metastasis by promoting EMT, suggesting that CDT1 played a pivotal role in ALL metastasis and may serve as a novel prognostic biomarker and therapeutic target.


Assuntos
Transição Epitelial-Mesenquimal , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética
6.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
7.
Nat Commun ; 12(1): 4843, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376693

RESUMO

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Repetições de Microssatélites/genética , Proteínas de Ligação a Telômeros/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades , DNA Polimerase I/genética , DNA Primase/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
8.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445562

RESUMO

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway; however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3high therapeutic-refractory cancer.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Epidérmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Complexo do Signalossomo COP9/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeo Hidrolases/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
9.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360715

RESUMO

Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells; however, its function in vivo is not well understood due to its early embryonic lethality in null mice exhibiting spontaneous DNA damage, cell cycle delays, and defects in check point activation. Here, we generated germ cell-specific Prmt1 knock-out (KO) mice to evaluate the function of PRMT1 in spermatogenesis. Our findings demonstrate that PRMT1 is vital for male fertility in mice. Spermatogenesis in Prmt1 KO mice was arrested at the zygotene-like stage of the first meiotic division due to an elevated number of DNA double-strand breaks (DSBs). There was a loss of methylation in meiotic recombination 11 (MRE11), the key endonuclease in MRE11/RAD50/NBS 1 (MRN) complex, resulting in the accumulation of SPO11 protein in DSBs. The ATM-mediated negative feedback control over SPO11 was lost and, consequently, the repair pathway of DSBs was highly affected in PRMT1 deficient male germ cells. Our findings provide a novel insight into the role of PRMT1-mediated asymmetric demethylation in mouse spermatogenesis.


Assuntos
Células Germinativas/enzimologia , Meiose , Proteína-Arginina N-Metiltransferases/metabolismo , Espermatogênese , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1129-1135, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362492

RESUMO

OBJECTIVE: To investigate the effect of CDK1 interference regulation of PLK1, Aurora B and TRF1 on the proliferation of leukemia cells. METHODS: The human myelogenous leukemia cell line HL-60 was selected as the research object, and the effect of TRF1 expression and its changes on cell proliferation and cycle was investigated by regulating intracellular CDK1 expression. The objects were divided into 5 groups, including control group, shRNA-NC group, CDK1-shRNA group, pcDNA group and pcDNA-CDK1 group. RT-PCR was used to detect the CDK1 expression of cells in each group; colony formation was used to detect the proliferation of the cells. Western blot was used to detect the expression of CDK1, PLK1, Aurora B, TRF1, and cyclin p53, p27, cyclinA. RESULTS: The phosphorylation level of PLK1, Aurora B and the expression of TRF1 in the CDK1-shRNA group were significantly down-regulated as compared with those in the control group (P<0.05). Compared with the control group, the cells in CDK1-shRNA group showed lower clone formation rate, the increasing of cycle-associated proteins p53 and p27 and the decreasing of cyclinA expression (P<0.05). It was shown that interfered CDK1 expression could inhibit the proliferation of HL-60 cells and prolong the time that they enter mitosis, thereby extending the cell cycle. Compared with the control group, the overexpressed CDK1 in the pcDNA-CDK1 group made the phosphorylation level of PLK1, Aurora B, and TRF1 expression increase significantly (P<0.05), also the colony formation rate (P<0.05). The cycle-related proteins p53 and p27 was down-regulated, while cyclinA expression was up-regulate significantly (P<0.05). The results indicted that overexpressed CDK1 could stimulate adverse reactions, thereby promoting the proliferation of HL-60 cells and shortening the cell cycle. CONCLUSION: Knocking out CDK1 can inhibit the phosphorylation of PLK1 and Aurora B and negatively regulate TRF1, thereby inhibiting the proliferation of leukemia cells.


Assuntos
Proteínas de Ciclo Celular , Leucemia , Proteína Quinase CDC2 , Proteínas de Ciclo Celular/genética , Proliferação de Células , Humanos , Mitose , Fosforilação , Proteínas Proto-Oncogênicas/genética
11.
J Cell Sci ; 134(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34432036

RESUMO

The budding yeast phosphatase Cdc14 has a central role in mitotic exit and cytokinesis. Puzzlingly, a uniform picture for the three human CDC14 paralogues CDC14A, CDC14B and CDC14C in cell cycle control has not emerged to date. Redundant functions between the three CDC14 phosphatases could explain this unclear picture. To address the possibility of redundancy, we tested expression of CDC14 and analysed cell cycle progression of cells with single and double deletions in CDC14 genes. Our data suggest that CDC14C is not expressed in human RPE1 cells, excluding a function in this cell line. Single- and double-knockouts (KO) of CDC14A and CDC14B in RPE1 cells indicate that both phosphatases are not important for the timing of mitotic phases, cytokinesis and cell proliferation. However, cycling CDC14A KO and CDC14B KO cells show altered ciliogenesis compared to wild-type cells. The cilia of cycling CDC14A KO cells are longer, whereas CDC14B KO cilia are more frequent and disassemble faster. In conclusion, this study demonstrates that the cell cycle functions of CDC14 proteins are not conserved between yeast and human cells.


Assuntos
Fosfatases de Especificidade Dupla , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Citocinese/genética , Fosfatases de Especificidade Dupla/genética , Humanos , Mitose , Proteínas Tirosina Fosfatases/genética
12.
Nat Commun ; 12(1): 4918, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389719

RESUMO

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.


Assuntos
Nucléolo Celular/genética , Reparo do DNA , DNA Ribossômico/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteína com Valosina/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Dano ao DNA , DNA Ribossômico/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Técnicas do Sistema de Duplo-Híbrido , Proteína com Valosina/metabolismo
13.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208772

RESUMO

Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic responses often involve human diseases. The importance of regulating angiogenesis in inflammatory diseases has been demonstrated through some successful cases of anti-angiogenesis therapies in related diseases, including arthritis, but it has been reported that some synthetic types of antiangiogenic drugs have potential side effects. In recent years, the importance of finding alternative strategies for regulating angiogenesis has begun to attract the attention of researchers. Therefore, identification of natural ingredients used to prevent or treat angiogenesis-related diseases will play a greater role. Isookanin is a phenolic flavonoid presented in Bidens extract, and it has been reported that isookanin possesses some biological properties, including antioxidative and anti-inflammatory effects, anti-diabetic properties, and an ability to inhibit α-amylase. However, its antiangiogenic effects and mechanism thereof have not been studied yet. In this study, our results indicate that isookanin has an effective inhibitory effect on the angiogenic properties of microvascular endothelial cells. Isookanin shows inhibitory effects in multiple stages of PGE2-induced angiogenesis, including the growth, proliferation, migration, and tube formation of microvascular endothelial cells. In addition, isookanin induces cell cycle arrest in S phase, which is also the reason for subsequent inhibition of cell proliferation. The mechanism of inhibiting angiogenesis by isookanin is related to the inhibition of PGE2-mediated ERK1/2 and CREB phosphorylation. These findings make isookanin a potential candidate for the treatment of angiogenesis-related diseases.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Chalconas/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Modelos Biológicos , Fosforilação
14.
Nat Commun ; 12(1): 4601, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326322

RESUMO

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.


Assuntos
Proteínas de Ciclo Celular/genética , Cinesina/genética , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Sistemas CRISPR-Cas , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Cinesina/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Neoplasias da Próstata/metabolismo , Taxa de Sobrevida
15.
Nat Commun ; 12(1): 4605, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326328

RESUMO

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Espermatogênese/fisiologia , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cristalografia por Raios X/métodos , Feminino , Recombinação Homóloga , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Meiose , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência
17.
Mol Cell ; 81(16): 3262-3274.e3, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34214466

RESUMO

N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Glicina/química , Conformação Proteica , Receptores de Citocinas/ultraestrutura , Sequência de Aminoácidos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Glicina/genética , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Ligação Proteica/genética , Domínios Proteicos/genética , Proteólise , Receptores de Citocinas/química , Receptores de Citocinas/genética , Especificidade por Substrato , Ubiquitina/genética
18.
Cell Prolif ; 54(8): e13096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240779

RESUMO

OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.


Assuntos
Edição de Genes , Piruvato Quinase/metabolismo , Processamento Alternativo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação para Baixo , Éxons , Feminino , Células HCT116 , Humanos , Mutagênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/genética , Regulação para Cima , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Theriogenology ; 172: 239-254, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34298284

RESUMO

Meiotic recombination is key to the repair of DNA double-strand break damage, provide a link between homologs for proper chromosome segregation as well as ensure genetic diversity in organisms. Defects in recombination often lead to sterility. The ubiquitously expressed Rad51 and the meiosis-specific DMC1 are two closely related recombinases that catalyze the key strand invasion and exchange step of meiotic recombination. This study cloned and sequenced the coding region of cattle-yak Rad51 and determined its mRNA and protein expression levels, evaluated its molecular and evolutionary relationship as well as evaluated the histo-morphological structure of testes in the yellow cattle, yak and the sterile cattle-yak hybrid. The Rad51 gene was amplified using PCR, cloned and sequenced using testicular cDNA from yak and cattle-yak. Real-time PCR was used to examine the expression levels of Rad51/DMC1 mRNA in the cattle, yak and cattle-yak testis while western blotting, immunofluorescence and immunohistochemistry were used to assess the protein expression and localization of Rad51/DMC1 protein in the testicular tissue sections. The results revealed that the mRNA and protein expression of Rad51 and DMC1 are extremely low in the male cattle-yak testis with a corresponding higher incidence of germ cell apoptosis. There was also thinning of the germinal epithelium possibly due to the depletion of the germ cells leading to the widening of the lumen area of the cattle-yak seminiferous tubule. Our findings provide support for the hypothesis that the low expression of Rad51 and DMC1 may contribute to the male hybrid sterility in the cattle-yak.


Assuntos
Reparo do DNA , Testículo , Animais , Bovinos/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Masculino , Meiose , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Testículo/metabolismo
20.
Mol Cell ; 81(15): 3082-3095.e6, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34197738

RESUMO

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos , Animais , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas , Coativador 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
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