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1.
Biomolecules ; 11(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34439792

RESUMO

The recent pandemic we are experiencing caused by the coronavirus disease 2019 (COVID-19) has put the world's population on the rack, with more than 191 million cases and more than 4.1 million deaths confirmed to date. This disease is caused by a new type of coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A massive proteomic analysis has revealed that one of the structural proteins of the virus, the E protein, interacts with BRD2 and BRD4 proteins of the Bromodomain and Extra Terminal domain (BET) family of proteins. BETs are essential to cell cycle progression, inflammation and immune response and have also been strongly associated with infection by different types of viruses. The fundamental role BET proteins play in transcription makes them appropriate targets for the propagation strategies of some viruses. Recognition of histone acetylation by BET bromodomains is essential for transcription control. The development of drugs mimicking acetyl groups, and thereby able to displace BET proteins from chromatin, has boosted interest on BETs as attractive targets for therapeutic intervention. The success of these drugs against a variety of diseases in cellular and animal models has been recently enlarged with promising results from SARS-CoV-2 infection studies.


Assuntos
COVID-19/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , COVID-19/imunologia , COVID-19/virologia , Proteínas do Envelope de Coronavírus/metabolismo , Humanos , Imunidade Inata , Ligação Proteica
2.
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
3.
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
4.
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
5.
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
6.
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
7.
Nutrients ; 13(7)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34371875

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) have been recognized to cause neurobehavioral dysfunctions and disorder of cognition and behavioral patterns in childhood. Momordica charantia L. (MC) has been widely known for its nutraceutical and health-promoting properties. To date, the effect of MC for the prevention and handling of PAHs-induced neurotoxicity has not been reported. In the current study, the neuroprotective effects of MC and its underlying mechanisms were investigated in mouse hippocampal neuronal cell line (HT22); moreover, in silico analysis was performed with the phytochemicals MC to decipher their potential function as neuroprotectants. MC was demonstrated to possess neuroprotective effect by reducing reactive oxygen species' (ROS') production and down-regulating cyclin D1, p53, and p38 mitogen-activated protein kinase (MAPK) protein expressions, resulting in the inhibition of cell apoptosis and the normalization of cell cycle progression. Additionally, 28 phytochemicals of MC and their competence on inhibiting cytochrome P450 (CYP: CYP1A1, CYP1A2, and CYP1B1) functions were resolved. In silico analysis of vitamin E and stigmasterol revealed that their binding to either CYP1A1 or CYP1A2 was more efficient than the binding of each positive control (alizarin or purpurin). Together, MC is potentially an interesting neuroprotectant including vitamin E and stigmasterol as probable active components for the prevention for PAHs-induced neurotoxicity.


Assuntos
Hipocampo/efeitos dos fármacos , Momordica charantia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Estigmasterol/farmacologia , Vitamina E/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Momordica charantia/química , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Estigmasterol/isolamento & purificação , Vitamina E/isolamento & purificação
8.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361115

RESUMO

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Cardiolipinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Clostridioides difficile/metabolismo , Lipídeos de Membrana/metabolismo , Clostridioides difficile/crescimento & desenvolvimento , Transporte Proteico
9.
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
10.
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
11.
Nature ; 596(7870): 138-142, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290405

RESUMO

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.


Assuntos
Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separase/química , Separase/metabolismo , Motivos de Aminoácidos , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/ultraestrutura , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinases relacionadas a CDC2 e CDC28/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Microscopia Crioeletrônica , Ciclina B1/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Securina/ultraestrutura , Separase/antagonistas & inibidores , Separase/ultraestrutura , Especificidade por Substrato
12.
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
13.
Mol Cell ; 81(15): 3065-3081.e12, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34297911

RESUMO

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Transcrição Genética/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Laminas/genética , Laminas/metabolismo , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos
14.
Anticancer Res ; 41(7): 3597-3606, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230156

RESUMO

AIM: To evaluate the association between bromodomain-containing protein 4 (BRD4) expression and clinicopathological factors and prognosis in human breast cancer specimens. PATIENTS AND METHODS: We used tissue microarrays constructed from samples of patients (n=183) who underwent surgery. We validated the association between BRD4 expression and prognosis in solid tumours, including breast cancer, using The Cancer Genome Atlas (TCGA) database. RESULTS: Immunohistochemical staining showed that BRD4 was widely distributed in breast cancer tissues. BRD4 was strongly expressed in 19.7% of patients but BRD4 staining intensity was not correlated with other clinicopathological factors. Most importantly, patients with a strong BRD4 expression had a significantly longer disease-specific survival than those with a weak BRD4 expression (100.0% vs. 91.3% at 5 years, p=0.027). mRNA expression analysis showed similar results (91.2% vs. 80.2% at 6 years, p=0.047). CONCLUSION: Strong BRD4 expression was associated with a significantly better prognosis in breast cancer tumours.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/metabolismo
15.
Nat Commun ; 12(1): 4441, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290255

RESUMO

BRD4, a Bromodomain and Extraterminal (BET) protein family member, is a promising anti-cancer drug target. However, resistance to BET inhibitors targeting BRD4 is common in solid tumors. Here, we show that cancer-associated fibroblast (CAF)-activated stromal signaling, interleukin-6/8-JAK2, induces BRD4 phosphorylation at tyrosine 97/98 in colorectal cancer, resulting in BRD4 stabilization due to interaction with the deubiquitinase UCHL3. BRD4 phosphorylation at tyrosine 97/98 also displays increased binding to chromatin but reduced binding to BET inhibitors, resulting in resistance to BET inhibitors. We further show that BRD4 phosphorylation promotes interaction with STAT3 to induce chromatin remodeling through concurrent binding to enhancers and super-enhancers, supporting a tumor-promoting transcriptional program. Inhibition of IL6/IL8-JAK2 signaling abolishes BRD4 phosphorylation and sensitizes BET inhibitors in vitro and in vivo. Our study reveals a stromal mechanism for BRD4 activation and BET inhibitor resistance, which provides a rationale for developing strategies to treat CRC more effectively.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Cromatina/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Janus Quinase 2/metabolismo , Fosforilação , Domínios Proteicos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Microambiente Tumoral , Ubiquitina Tiolesterase/metabolismo
16.
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
17.
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
18.
Nature ; 596(7870): 133-137, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34234345

RESUMO

The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Ilhas de CpG/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/química , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Regulação da Expressão Gênica , Genes Essenciais , Humanos , Camundongos , Imagem Individual de Molécula
19.
Nat Commun ; 12(1): 4105, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215755

RESUMO

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Dermatite/metabolismo , Endorribonucleases/metabolismo , Inflamação/metabolismo , Células Mieloides/metabolismo , Ribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CCL2 , Células Dendríticas , Endorribonucleases/deficiência , Endorribonucleases/genética , Epigenômica , Humanos , Imiquimode , Inflamação/patologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Psoríase , Ribonucleases/deficiência , Ribonucleases/genética , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
20.
Science ; 373(6551)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244384

RESUMO

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.


Assuntos
Proteínas de Ciclo Celular/genética , Síndrome de Down/genética , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide/genética , Pré-Leucemia/genética , Animais , Antígenos CD34/análise , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Transformação Celular Neoplásica , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Síndrome de Down/complicações , Feminino , Fator de Transcrição GATA1/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Fígado/embriologia , Masculino , Megacariócitos/fisiologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Pré-Leucemia/metabolismo , Pré-Leucemia/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores
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