RESUMEN
In brief: Aberration in cell cycle progression is one of the essential mechanisms underlying tumorigenesis, making regulators of cell cycle reasonable anti-cancer therapeutic targets. Here, we dissected the regulatory mechanism involving the novel axis ZNF146/TFDP1/DEPDC1B in the cell cycle in ovarian cancer. Abstract: Ovarian cancer (OC) is the third most common kind of gynecological tumor, in addition to being the most lethal. Transcription factor Dp-1 (TFDP1) functions as a binding partner for E2F transcription factors, and its target genes include those involved in DNA synthesis, cell cycle, and apoptosis. However, the regulatory role of TFDP1 in OC remains incompletely understood. This study aimed to investigate the role and mechanism of TFDP1 in OC. TFDP1 was highly expressed in the ovarian epithelial tissues of OC patients, and the expression of TFDP1 in OC cells was higher than that in normal ovarian epithelial cells. Silencing of TFDP1 inhibited the biological activity of OC cells and hindered cell cycle entry. Zinc finger protein 146 (ZNF146) knockdown induced cell cycle arrest at the G0/G1 phase and tumor growth by blocking TFDP1 transcription, which was overturned by ectopic expression of TFDP1. TFDP1 stimulated DEP domain-containing protein 1B (DEPDC1B) expression through transcriptional activation. DEPDC1B increased the proportion of OC cells in the G2/M phase and potentiated tumor malignant progression in nude mice inhibited by sh-ZNF146. Taken together, these findings demonstrate that ZNF146 participates in TFDP1/DEPDC1B activation and plays a vital role in the cell cycle in OC.
Asunto(s)
Ciclo Celular , Ratones Desnudos , Neoplasias Ováricas , Factor de Transcripción DP1 , Animales , Femenino , Humanos , Ratones , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Proteínas Activadoras de GTPasa , Ratones Endogámicos BALB C , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Factor de Transcripción DP1/metabolismo , Factor de Transcripción DP1/genéticaRESUMEN
Thymidine kinase 1 (TK1) level is an independent survival prognostic factor for both premalignant and malignant cervical pathologies. Herein, this study sought to probe the impacts of TK1 on cervical cancer (CC) progression and its underlying mechanism. Transcription factor Dp-1 (TFDP1) and TK1 expression was assessed using qRT-PCR in CC cell lines. After ectopic expression and knockdown experiments, cell counting kit-8 and colony formation assays were adopted to measure cell proliferation, western blot to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins, and Transwell assays to assess cell invasion and migration. The binding of TFDP1 to TK1 was predicted by bioinformatic sites and verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Tumor xenograft experiments in nude mice were performed to validate the influence of TFDP1/TK1 on CC progression in vivo. CC cells had high TK1 and TFDP1 expression. TFDP1 or TK1 knockdown restrained CC cell EMT, invasion, migration, and proliferation. TFDP1 facilitated TK1 expression in CC via transcription. Overexpression of TK1 counteracted the suppressive impacts of TFDP1 knockdown on CC cell malignant behaviors. Moreover, TFDP1 knockdown depressed CC growth in vivo by downregulating TK1. TFDP1 knockdown restricted proliferation and EMT in CC by downregulating TK1 expression.
Asunto(s)
Neoplasias del Cuello Uterino , Humanos , Animales , Ratones , Femenino , Neoplasias del Cuello Uterino/genética , Factor de Transcripción DP1 , Transición Epitelial-Mesenquimal , Ratones Desnudos , Proliferación CelularRESUMEN
The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1â¼E2F8), expression of activator E2Fs (E2F1â¼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.
Asunto(s)
Factor de Transcripción E2F1 , Regulación de la Expresión Génica , Humanos , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Genes Supresores de Tumor , Proteínas de Ciclo Celular/metabolismo , Factor de Transcripción E2F3/metabolismo , Factor de Transcripción DP1/genética , Factor de Transcripción DP1/metabolismoRESUMEN
Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743, on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743-mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse.
Asunto(s)
Trastornos Relacionados con Cocaína , Cocaína , VIH-1 , Animales , Cocaína/metabolismo , Cocaína/farmacología , Trastornos Relacionados con Cocaína/tratamiento farmacológico , Cricetinae , Cricetulus , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Doxiciclina , Humanos , Ratones , Ratones Transgénicos , Recompensa , Transactivadores , Factor de Transcripción DP1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5' flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes (2.167 ± 0.085, 2.341 ± 0.104, 2.529 ± 0.107 and 2.189 ± 0.070 for CC and 2.052 ± 0.047, 2.206 ± 0.057, 2.341 ± 0.056 and 2.160 ± 0.039 for GC) were significantly higher (p < 0.05) than those with the GG genotype (1.893 ± 0.051, 2.027 ± 0.064, 2.107 ± 0.061 and 1.74 ± 0.05). The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT−qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats.
Asunto(s)
Cabras , Células de la Granulosa , Animales , Proliferación Celular , Ciclinas/genética , Femenino , Cabras/genética , Mutación/genética , Regiones Promotoras Genéticas/genética , Factor de Transcripción DP1/genética , Regulación hacia ArribaRESUMEN
The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136-3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141-10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.
Asunto(s)
Leiomiosarcoma , Factores de Transcripción E2F/metabolismo , Glucólisis/genética , Humanos , Leiomiosarcoma/genética , ARN Nuclear Pequeño , Factor de Empalme U2AF , Factor de Transcripción DP1/genética , Factor de Transcripción DP1/metabolismoRESUMEN
Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b, and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b, the transcription factor TFDP1, which normally binds to the promoter of Trp53, cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a. Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.
Asunto(s)
Epigénesis Genética , Proteína p53 Supresora de Tumor , Animales , Desarrollo Embrionario , Femenino , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Embarazo , Transducción de Señal , Factor de Transcripción DP1 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Among eutherian (placental) mammals, placental embedding into the maternal endometrium exhibits great differences, from being deeply invasive (e.g., humans) to noninvasive (e.g., cattle). The degree of invasion of placental trophoblasts is positively correlated with the rate of cancer malignancy. Previously, we have shown that fibroblasts from different species offer different levels of resistance to the invading trophoblasts as well as to cancer cell invasion. Here we present a comparative genomic investigation revealing cis-regulatory elements underlying these interspecies differences in invasibility. We identify transcription factors that regulate proinvasibility and antiinvasibility genes in stromal cells. Using an in vitro invasibility assay combined with CRISPR-Cas9 gene knockout, we found that the transcription factors GATA2 and TFDP1 strongly influence the invasibility of endometrial and skin fibroblasts. This work identifies genomic mechanisms explaining species differences in stromal invasibility, paving the way to therapies targeting stromal characteristics to regulate placental invasion, wound healing, and cancer dissemination.
Asunto(s)
Endometrio/metabolismo , Trofoblastos/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Endometrio/patología , Femenino , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Técnicas de Inactivación de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Factor de Transcripción DP1/metabolismo , Trofoblastos/patologíaRESUMEN
Microglia and macrophages initiate and orchestrate the innate immune response to central nervous system (CNS) virus infections. Microglia initiate neurotropic coronavirus clearance from the CNS, but the role of infiltrating macrophages is not well understood. Here, using mice lacking cell-specific expression of DP1, the receptor for prostaglandin D2 (PGD2), we delineate the relative roles of PGD2 signaling in microglia and macrophages in murine coronavirus-infected mice. We show that the absence of PGD2/DP1 signaling on microglia recapitulated the suboptimal immune response observed in global DP1-/- mice. Unexpectedly, the absence of the DP1 receptor on macrophages had an opposite effect, resulting in enhanced activation and more rapid virus clearance. However, microglia are still required for disease resolution, even when macrophages are highly activated, in part because they are required for macrophage recruitment to sites of infection. Together, these results identify key differences in the effects of PGD2/DP1 signaling on microglia and macrophages and illustrate the complex relationship between the two types of myeloid cells. IMPORTANCE Current understanding about the roles of microglia versus macrophages in viral encephalitis is limited. We previously showed that the signaling of a single prostaglandin, PGD2, through its DP1 receptor on myeloid cells is critical for optimal immune responses in infected mice. Here, we demonstrate that the specific ablation of the DP1 receptor on macrophages and microglia had markedly different effects on outcomes. DP1-/- macrophages exhibited greater phagocytic properties than controls, resulting in enhanced kinetics of virus clearance, while DP1 absence on microglia resulted in increased lethality. Microglia were still required for protection, even when DP1 was not expressed on macrophages. These results suggest that therapeutic strategies directed at specific myeloid subsets in the brain may be useful in the context of viral infections.
Asunto(s)
Macrófagos/metabolismo , Microglía/metabolismo , Virus de la Hepatitis Murina/patogenicidad , Prostaglandina D2/metabolismo , Animales , Encefalitis/virología , Ratones , Fagocitosis , Transducción de Señal , Factor de Transcripción DP1/metabolismoRESUMEN
The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.
Asunto(s)
ADN/química , ADN/metabolismo , Factor de Transcripción E2F1/química , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción DP1/química , Factor de Transcripción DP1/metabolismo , Ciclo Celular , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Fosforilación , Unión Proteica , Conformación ProteicaRESUMEN
Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.
Asunto(s)
Síndromes Mielodisplásicos , Neoplasias , Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Humanos , Masculino , Síndromes Mielodisplásicos/tratamiento farmacológico , Análisis de Secuencia de ARN , Testículo , Factor de Transcripción DP1RESUMEN
BACKGROUND: The epithelial-mesenchymal transition (EMT) is a pivotal process for fibrotic disease, embryonic development, and wound healing. Moreover, some evidence has proven that the disorder of EMT also plays an important role in carcinogenesis, especially invasion and metastasis of various tumors (Ritchie et al., 2015). Additionally, gastric adenocarcinoma (GAC) is a common gastrointestinal malignancy which is the fourth most commonly diagnosed tumor. Our study is aimed at identifying the prognostic value of EMT-related genes in gastric adenocarcinoma. METHODS: Firstly, high-throughput and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. 99 differentially expressed EMT-related genes (ERGs) were obtained in these gastric adenocarcinoma data. Secondly, GO and KEGG enrichment analyses show that EMT may promote gastric carcinogenesis. Next, 10 ERGs associated with prognosis of gastric adenocarcinoma patients are screened out by univariate Cox regression, and 6 pivotal prognostic ERGs (MMP8, MMP11, TFDP3, MYB, F2, and CNTN1) are identified through multivariate Cox regression. These 6 genes are confirmed with significant prognostic value in gastric adenocarcinoma through overall survival (OS) analysis. Finally, a risk score formula is constructed and tested in another gastric adenocarcinoma cohort from GEO. RESULTS: 99 differentially expressed EMT-related genes (ERGs) and their enriched pathways are identified. 10 ERGs are strongly related to the prognosis of GAC patients. A risk score formula of 6 prognosis-related ERGs used to predict the prognosis of gastric adenocarcinoma patients is identified and tested (risk score = 0.448115∗expression value of MMP8 + 0.378892∗expression value of MMP11 - 0.3226∗expression value of MYB + 1.322812∗expression value of TFDP3 + 0.325063∗expression value of F2 + 0.334197∗expression value of CNTN1). CONCLUSION: This study provides a potential prognostic signature for predicting prognosis of gastric adenocarcinoma patients and molecular insights of EMT in gastric adenocarcinoma, and the formula focusing on the prognosis of gastric adenocarcinoma can be effective.
Asunto(s)
Adenocarcinoma/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Metaloproteinasa 11 de la Matriz , Metaloproteinasa 8 de la Matriz , Pronóstico , Proteínas Proto-Oncogénicas c-myb , Factores de Riesgo , Neoplasias Gástricas/patología , Análisis de Supervivencia , Factor de Transcripción DP1RESUMEN
The green alga Chlamydomonas proliferates by "multiple fission": a long G1 with >10-fold cell growth followed by multiple rapid divisions. Cells above a critical size threshold are "committed" and will divide independent of light and further cell growth. The number of divisions carried out depends on the initial size of the committed mother cell. Here, I show that CDKA1, the ortholog of the yeast and animal mitotic inducer CDK1, regulates the critical size for commitment. The Rb/E2F/Dp1 pathway regulates division number as well as commitment size. Epistasis analysis indicated that CDKA1 and Rb/E2F/Dp1 regulate multiple fission by distinct mechanisms. Rb-E2F/Dp1 regulates G1/S gene expression in animals and land plants. Transcriptome analysis showed that mat3 or dp1 disruption altered regulation of a large group of cell-division-associated genes with respect to cell size, but not with respect to synchronization timing. In contrast, cdka1 inactivation disturbed both temporal and cell-size regulation of expression. These defects were enhanced by double inactivation of cdka1 and dp1, suggesting interaction between CDKA1 and the Rb-E2F/Dp1 pathways in regulating cell-cycle-specific gene expression and cell-cycle initiation. In the context of a theoretical model for regulation of Chlamydomonas multiple fission, these results suggest that CDKA1 may promote a switch into a division-competent state, and E2F/Dp1 may promote maintenance of this state.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Chlamydomonas reinhardtii/metabolismo , Factores de Transcripción E2F/metabolismo , Proteína Quinasa CDC2/genética , Factores de Transcripción E2F/genética , Regulación de la Expresión Génica , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción DP1/genética , Factor de Transcripción DP1/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: It is important to establish cancer stem cell (CSC)-targeted therapies to eradicate cancer. As it is a CSC marker, we focused on Kruppel-like factor 5 (KLF5) in this study. METHODS: We searched for candidate microRNAs (miRNAs) that inhibited KLF5 expression by in silico analyses and screened them in colon cancer cell lines. RESULTS: We identified one promising miRNA, miR-4711-5p, that downregulated KLF5 expression by direct binding. This miRNA suppressed cell proliferation, migration and invasion ability, as well as stemness, including decreased stem cell marker expression, reactive oxygen species activity and sphere formation ability. MiR-4711-5p inhibited the growth of DLD-1 xenografts in nude mice with no adverse effects. We found that miR-4711-5p provoked G1 arrest, which could be attributed to direct binding of miR-4711-5p to TFDP1 (a heterodimeric partner of the E2F family). Our findings also suggested that direct binding of miR-4711-5p to MDM2 could upregulate wild-type p53, leading to strong induction of apoptosis. Finally, we found that miR-4711-5p had a potent tumour-suppressive effect compared with a putative anti-oncomiR, miR-34a, in tumour cell cultures derived from five patients with colorectal cancer. CONCLUSIONS: Our data suggest that miR-4711-5p could be a promising target for CSC therapy.
Asunto(s)
Neoplasias del Colon/terapia , Factores de Transcripción de Tipo Kruppel/metabolismo , MicroARNs/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factor de Transcripción DP1/metabolismo , Animales , Ciclo Celular/genética , Proliferación Celular/fisiología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Células HT29 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Factor de Transcripción DP1/genéticaRESUMEN
BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.
Asunto(s)
Factor de Transcripción E2F1/metabolismo , Neoplasias Hepáticas/genética , Estatmina/genética , Factor de Transcripción DP1/metabolismo , alfa Carioferinas/metabolismo , Factor de Transcripción E2F1/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Transducción de Señal/genética , Estatmina/metabolismo , Factor de Transcripción DP1/genética , Células Tumorales Cultivadas , alfa Carioferinas/genéticaRESUMEN
The eukaryotic GINS, Cdc45, and minichromosome maintenance proteins form an essential complex that moves with the DNA replication fork. The GINS protein complex has also been reported to associate with DNA polymerase. In archaea, the third domain of life, DNA polymerase D (PolD) is essential for DNA replication, and the genes encoding PolDs exist only in the genomes of archaea. The archaeal GAN (GINS-associated nuclease) is believed to be a homolog of the eukaryotic Cdc45. In this study, we found that the Thermococcus sp. 4557 DP1 (small subunit of PolD) interacted with GINS15 in vitro, and the 3'-5' exonuclease activity of DP1 was inhibited by GINS15. We also demonstrated that the GAN, GINS15, and DP1 proteins interact to form a complex adapting a GAN-GINS15-DP1 order. The results of this study imply that the complex constitutes a core of the DNA replisome in archaea.
Asunto(s)
Proteínas Arqueales/metabolismo , Mapas de Interacción de Proteínas , Multimerización de Proteína , Thermococcus/enzimología , Factor de Transcripción DP1/metabolismo , Replicación del ADNRESUMEN
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.
Asunto(s)
Complejo Represivo Polycomb 1/genética , Transcripción Genética , Ubiquitina-Proteína Ligasas/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cromatina/genética , Proteínas de Unión al ADN/genética , Factor de Transcripción E2F6/genética , Heterocromatina/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/genética , Proteínas Represoras/genética , Factor de Transcripción DP1/genética , Factores de Transcripción/genética , Factores Estimuladores hacia 5'/genéticaRESUMEN
The field of endometriosis etiopathogenesis aims to identify the origin of disease in endometrial disorders. Changes in gene and protein expression related to cell adhesion, collagenases, and, mainly, cell cycle regulators have been identified. We set out to analyze the expression of the transcription factor DP-1 (TFDP1) gene, which encodes a protein that controls the G1/S phase passage of the cell cycle, in the endometrium of women with deep infiltrating endometriosis (DIE). Samples of endometrium from both endometriosis-affected women and healthy women were collected, cultured and maintained at the Cell Bank of the Pelvic Pain and Endometriosis Unit of the Federal University of Sao Paulo. This study analyzed five samples from the endometrium cell culture of healthy patients (i.e. no pelvic disease, as determined by means of laparoscopic tubal ligation) and six samples from women diagnosed with DIE. Samples were evaluated for TFDP1 gene expression by real-time PCR. We observed a downregulation of TFDP1 in the endometrium cells of women with DIE when compared to the control (a fold-change of -2.05, p value=.011). The TFDP1 gene is part of the cell cycle pathway, but its function is not yet clear. Additional studies are necessary to clarify the function of TFDP1 in endometriosis etiopathogenesis.
Asunto(s)
Endometriosis/metabolismo , Endometrio/metabolismo , Enfermedades Peritoneales/metabolismo , Factor de Transcripción DP1/metabolismo , Adulto , Regulación hacia Abajo , Endometriosis/genética , Endometriosis/patología , Endometrio/patología , Femenino , Humanos , Enfermedades Peritoneales/genética , Enfermedades Peritoneales/patología , Factor de Transcripción DP1/genéticaRESUMEN
PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
Asunto(s)
Proteínas de Unión al ADN/ultraestructura , Factor de Transcripción DP1/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico , Microscopía por Crioelectrón/métodos , ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Dominios Proteicos/genética , Subunidades de Proteína/metabolismo , Pyrococcus abyssi/metabolismo , Pyrococcus abyssi/ultraestructura , Factor de Transcripción DP1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Karyopherin alpha 2 (KPNA2) is a nuclear import factor that is elevated in multiple cancers. However, its molecular regulation at the transcriptional levels is poorly understood. Here we found that KPNA2 was significantly upregulated in gallbladder cancer (GBC), and the increased levels were correlated with short survival of patients. Gene knocking down of KPNA2 inhibited tumor cell proliferation and migration in vitro as well as xenografted tumor development in vivo. A typical transcription factor E2F1 associated with its DNA-binding partner DP1 bond to the promoter region of KPNA2 and induced KPNA2 expression. In contrast, an atypical transcription factor E2F7 competed against DP1 and blocked E2F1-induced KPNA2 gene activation. Mutation of the dimerization residues of E2F7 or DNA-binding domain of E2F1 abolished the suppressive effects of E2F7 on KPNA2 gene expression. In addition, KPNA2 mediated nuclear localization of E2F1 and E2F7, where they in turn controlled KPNA2 expression. Taken together, our data provided mechanistic insights into divergently transcriptional regulation of KPNA2, thus pointing to KPNA2 as a potential target for cancer therapy.