Search

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

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

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.

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.

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 [sup3/supH]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [sup3/supH]DA uptake and decreased the cocaine-mediated dissociation of [sup3/supH]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.

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.

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.

The transcription factor Dp1, as a binding partner, often forms a dimerization complex with typical E2F to play a central role in regulating gene expression during G1/S cell cycle progression. In this study, a full-length dp1 cDNA (Pcdp1) was successfully cloned and characterized from the large yellow croaker Pseudosciaena crocea. The nucleotidic sequence of Pcdp1 is 1,427 bp long with an open reading frame (ORF) of 1,239 bp encoding a putative protein of 412 amino acids, a 5′-untranslated region of 116 bp and a 3′-untranslated region of 70 bp. Prediction of protein domains showed that PcDp1 contains a DNA-binding domain (DBD) with a DEF box, a dimerization domain and an acidic region at C terminus with transcription activity. Homology comparisons indicated that PcDp1 shared the highest sequence identity of 98.55% with Oreochromis niloticus dp1, followed by 88.72% identity with Danio rerio dp1 and a relatively low identity of 78.91–80.55% with its mammalian and amphibian counterparts. The mRNA of Pcdp1 showed ubiquitously expression in all analyzed tissues, with the highest level of expression in the body kidney. Moderate expression levels of Pcdp1 was found in several immune-related tissues including the gills, head kidney and liver, indicating that PcDp1 might play an important role in osmotic pressure regulation and immune response of the large yellow croaker. The subcellular localization of PcDp1 revealed that it is mainly distributed in the cytoplasm both in COS-7 and parenchymal cells of the spleen, head kidney and kidney tissues. Furthermore, the recombinant PcDp1 exhibited DNA-binding activity to E2F site in vitro. In conclusion, these results indicated that PcDp1 may participate in immune regulation and provide a foundation for further study of the regulatory mechanism of Dp1 in teleosts., Large yellow croaker (Pseudosciaena crocea); Transcription factor Dp1; Tissue distribution; Subcellular localization; DNA-binding activity.

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.

The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.

COMMD protein family is an evolutionarily conserved gene family implicated in a number of critical processes including inflammation, copper homeostasis, sodium balance, endosomal sorting and cancer. In an effort to profile the expression pattern of COMMD family in several non-small cell lung cancer (NSCLC) cell lines, we found that compared with that in human bronchial epithelial (HBE) cells, the mRNA expression levels of five COMMD genes including COMMD3, COMMD4, COMMD5, COMMD6 and COMMD8 were significantly down-regulated, whereas COMMD9 was up-regulated in NSCLC cell lines. Here we reported that the expression of COMMD9 protein was significantly increased in various NSCLC cell lines and tissue samples. SiRNA-induced knocking down of COMMD9 inhibited proliferation and migration, arrested cell cycle at G1/S transition and induced autophagy in NSCLC cells. Mechanistically, COMMD9 interacted with the TFDP1 through COMM domain, and DNA-binding domain of TFDP1 was required for this interaction. Moreover, decreased expression COMMD9 attenuated TFDP1/E2F1 activation accompanied with enhanced p53 signaling pathway. Taken together, these findings demonstrate that COMMD9 participates in TFDP1/E2F1 activation and plays a critical role in non-small cell lung cancer.

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.

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.

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.

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., A cryo-EM reconstruction of the archaeal PolD DNA polymerase assembly bound to DNA defines the minimal catalytic fold for two-barrel DNA and RNA polymerases and suggests a scenario for the evolution of the eukaryotic replicative apparatus., Author summary PolD is an unusual archaeal replicative DNA polymerase (DNAP) in that it has all the functional properties of a replicative DNAP but with a catalytic core reminiscent of an RNA polymerase (RNAP). We now describe the cryo–electron microscopy (cryo-EM) structure of a DNA-bound heterodimeric DP1–DP2 PolD complex from the archaeon Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD with RNAPs extends their structural similarities and highlights the minimal catalytic core shared by all cellular transcriptases. Elucidating the structure of the interface between PolD’s DP1 and DP2 subunits, which is conserved in all eukaryotic replicative DNA polymerases, clarifies their evolutionary relationships with PolD and sheds light on the process of domain acquisition and exchange that occurred during the evolution of the eukaryotic replisome.

Summary 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., Graphical Abstract, Highlights • PRC1 complexes retain high target specificity with little compensatory functions • PCGF2 defines repressive signatures and PCGF3 and PCGF6 transcription active states • RING1A/B is dispensable for the assembly and recruitment of PCGF3 and PCGF6 complexes • PCGF3 and PCGF6 complexes bind target genes through specific DNA binding activities, PRC1 encompasses different sub-complexes with distinct biochemical properties. Scelfo et al. dissect these activities, highlighting their little redundancy and high functional specificity. PRC1 sub-complexes associate specifically with transcriptional repression or activation, can function independently of the core RING1A/B enzymes, and can be recruited to chromatin by specific DNA binding activities.

The heterodimeric transcription factor E2F1-DP1 plays crucial roles in coordinating gene expression during G1/S cell cycle progression. For transcriptional activation, the transactivation domain (TAD) of E2F1 is known to interact with the TATA-binding protein of TFIID and the p62 subunit of TFIIH. It is generally believed that DP1 facilitates E2F1 binding to target DNA and does not possess a TAD. Here, we show that an acidic region of DP1, whose function has remained elusive, binds to the plekstrin homology (PH) domain of p62 with higher affinity than that of E2F1 and contributes to transcriptional activation. The structure of the complex revealed that DP1 forms a twisted U-shaped, string-like conformation and binds to the surface of the PH domain by anchoring Phe403 into a pocket in the PH domain. The transcriptional activity of E2F1-DP1 was reduced when Phe403 of DP1 was mutated. These findings indicate that the acidic region of DP1 acts as a TAD by contacting TFIIH.

// Ming Chu 1, 2 , Kailin Yin 1, 2 , Yujun Dong 3 , Pingzhang Wang 1, 2 , Yun Xue 4 , Peng Zhou 1, 2 , Yuqi Wang 1, 2 , Yuedan Wang 1, 2 1 Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing, China 2 Key Laboratory of Medical Immunology, Ministry of Health, Beijing, China 3 Department of Hematology, Peking University First Hospital, Beijing, China 4 Department of Anesthesiology, Fuling Center Hospital of Chongqing City, Chongqing, China Correspondence to: Yuedan Wang, email: wangyuedan@bjmu.edu.cn Ming Chu, email: famous@bjmu.edu.cn Keywords: TFDP3, E2F1, chemoresistance, minimal residual disease, childhood T-ALL Received: September 06, 2016 Accepted: November 08, 2016 Published: November 26, 2016 ABSTRACT Acquired drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. In this study, a novel gene therapy target for childhood T-ALL to overcome chemoresistance was discovered: TFDP3 increased in the minimal residual disease (MRD) positive childhood T-ALL patients. Then, we established a preclinical model of resistance to induction therapy to examine the functional relevance of TFDP3 to chemoresistance in MRD derived from Jurkat/E6-1. Jurkat xenografts in NOD/SCID mice were exposed to a four drug combination (VXLD) of vincristine (VCR), dexamethasone (DEX), L-asparaginase (L-asp) and daunorubicin (DNR). During the 4-week VXLD treatment, the level of TFDP3 increased 4-fold. High expression of TFDP3 was identified in the re-emerging lines (Jurkat/MRD) with increased chemoresistance, which is correlated with partially promoter demethylation of TFDP3 . Downregulation of TFDP3 by RNA interference reversed chemoresistance in Jurkat/MRD accompanied by reinstated E2F1 activity that coincided with increased levels of p53, p73, and associated proapoptotic target genes. Importantly, TFDP3 silencing in vivo induced apparent benefit to overcome chemoresistance in combination with VXLD treatment. Collectively, TFDP3 confers chemoresistance in MRD within childhood T-ALL, indicating that TFDP3 is a potential gene therapy target for residual cancer.

Lung cancer is still the leading cause of malignant deaths in the world. It is of great importance to find novel functional genes for the tumorigenesis of lung cancer. We demonstrated that Rac3 could promote cell proliferation and inhibit apoptosis in lung adenocarcinoma cell line A549 previously. The aim of this study was to investigate the function and mechanism of Rac3 in lung adenocarcinoma cell lines. Immunohistochemistry staining was performed in 107 lung adenocarcinoma tissues and matched non-tumor tissues. Multivariate analysis and Kaplan-Meier analysis were used to investigate the correlation between Rac3 expression and the clinical outcomes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry analysis were employed to determine the proliferative ability, cell cycle distribution, and apoptosis in H1299 and H1975 cell lines. Gene expression microarray and pathway analysis between the Rac3-siRNA group and the control group in A549 cells were performed to investigate the pathways and mechanism of Rac3 regulation. Rac3 was shown to be positively expressed in lung adenocarcinoma tissues, and the expression of Rac3 associates with longer survival in lung adenocarcinoma patients. Silencing of Rac3 significantly induced cell growth inhibition, colony formation decrease, cell cycle arrest, and apoptosis of lung adenocarcinoma cell lines, which accompanied by obvious downregulation of CCND1, MYC, and TFDP1 of cell cycle pathway involving in the tumorigenesis of lung adenocarcinoma based on the gene expression microarray. In conclusion, these findings suggest that Rac3 has the potential of being a therapeutic target for lung adenocarcinoma.

Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.

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.

The DP proteins are components of the E2F transcription factor. They form heterodimers with the E2F proteins and these complexes bind efficiently to E2F response elements in promoters of genes that are involved in cell cycle regulation. The properties of the DP proteins are less documented than those of their E2F counterpart and the present work was aimed at characterizing avian DP genes (named chDP) and their products. Here we describe the cloning of the chicken homologues of the mammalian DP-1 and DP-2 proteins. This work also suggests that DP-2 isoforms have an additional 60 amino acid extension at the N-terminus compared to its human counterpart. Gel-shift assays and coimmunoprecipitation show that both DP-1 and DP-2 dimerize to chE2F-1 and activate transcription efficiently, as demonstrated by transient expression assays. However, contrary to the expression patterns exhibited by E2F-1 during the cell cycle or during neuroretina development, DP member's expression appears more invariant, suggesting that E2F activity is limited by the availability of the E2F proteins.

Cancer/testis antigen TFDP3 belongs to the transcription factor DP(TFDP) family. It can bind to E2F family molecules to form a heterodimeric transcription factor E2F/TFDP complex. The complex is an important regulatory activator of cell cycle, involved in the regulation of cell proliferation, differentiation, apoptosis and other important physiological activities. In addition, TFDP3 has also been found to be a tumor-associated antigen that only expresses in malignant tumor tissue and normal testicular tissue; Thus, it is closely related to tumor occurrence and development. In this study, our group investigated the expression of TFDP3 in mononuclear cell samples from a variety of tissue-derived malignant tumors, breast cancer and benign breast lesions. The results show that TFDP3 is expressed in the malignant form of various tissues. Moreover, our recent research had focused on the ability of TFDP3 to influence the drug resistance and apoptosis of tumor cells. To further clarify the mechanisms involved in tumor resistance, this study also examined the expression of TFDP3 and tumor cell autophagy regulation; Autophagy helps cells cope with metabolic stress (such as in cases of malnutrition, growth factor depletion, hypoxia or hypoxia) removes erroneously folded proteins or defective organelles to prevent the accumulation of abnormal proteins; and removes intracellular pathogens. Our results showed that TFDP3 expression can induce autophagy by up-regulating the expression of autophagic key protein LC3(MAP1LC3) and increasing the number of autophagosomes during chemotherapy of malignant tumors. Then, DNA and organelles damage caused by the chemotherapy medicine are repaired. Thus, TFDP3 contributes toward tumor cell resistance. When siRNA inhibits TFDP3 expression, it can reduce cell autophagy, improving the sensitivity of tumor cells to chemotherapy drugs.

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.

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.

Background: For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow. Results: By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant. Conclusions: From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development. Developmental Dynamics 244:651–668, 2015. © 2015 Wiley Periodicals, Inc.

TFDP3, also be known as HCA661, was one of the cancer-testis antigens, which only expressed in human tissues. The recent researches about TFDP3 mostly focused on its ability to control the drug resistance and apoptosis of tumor cells. However, the role of TFDP3 in the progress of the cell cycle is rarely involved. In this study, we examined the expression of TFDP3 in human liver tissues firstly. After that, we detect the expression of TFDP3 at the RNA level and protein level in L-02 cell line and HepG2 cell line, and the location of TFDP3 was defined by immunofluorescence technique. Furthermore, we synchronized the cells to G1 phase, S phase and G2 phase, and arrested cell mitosis. The localization of TFDP3 and co-localization with E2F1 molecules in different phases of hepatocyte lines. Finally, TFDP3 gene knockout was performed on L-02 and HepG2 cell lines, and detected the new cell cycles by flow cytometry. The result showed that the expression of TFDP3 molecule is negative in normal liver tissue, but positive in immortalized human hepatocyte cell line, and the expression level is lower than in hepatocellular carcinoma cell line. The expression level of TFDP3 was in the dynamic change of L-02 and HepG2 cell lines, and was related to the phase transition. TFDP3 can bind to E2F1 molecule to form E2F/TFDP3 complex; and the localizations of TFDP3 and E2F1 molecules and the co-localization were different in different phases of cell cycle in the nucleus and cytoplasm, which indicated that the E2F/TFDP3 complex involved in the process of regulating the cell cycle. By knocking down the TFDP3 expression level in L-02 and HepG2 cell lines, the cell cycle would be arrested in S phase, which confirmed that TFDP3 can be a potential target for tumor therapy.

Breast cancer remains a lethal disease to women due to lymph node metastasis, the tumor microenvironment, secondary resistance and other unknown factors. Several important transcription factors involved in this disease, such as PTEN, p53 and beta-catenin, have been identified and researched in-depth as candidates for targeted therapy in breast cancer. TFDP3 is a new, promising candidate for transcriptional regulation in breast cancer, although it was first identified in hepatocellular carcinoma. Here, we demonstrate that TFDP3 is expressed in a variety of malignancies, normal testis tissue and breast cancer cell lines and thus provide evidence that TFDP3 is a cancer-testis antigen. We illustrate that overexpression or silencing TFDP3 interferes with epithelial-mesenchymal transition but does not influence cell proliferation, indicating that the TFDP3 protein acts as a transcription factor during epithelial-mesenchymal transition. These data highlight that TFDP3 is expressed in breast cancer, that it is a member of the cancer-testis antigen family and that it functions as a regulator in epithelial-mesenchymal transition.

During lytic infections HHV-6A and HHV-6B disrupt E2F1-Rb complexes by Rb degradation, releasing E2F1 and driving the infected cells toward the S-phase. Whereas upon infection E2F1 and its cofactor DP1 were up-regulated, additional E2F responsive genes were expressed differentially in various cells. E2F binding sites were identified in promoters of several HHV-6 genes, including the U27 and U79 associated with viral DNA replication, revealing high dependence on the binding site and the effect of the E2F1 transcription factor. Viral genes regulation by E2F1 can synchronize viral replication with the optimal cell cycle phase, enabling utilization of host resources for successful viral replication. Furthermore, it was found that infection by roseoloviruses leads to cell cycle arrest, mostly in the G2/M-phase.

Prostaglandin D2 (PGD2) causes cough and levels are increased in asthma suggesting that it may contribute to symptoms. Although the prostaglandin D2 receptor 2 (DP2) is a target for numerous drug discovery programmes little is known about the actions of PGD2 on sensory nerves and cough.We used human and guinea pig bioassays, in vivo electrophysiology and a guinea pig conscious cough model to assess the effect of prostaglandin D2 receptor (DP1), DP2 and thromboxane receptor antagonism on PGD2 responses.PGD2 caused cough in a conscious guinea pig model and an increase in calcium in airway jugular ganglia. Using pharmacology and receptor-deficient mice we showed that the DP1 receptor mediates sensory nerve activation in mouse, guinea pig and human vagal afferents. In vivo, PGD2 and a DP1 receptor agonist, but not a DP2 receptor agonist, activated single airway C-fibres. Interestingly, activation of DP2 inhibited sensory nerve firing to capsaicin in vitro and in vivo.The DP1 receptor could be a therapeutic target for symptoms associated with asthma. Where endogenous PGD2 levels are elevated, loss of DP2 receptor-mediated inhibition of sensory nerves may lead to an increase in vagally associated symptoms and the potential for such adverse effects should be investigated in clinical studies with DP2 antagonists.

Re-sequencing of target genes is a highly effective approach for identifying mutations in cancers. Mutations, including indels (insertions, deletions, and the combination of the two), play important roles in carcinogenesis. Combining genomic DNA capture using high-density oligonucleotide microarrays (NimbleGen, Inc.) with next-generation high-throughput sequencing, we identified approximately 1600 indels for colorectal cancers in the Chinese population. Among them, 5 indels were localized to exonic regions of genes, including the TFDP1 (transcription factor Dp-1) gene. TFDP1 is an important transcription factor that coordinates with E2F proteins, thereby promoting transcription of E2F target genes and regulating the cell cycle and differentiation. We report here the identification of a recurrent frame-shift indel mutation (named indel84) in the TFDP1 gene in colorectal cancers by next-generation sequencing. We found in a validation set that TFDP1 indel84 is present in 70% of colorectal cancer (CRC) tissues. Wild-type TFDP1 encodes a protein of 410 amino acids with a potential DNA binding site at its N-terminal followed by several functional protein domains. The TFDP1 indel cDNA would generate an alternative TFDP1 protein missing the first 120 amino acids and potentially affecting the DNA binding domain. We further demonstrated that the TFDP1 indel84 mutation generated a gain-of-function phenotype by increasing cell proliferation, migration, and invasion of CRC cells. Our study identified a key molecular event for CRC that might have great diagnostic and therapeutic potentials.

The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. Conclusion: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression. (Hepatology 2014;60:1241–1250)

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1.

E2F activity is negatively regulated by retinoblastoma protein (pRb) through binding to the E2F-1 subunit. Within the E2F heterodimer, DP proteins are E2F partner subunits that allow proper cell cycle progression. In contrast to the other DP proteins, the newest member of the family, DP-4, downregulates E2F activity. In this study we report an unexpected role for DP-4 in regulating E2F-1 activity during the DNA damage response. Specifically, DP-4 is induced in DNA-damaged cells, upon which it binds to E2F-1 as a non-DNA-binding E2F-1/DP-4 complex. Consequently, depleting DP-4 in cells re-instates E2F-1 activity that coincides with increased levels of chromatin-bound E2F-1, E2F-1 target gene expression and associated apoptosis. Mutational analysis of DP-4 highlighted a C-terminal region, outside the DNA-binding domain, required for the negative control of E2F-1 activity. Our results define a new pathway, which acts independently of pRb and through a biochemically distinct mechanism, involved in negative regulation of E2F-1 activity.

The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53.

The present investigation was undertaken to evaluate the antimicrobial and antioxidant activities of the wild edible mushroom Agaricus lanipes, and also to investigate its cytotoxicity and potential and possible apoptotic effect against the A549 lung cancer cell line in in vitro conditions. Total antioxidant capacity, total phenolic content, total oxidant status, total antioxidant status, lipid hydroperoxides, and total free –SH levels of A. lanipes were found to be 4.55 mg T/g, 14.6 mg GA equivalent/g, 3.10 mg H2O2 equivalent/g, 2.25 mg H2O2 equivalent/g, and 1.90 µmol/g, respectively. The methanolic extract of A. lanipes had relatively strong antimicrobial activity against seven tested microorganism strains. It also had high anti-proliferative potency and strong pro-apoptotic effects, and this mushroom used as a daily nutrient could be a source for new drug developments and treatment in cancer therapies, and could be a guide for studies in this area. © 2017, Springer Science+Business Media Dordrecht.

Rubimetide (Met-Arg-Trp), which had been isolated as an antihypertensive peptide from an enzymatic digest of spinach ribulose-bisphosphate carboxylase/oxygenase (Rubisco), showed anxiolytic-like activity prostaglandin (PG) D2-dependent manner in the elevated plus-maze test after administration at a dose of 0.1mg/kg (ip.) or 1mg/kg (p.o.) in male mice of ddY strain. In this study, we found that rubimetide has weak affinities for the FPR1 and FPR2, subtypes of formyl peptide receptor (FPR). The anxiolytic-like activity of rubimetide (0.1mg/kg, ip.) was blocked by WRW4, an antagonist of FPR2, but not by Boc-FLFLF, an antagonist of FPR1, suggesting that the anxiolytic-like activity was mediated by the FPR2. Humanin, an endogenous agonist peptide of the FPR2, exerted an anxiolytic-like activity after intracerebroventricular (icv) administration, which was also blocked by WRW4. MMK1, a synthetic agonist peptide of the FPR2, also exerted anxiolytic-like activity. Thus, FPR2 proved to mediate anxiolytic-like effect as the first example of central effect exerted by FPR agonists. As well as the anxiolytic-like activity of rubimetide, that of MMK1 was blocked by BW A868C, an antagonist of the DP1-receptor. Furthermore, anxiolytic-like activity of rubimetide was blocked by SCH58251 and bicuculline, antagonists for adenosine A2A and GABAA receptors, respectively. From these results, it is concluded that the anxiolytic-like activities of rubimetide and typical agonist peptides of the FPR2 were mediated successively by the PGD2-DP1 receptor, adenosine-A2A receptor, and GABA-GABAA receptor systems downstream of the FPR2.

Despite extensive research, the prognosis of high-grade glioblastoma multiforme (GBM) has improved only slightly because of the limited response to standard treatments. Recent advances (discoveries of molecular biomarkers) provide new opportunities for the treatment of GBM. The aim of the present study was to identify diagnostic biomarkers of high-grade GBM. First, we combined 3 microarray expression datasets to screen them for genes differentially expressed in patients with high-grade GBM relative to healthy subjects. Next, the target network was constructed via the empirical Bayesian coexpression approach, and centrality analysis and a molecular complex detection (MCODE) algorithm were performed to explore hub genes and functional modules. Finally, a validation test was conducted to verify the bioinformatic results. A total of 277 differentially expressed genes were identified according to the criteria P < 0.05 and |log2(fold change)| ≥ 1.5. These genes were most significantly enriched in the cell cycle pathway. Centrality analysis uncovered 9 hub genes; among them, TFDP1 showed the highest degree of connectivity (43) and is a known participant in the cell cycle pathway; this finding pointed to the important role of TFDP1 in the progression of high-grade GBM. Experimental validation mostly supported the bioinformatic results. According to our study results, the gene TFDP1 and the cell cycle pathway are strongly associated with high-grade GBM; this result may provide new insights into the pathogenesis of GBM.

Through data mining the Stanford Microarray Database, the stathmin 1 (STMN1) transcript was found to be frequently upregulated in the hepatocellular carcinoma (HCC) with low alpha-fetoprotein level. The molecular mechanism of STMN1 upregulation in HCCs remained unclear.Quantitative RT-PCR, immunoblotting, immunohistochemistry, and transfection of expression or small hairpin RNA interference plasmids, chromatin immunoprecipitation (ChIP), and quantitative ChIP assays were performed in HCC specimens or 2 distinct HCC-derived cell lines. Dual luciferase assay and site-directed mutagenesis were applied to analyze the activities of STMN1 proximal promoter region.STMN1 mRNA and proteins were significantly associated with several clinicopathological features. High STMN1 or E2F1 immunoexpression was predictive of poor overall survival (OS) rate (P.01). In HCC-derived cell lines, E2F1 was elevated before STMN1 mRNA during the cell cycle. Exogenous expression of E2F1 or both transcription factor DP-1 (TFDP1) and E2F1 genes induced E2F1 and STMN1 mRNA (P.01). Knockdown of the E2F1 gene suppressed E2F1 and STMN1 mRNA and E2F1 and STMN1 protein levels (P.05). The promoter activity of STMN1 gene increased with overexpression of both E2F1 and TFDP1 genes (P.05); however, it decreased when mutations were introduced in the E2F1-binding sites (P.05).Upregulation of E2F1 and STMN1 proteins associate with worse outcomes in patients with HCC. E2F1 significantly correlates with STMN1 protein level in HCC lesions and in vitro transactivation assays, suggesting that STMN1 gene is transactivated by the E2F1 protein.

Gastrointestinal stromal tumors feature a wide spectrum of biologic behavior, ranging from benign to extremely malignant. To determine the role of p16(INK4a) alteration in progression of gastrointestinal stromal tumors of the stomach, we have investigated protein expression and gene methylation in correlation with clinicopathologic factors and survival. In addition to immunohistochemical analysis of p16(INK4a) in a series of 95 cases, real-time quantitative methylation specific polymerase chain reaction for p16(INK4a) and immunostaining for cyclin D1, cyclin E, pRb, DP-1, E2F-1, and Ki-67 were also evaluated in randomly selected samples. The p16(INK4a) labeling indices ranged from 0% to 74% (median, 21%), demonstrating a significant inverse correlation with size (P = .046). On univariate (P = .003) and multivariate (P = .067) analyses, loss of p16(INK4a) expression increased the likelihood of a poor tumor-related survival. In addition, size (P = .036) and the mitotic index (P = .005) had independent prognostic influence. The p16(INK4a) methylation index, which ranged from 0% to 100% (median, 17%), was significantly higher in larger tumors (P < .001) and in high-risk category lesions (P = .001) and inversely correlated with protein expression. Hierarchical cluster analysis based on expression of p16(INK4a) network members identified 2 clusters in 27 randomly selected tumor samples, containing 11 and 16 tumors each. Former cluster samples demonstrated higher risk category (P = .022), higher p16(INK4a) methylation (P < .001), and more reduced pRb expression (P < .018). In addition, p16(INK4a) network members clustered into 2 groups: (1) showing down-regulated p16(INK4a) protein and up-regulating of both cyclin D1 and DP-1 and (2) down-regulated pRb and up-regulated E2F-1. We conclude that p16(INK4a) alteration has an important role in progression of gastrointestinal stromal tumors of the stomach. Furthermore, the study provides a possible link between regulation of p16(INK4a) network members and gastrointestinal stromal tumors.

In a variety of neurodegenerative disease, despite the frequent correlation of neuronal cell cycle and cell death in the same neuronal populations, the mechanistic pathway linking the two remains undefined. One possible link is the atypical cyclin dependent kinase, Cdk5. Cdk5 exerts a double protective function in neurons, first by suppressing the cell cycle in the nucleus and second by suppressing cell death in the cytoplasm. Cdk5 transport between nucleus and cytoplasm serves to regulate the balance between these two events. Cdk5 nuclear localization relies on its interaction with p27, and its cell cycle suppression activity is achieved by direct binding to E2F1, disrupting the DP1-E2F1 dimer and its DNA binding ability. To bind to E2F1, Cdk5 does not need to be catalytically active but it does require a physical association with both p27 and its cyclin-like activator, p35. Because of this requirement, the proper levels and locations of p27 and p35 are characteristics that endow a neuron a unique form of cell cycle regulation that uses Cdk5 in a non-catalytic role. The findings offer cautionary notes to any strategy aimed at blocking Cdk5 activity as a means of combating neurodegenerative disease. To the extent that these approaches either directly or indirectly influence Cdk5 levels or location, they may produce unexpected and possibly unwanted consequences.

SUMMARY The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy., Graphical Abstract, In Brief Uncontrolled proliferation is a hallmark of tumorigenesis and is associated with perturbed transcriptomic profile. Fulciniti et al. explored the interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma, reporting the existence of two distinct regulatory axes that can be synergistically targeted to impact myeloma growth and survival.

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit—RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle–regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.