Cell Cycle Transcriptional Regulation Research Articles

Cell-cycle regulated transcription associates with DNA replication timing in yeast and human

BackgroundEukaryotic DNA replication follows a specific temporal program, with some genomic regions consistently replicating earlier than others, yet what determines this program is largely unknown. Highly transcribed regions have been observed to replicate in early S-phase in all plant and animal species studied to date, but this relationship is thought to be absent from both budding yeast and fission yeast. No association between cell-cycle regulated transcription and replication timing has been reported for any species.ResultsHere I show that in budding yeast, fission yeast, and human, the genes most highly transcribed during S-phase replicate early, whereas those repressed in S-phase replicate late. Transcription during other cell-cycle phases shows either the opposite correlation with replication timing, or no relation. The relationship is strongest near late-firing origins of replication, which is not consistent with a previously proposed model—that replication timing may affect transcription—and instead suggests a potential mechanism involving the recruitment of limiting replication initiation factors during S-phase.ConclusionsThese results suggest that S-phase transcription may be an important determinant of DNA replication timing across eukaryotes, which may explain the well-established association between transcription and replication timing.

Mechanisms of antitumor activity by melanoxoin on human non-small cell lung cancer cells

Cancer is a leading cause of death worldwide. The standard treatments of cancer include surgery, chemotherapy, radiation therapy and the newer targeted therapy. Till now, there are various anticancer drugs, which derived from natural products. In this study, we screened a serious of compounds isolated from Pterocarpus santalinus to identify their cell cytotoxicity. We performed the MTT assay to evaluate the cytotoxic effects of test compounds against several human cancer cell lines. Among them, melanoxoin showed the highest cytotoxic effects with a IC50 of 1.98µg/ml in the human non-small cell lung cancer H1299 cells. We next used Human OneArray® v5 to comprehensive analysis the DNA transcription of H1299 cells. It showed that melanoxoin regulated the transcription of several cell cycle regulators in the H1299 cells. Cell cycle analysis by flow cytometry revealed melanoxoin causes G2/M arrest and then increased tumor cell apoptosis. The neutral comet assay revealed that the treatment with melanoxoin induced significant DNA damage in H1299 cells. Therefore, we are interested in the development the molanoxoin as a new source of anti-cancer drug.

Chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy

To identify the upstream signals of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy (TLE), we evaluated by immunohistochemistry and confocal microscopy brain tissues of 13 TLE patients and 5 control patients regarding expression of chemokines and cell-cycle proteins. The chemokine RANTES (CCR5) and other CC-chemokines and apoptotic markers (caspase-3, -8, -9) were expressed in lateral temporal cortical and hippocampal neurons of TLE patients, but not in neurons of control cases. The chemokine RANTES is usually found in cytoplasmic and extracellular locations. However, in TLE neurons, RANTES was displayed in an unusual location, the neuronal nuclei. In addition, the cell-cycle regulatory transcription factor E2F1 was found in an abnormal location in neuronal cytoplasm. The pro-inflammatory enzyme cyclooxygenase-2 and cytokine interleukin-1β were expressed both in neurons of patients suffering from temporal lobe epilepsy and from cerebral trauma. The vessels showed fibrin leakage, perivascular macrophages and expression of IL-6 on endothelial cells. In conclusion, the cytoplasmic effects of E2F1 and nuclear effects of RANTES might have novel roles in neuronal apoptosis of TLE neurons and indicate a need to develop new medical and/or surgical neuroprotective strategies against apoptotic signaling by these molecules. Both RANTES and E2F1 signaling are upstream from caspase activation, thus the antagonists of RANTES and/or E2F1 blockade might be neuroprotective for patients with medically intractable temporal lobe epilepsy. The results have implications for the development of new medical and surgical therapies based on inhibition of chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy.

Cross Talk Between Estradiol and mTOR Kinase in the Regulation of Ovarian Granulosa Proliferation

Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles.

Targeted Deletion of Nrf2 Reduces Urethane-Induced Lung Tumor Development in Mice

Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2 +/+ and Nrf2 -/- mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2 -/- mice compared to Nrf2 +/+ mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2 -/- mice than in Nrf2 +/+ mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2 +/+ mice relative to Nrf2 -/- mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

Tumor Suppressor Menin Represses Paired Box Gene 2 Expression via Wilms Tumor Suppressor Protein-Polycomb Group Complex

Tumor suppressor menin, the product of the MEN1 gene, plays a key role in controlling histone 3 lysine 4 trimethylation (H3K4me3) and gene transcription, which can regulate proliferation, apoptosis, and differentiation. However, little is known as to whether menin controls gene expression and cell proliferation and survival via regulating Polycomb group (PcG) protein complex/H3K27me3. Here we show that menin specifically represses transcription factor Paired box gene 2 (Pax2) through PcG-mediated H3K27me3 and Wilms tumor suppressor protein (WT1), a zinc finger domain-containing DNA-binding protein. Menin does not directly bind to the Pax2 locus, instead, it up-regulates WT1 expression. WT1 recruits PcG complex to the Pax2 promoter and represses expression of Pax2 through PcG-dependent H3K27me3. Moreover, WT1 also interacts with DNA methyltransferase 1 (DNMT1), and recruits DNMT1 to the Pax2 promoter, resulting in hypermethylation of CpG in the Pax2 promoter. Together, these studies have uncovered a novel epigenetic mechanism whereby menin regulates H3K27me3 and promoter DNA methylation via WT1 and suggest that WT1 protein plays an important, yet previously unappreciated role in regulating the function of the menin/PcG axis, H3K27 methylation, and DNA methylation, resulting in repression of gene transcription.

The Cell Cycle Regulated Transcriptome of Trypanosoma brucei

Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a “double-cut” elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

Phosphorylation of the MBF Repressor Yox1p by the DNA Replication Checkpoint Keeps the G1/S Cell-Cycle Transcriptional Program Active

BackgroundIn fission yeast Schizosaccharomyces pombe G1/S cell-cycle regulated transcription depends upon MBF. A negative feedback loop involving Nrm1p and Yox1p bound to MBF leads to transcriptional repression as cells exit G1 phase. However, activation of the DNA replication checkpoint response during S phase results in persistent expression of MBF-dependent genes.Methodology/Principal FindingsThis report shows that Yox1p binding to MBF is Nrm1-dependent and that Yox1p and Nrm1p require each other to bind and repress MBF targets. In response to DNA replication stress both Yox1p and Nrm1p dissociate from MBF at promoters leading to de-repression of MBF targets. Inactivation of Yox1p is an essential part of the checkpoint response. Cds1p (human Chk2p) checkpoint protein kinase-dependent phosphorylation of Yox1p promotes its dissociation from the MBF transcription factor. We establish that phosphorylation of Yox1p at Ser114, Thr115 is required for maximal checkpoint-dependent activation of the G1/S cell-cycle transcriptional program.Conclusions/SignificanceThis study shows that checkpoint-dependent phosphorylation of Yox1p at Ser114, Thr115 results in de-repression of the MBF transcriptional program. The remodeling of the cell cycle transcriptional program by the DNA replication checkpoint is likely to comprise an important mechanism for the avoidance of genomic instability.

Primers on Molecular Pathways —The NFAT Transcription Pathway in Pancreatic Cancer

The calcineurin-responsive nuclear factor of activated T cells (NFAT) family of transcription factors was originally identified as a group of inducible nuclear proteins, which regulate transcription during T lymphocyte activation. However, following their initial discovery, a multitude of studies quickly established that NFAT proteins are also expressed in cells outside the immune system, where they participate in the regulation of the expression of genes influencing cell growth and differentiation. Ectopic activation of individual NFAT members is now recognized as an important aspect for oncogenic transformation in several human malignancies, most notably in pancreatic cancer. Sustained activation of the Ca2+/calcineurin/NFAT signaling pathway has emerged as a powerful regulatory principle governing pancreatic cancer cell growth. Activated NFAT proteins form complexes with key oncogenic proteins to regulate the transcription of master cell cycle regulators and proteins with functions in cell survival, migration and angiogenesis. This review pays particular attention to recent advances in our understanding of how the NFAT transcription pathway controls gene expression during development and progression of pancreatic cancer.

Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Abstract 1252: Interacting role of the androgen receptor and the corepressor Alien in the proliferation inhibition of prostate cancer cells

Abstract Androgen receptor (AR) plays a central role in normal prostate growth and in prostate cancer development. The goal of this study was to identify negative regulatory factor(s) that contribute to proliferation inhibition of prostate cancer cells. The biphasic proliferation pattern of LNCaP human prostate cancer cells at low versus high androgen concentrations was utilized to examine regulatory factors involved in AR-mediated regulation of E2F1 gene transcription. E2F1 is a key cell cycle regulatory transcription factor and an important proliferation stimulator of prostate cancer cells. LNCaP cells are known to be stimulated by ≤ 1 nM androgen, while the cells are arrested at G1/S at high androgen (≥10 nM). Quantitative RT-PCR revealed increased expression of E2F1 and E2F1 target genes (such as cyclin A and the antiapoptotic protein Bcl2) in LNCaP cells treated with 0.1nM R1881 (a synthetic androgen). This up regulation was markedly reduced at the 50 nM R1881 treatment condition. The E2F1 promoter activity showed a similar biphasic regulation by low versus high androgen. Functional analysis of deleted E2F1 promoter fragments identified an androgen responsive region (ARR) in the E2F1 upstream promoter. Chromatin immunoprecipitation (ChIP) of LNCaP cells treated with 0.1nM R1881 showed co-recruitment of AR and the p160 coactivator SRC3 to the ARR in parallel to E2F1 gene induction and increased cell proliferation. AR and SRC3 occupancy at ARR was abrogated by 50 nM R1881 treatment, in parallel to de-induction of E2F1 gene expression. Proliferation inhibition at 50 nM R1881 and loss of AR occupancy at ARR was prevented by SRC3 over expression. Reciprocally, proliferation stimulation by 0.1nM R1881 was lost in SRC3-silenced cells. In further novel findings, we show that the corepressor protein Alien (also known as TRIP15) strongly interacted with AR at 50 nM R1881, while interaction of AR with Alien was much weaker at 0.1 nM R1881. Androgen dose-dependent differential interaction between AR and Alien was demonstrated by co-immunoprecipitation and GST pull down assays of LNCaP cell nuclear extracts. Alien was absent constitutively from ARR in the E2F1 promoter at all conditions (i.e. LNCaP cells treated with vehicle, 0.1 nM R1881 or 50 nM R1881). This suggests that Alien interacts with AR in the nucleoplasm, not in a chromatin environment of the E2F1 promoter. Stable knockdown of Alien prevented proliferation inhibition of LNCaP cells at 50 nM R1881. We conclude that the corepressor Alien/TRIP15 negatively regulates proliferation of prostate cancer cells by sequestering AR in the nucleoplasm, which in turn leads to interference with E2F1 gene induction by androgen-activated AR. In ongoing studies we are utilizing a xenograft tumor model in immune-compromised mice to validate the inhibitory role of the corepressor Alien/TRIP15 in the proliferation of human prostate cancer cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1252.

Cell Cycle Control of Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by CTCF-Cohesin Interactions

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is characterized by the highly regulated transcription of a few viral genes essential for genome maintenance and host cell survival. A major latency control region has been identified upstream of the divergent promoters for the multicistronic transcripts encoding LANA (ORF73), vCyclin (ORF72), and vFLIP (ORF71) and for the complementary strand transcript encoding K14 and vGPCR (ORF74). Previous studies have shown that this major latency control region is occupied by the cellular chromatin boundary factor CTCF and chromosome structural maintenance proteins SMC1, SMC3, and RAD21, which comprise the cohesin complex. Deletion of the CTCF-cohesin binding site caused an inhibition of cell growth and viral genome instability. We now show that the KSHV genes regulated by CTCF-cohesin are under cell cycle control and that mutation of the CTCF binding sites abolished cell cycle-regulated transcription. Cohesin subunits assembled at the CTCF binding sites and bound CTCF proteins in a cell cycle-dependent manner. Subcellular distribution of CTCF and colocalization with cohesins also varied across the cell cycle. Ectopic expression of Rad21 repressed CTCF-regulated transcription of KSHV lytic genes, and a Rad21-CTCF chimeric protein converted CTCF into an efficient transcriptional repressor of KSHV genes normally activated in the G(2) phase. We conclude that cohesins interact with CTCF in mid-S phase and repress CTCF-regulated genes in a cell cycle-dependent manner. We propose that the CTCF-cohesin complex plays a critical role in regulating the cell cycle control of viral gene expression during latency and that failure to maintain cell cycle control of latent transcripts inhibits host cell proliferation and survival.

D009 Frizzled 4 regulates vascular formation in mice

A role for Wnt via the Frizzled (Fzd) cell surface receptors has recently emerged in vascular cell development. Among the Fzd members, Frizzled4 (Fzd4) was recently implicated in vessel formation in adult retinal vascular network. Consistent with this finding, we aimed to evaluate the function of Fzd4 in adult vascular setting. MicroCT and immunohistochemistry approaches revealed that Fzd4 deletion altered dramatically coronary and arterial network formations in the heart and in the kidney with a significant decrease of the arterial branching. Micro-array analysis revealed that in hearts of Fzd4 KO neonate mice, the primary effect of Fzd 4 deletion was the down-regulation of the cell cycle-regulating transcription factor E2F1, with a decreased expression of a large number of E2F target genes. This observation indicated that loss of Fzd4 lead to cell cycle arrest. We conducted in vitro studies to confirm the role of Fzd4 on cell cyle response on murine embryonic fibroblasts (MEF) and endothelial cells (EC) isolated from KO Fzd4 vs Wt mice. Fzd4 KO reduced MEF and EC proliferation. Fzd4 siRNA-mediated knockdown (KD) in EC delayed G1 to S cell cycle entry compared to that in control EC (transition G1 to S at 12H, 3,5 % vs 14,5 % vs control EC). In vitro matrigel assay showed that Fzd4 KD reduced the differentiation of HUVEC to form capillary structures under VEGF A-stimulation (43±4 in Fzd4 KD EC vs 28± 7, in control EC, P These findings are the first to show that a frizzled member Fzd4 can repress E2F1 cell cycle progression in EC independently of the Wnt/β catenin canonical pathway and affects deeply vascular formation and branching.

Global control of cell-cycle transcription by coupled CDK and network oscillators

A significant fraction of the Saccharomyces cerevisiae genome is transcribed periodically during the cell division cycle, indicating that properly timed gene expression is important for regulating cell-cycle events. Genomic analyses of the localization and expression dynamics of transcription factors suggest that a network of sequentially expressed transcription factors could control the temporal programme of transcription during the cell cycle. However, directed studies interrogating small numbers of genes indicate that their periodic transcription is governed by the activity of cyclin-dependent kinases (CDKs). To determine the extent to which the global cell-cycle transcription programme is controlled by cyclin-CDK complexes, we examined genome-wide transcription dynamics in budding yeast mutant cells that do not express S-phase and mitotic cyclins. Here we show that a significant fraction of periodic genes are aberrantly expressed in the cyclin mutant. Although cells lacking cyclins are blocked at the G1/S border, nearly 70% of periodic genes continued to be expressed periodically and on schedule. Our findings reveal that although CDKs have a function in the regulation of cell-cycle transcription, they are not solely responsible for establishing the global periodic transcription programme. We propose that periodic transcription is an emergent property of a transcription factor network that can function as a cell-cycle oscillator independently of, and in tandem with, the CDK oscillator.

CARM1 Regulates Estrogen-Stimulated Breast Cancer Growth through Up-regulation of E2F1

Estrogen receptor alpha (ER alpha) mediates breast cancer proliferation through transcriptional mechanisms involving the recruitment of specific coregulator complexes to the promoters of cell cycle genes. The coactivator-associated arginine methyltransferase CARM1 is a positive regulator of ER alpha-mediated transcriptional activation. Here, we show that CARM1 is essential for estrogen-induced cell cycle progression in the MCF-7 breast cancer cell line. CARM1 is specifically required for the estrogen-induced expression of the critical cell cycle transcriptional regulator E2F1 whereas estrogen stimulation of cyclin D1 is CARM1 independent. Upon estrogen stimulation, the E2F1 promoter is subject to CARM1-dependent dimethylation on histone H3 arginine 17 (H3R17me2) in a process that parallels the recruitment of ER alpha. Additionally, we find that the recruitment of CARM1 and subsequent histone arginine dimethylation are dependent on the presence of the oncogenic coactivator AIB1. Thus, CARM1 is a critical factor in the pathway of estrogen-stimulated breast cancer growth downstream of ER alpha and AIB1 and upstream of the cell cycle regulatory transcription factor E2F1. These studies identify CARM1 as a potential new target in the treatment of estrogen-dependent breast cancer.

Systematic identification of cell cycle regulated transcription factors from microarray time series data

BackgroundThe cell cycle has long been an important model to study the genome-wide transcriptional regulation. Although several methods have been introduced to identify cell cycle regulated genes from microarray data, they can not be directly used to investigate cell cycle regulated transcription factors (CCRTFs), because for many transcription factors (TFs) it is their activities instead of expressions that are periodically regulated across the cell cycle. To overcome this problem, it is useful to infer TF activities across the cell cycle by integrating microarray expression data with ChIP-chip data, and then examine the periodicity of the inferred activities. For most species, however, large-scale ChIP-chip data are still not available.ResultsWe propose a two-step method to identify the CCRTFs by integrating microarray cell cycle data with ChIP-chip data or motif discovery data. In S. cerevisiae, we identify 42 CCRTFs, among which 23 have been verified experimentally. The cell cycle related behaviors (e.g. at which cell cycle phase a TF achieves the highest activity) predicted by our method are consistent with the well established knowledge about them. We also find that the periodical activity fluctuation of some TFs can be perturbed by the cell synchronization treatment. Moreover, by integrating expression data with in-silico motif discovery data, we identify 8 cell cycle associated regulatory motifs, among which 7 are binding sites for well-known cell cycle related TFs.ConclusionOur method is effective to identify CCRTFs by integrating microarray cell cycle data with TF-gene binding information. In S. cerevisiae, the TF-gene binding information is provided by the systematic ChIP-chip experiments. In other species where systematic ChIP-chip data is not available, in-silico motif discovery and analysis provide us with an alternative method. Therefore, our method is ready to be implemented to the microarray cell cycle data sets from different species. The C++ program for AC score calculation is available for download from URL .

Cut Mutant Drosophila Auditory Organs Differentiate Abnormally and Degenerate

The Drosophila antenna is a sophisticated structure that functions in both olfaction and audition. Previous studies have identified Homothorax, Extradenticle, and Distal-less, three homeodomain transcription factors, as required for specification of antennal identity. Antennal expression of cut is activated by Homothorax and Extradenticle, and repressed by Distal-less. cut encodes the Drosophila homolog of human CAAT-displacement protein, a cell cycle-regulated homeodomain transcription factor. Cut is required for normal development of external mechanosensory structures and Malphigian tubules (kidney analogs). The role of cut in the Drosophila auditory organ, Johnston's organ, has not been characterized. We have employed the FLP/FRT system to generate cut null clones in developing Johnston's organ. In cut mutants, the scolopidial subunits that constitute Johnston's organ differentiate abnormally and subsequently degenerate. Electrophysiological experiments confirm that adult Drosophila with cut null antennae are deaf. We find that cut acts in parallel to atonal, spalt-major, and spalt-related, which encode other transcription factors required for Johnston's organ differentiation. We speculate that Cut functions in conjunction with these factors to regulate transcription of as yet unidentified targets.

The effect of serum high-density lipoprotein on the growth rate of human endothelial progenitor cells

To study the effect of high-density lipoprotein (HDL) on the proliferation of endothelial progenitor cells (EPC) isolated from human umbilical cord blood; to further explore its effect on prevention and development of atherogenesis. EPC isolated by density gradient centrifugation were cultured in a M200 medium. Immunofluorescence staining for CD133, CD34, KDR and Factor VIII were adopted respectively as the specific markers for identification. The effect of HDL on EPC proliferation was estimated on the 7th day of cell cultivation using MTT assay, confocal microscopy and fluorescence activated cell sorting. HDL, when incubated with EPC, was able to promote remarkably the proliferation rate of EPC, dose- and time-dependent. HDL participated in the transcriptional regulation of cell cycle by affecting the regulatory proteins such as cyclin D1. A subtype of progenitor cells was isolated from human cord blood with a potential of differentiating into mature endothelial cells (known as endothelial progenitor cells). HDL plays an important role on EPC fluorescence activated cell sorting differentiation and proliferation. Further studies are required to identify the signal pathway and the molecular mechanism of HDL effect on EPC proliferation.

Modelling the network of cell cycle transcription factors in the yeast Saccharomyces cerevisiae.

BackgroundReverse-engineering regulatory networks is one of the central challenges for computational biology. Many techniques have been developed to accomplish this by utilizing transcription factor binding data in conjunction with expression data. Of these approaches, several have focused on the reconstruction of the cell cycle regulatory network of Saccharomyces cerevisiae. The emphasis of these studies has been to model the relationships between transcription factors and their target genes. In contrast, here we focus on reverse-engineering the network of relationships among transcription factors that regulate the cell cycle in S. cerevisiae.ResultsWe have developed a technique to reverse-engineer networks of the time-dependent activities of transcription factors that regulate the cell cycle in S. cerevisiae. The model utilizes linear regression to first estimate the activities of transcription factors from expression time series and genome-wide transcription factor binding data. We then use least squares to construct a model of the time evolution of the activities. We validate our approach in two ways: by demonstrating that it accurately models expression data and by demonstrating that our reconstructed model is similar to previously-published models of transcriptional regulation of the cell cycle.ConclusionOur regression-based approach allows us to build a general model of transcriptional regulation of the yeast cell cycle that includes additional factors and couplings not reported in previously-published models. Our model could serve as a starting point for targeted experiments that test the predicted interactions. In the future, we plan to apply our technique to reverse-engineer other systems where both genome-wide time series expression data and transcription factor binding data are available.

Recent advances in TGFβ‐regulated transcription during carcinogenesis

AbstractThe multifunctional activities of transforming growth factor‐beta (TGFβ) endow it with both tumor suppressor and tumor promoting activities, depending on the stage of carcinogenesis and the cellular activation state. In early tumor stages, TGFβ inhibits epithelial cell growth through induction of apoptosis and transcriptional regulation of cell cycle controlling genes. During tumor progression, however, many cancer cells escape from TGFβ‐induced growth inhibition and, instead, respond to TGFβ with increased malignancy. TGFβ stimulates tumor promotion particularly in those tumor cells in which Smad‐signaling remains functional and through transcriptional regulation of gene expression. Thus, loss of sensitivity to growth inhibition by TGFβ is not synonymous with complete loss of TGFβ signaling. Rather, it suggests that tumor cells gain advantage by selective inactivation of TGFβ tumor suppressor activities while retaining its tumor promoting functions. In this review we focus on novel aspects in TGFβ signaling and transcription and in particular on the role of Smad‐interacting transcription factors. We also summarize recent advances in both cytoplasmic and nuclear crosstalk mechanisms between Smad and non‐Smad signaling pathways and how this interaction results in the fine‐tuning of Smad‐mediated transcription during cancer progression. This knowledge will help to better understand the molecular mechanisms responsible for the switch of TGFβ from a tumor suppressor to a tumor promotor.