Cell Cycle Transcription Factors Research Articles

Determination of the physiological and pathological roles of E2F3 in adult tissues

While genetically engineered mice have made an enormous contribution towards the elucidation of human disease, it has hitherto not been possible to tune up or down the level of expression of any endogenous gene. Here we describe compound genetically modified mice in which expression of the endogenous E2f3 gene may be either reversibly elevated or repressed in adult animals by oral administration of tetracycline. This technology is, in principle, applicable to any endogenous gene, allowing direct determination of both elevated and reduced gene expression in physiological and pathological processes. Applying this switchable technology to the key cell cycle transcription factor E2F3, we demonstrate that elevated levels of E2F3 drive ectopic proliferation in multiple tissues. By contrast, E2F3 repression has minimal impact on tissue proliferation or homeostasis in the majority of contexts due to redundancy of adult function with E2F1 and E2F2. In the absence of E2F1 and E2F2, however, repression of E2F3 elicits profound reduction of proliferation in the hematopoietic compartments that is rapidly lethal in adult animals.

Aging: when the ubiquitin–proteasome machinery collapses

In mammalian cells, protein degradation is an essential and dynamic process that is crucial for survival, growth, differentiation and proliferation of cells. Tellingly, the majority of intracellular proteins are degraded via the ubiquitin–proteasome system (UPS). UPS-mediated protein degradation serves qualitative and quantitative roles within the cellular proteome. For instance, UPS specifically targets misfolded, aggregated, toxic, mutant and otherwise structurally abnormal proteins for destruction and hence prevent aggregation and accumulation of toxic proteins. Furthermore, several cellular regulatory proteins, including cell cycle regulators, transcription factors, DNA replication and DNA repair proteins are selectively targeted for degradation via UPS and thus contribute to maintaining protein homeostasis (proteostasis) and proper functional proteome. Concomitantly, the deregulation of proteostasis may lead to several pathological disorders including aging-associated pathologies. Remarkably, augmenting the proteasomal activity has been linked to longevity in model organisms and protect these organisms from symptoms linked to protein homeostasis disorders. Herein I comment briefly on the recent work revealing the pivotal role of ubiquitin–proteasome-mediated protein degradation with respect to regulating aging process in model organisms.

Identification of Genes Mediating Drosophila Follicle Cell Progenitor Differentiation by Screening for Modifiers of GAL4::UAS Variegation.

The Drosophila melanogaster ovarian follicle cell lineage provides a powerful system for investigating how epigenetic changes contribute to differentiation. Downstream from an epithelial stem cell, follicle progenitors undergo nine mitotic cell cycles before transitioning to the endocycle and initiating differentiation. During their proliferative phase, follicle progenitors experience Lsd1-dependent changes in epigenetic stability that can be monitored using GAL4::UAS variegation. Eventually, follicle progenitors acquire competence to respond to Delta, a Notch ligand present in the environment, which signals them to cease division and initiate differentiation. The time required to acquire competence determines the duration of mitotic cycling and hence the final number of follicle cells. We carried out a screen for dominant modifiers of variegation spanning nearly 70% of Drosophila euchromatin to identify new genes influencing follicle progenitor epigenetic maturation. The eight genes found include chromatin modifiers, but also cell cycle regulators and transcription factors. Five of the modifier genes accelerate the acquisition of progenitor competence and reduce follicle cell number, however, the other three genes affect follicle cell number in an unexpected manner.

A Kinase-Phosphatase Switch Transduces Environmental Information into a Bacterial Cell Cycle Circuit.

The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information.

The Interaction Mode of the Acidic Region of the Cell Cycle Transcription Factor DP1 with TFIIH

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.

Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells

Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.

A Computational Method for Identifying Yeast Cell Cycle Transcription Factors.

The eukaryotic cell cycle is a complex process and is precisely regulated at many levels. Many genes specific to the cell cycle are regulated transcriptionally and are expressed just before they are needed. To understand the cell cycle process, it is important to identify the cell cycle transcription factors (TFs) that regulate the expression of cell cycle-regulated genes. Here, we describe a computational method to identify cell cycle TFs in yeast by integrating current ChIP-chip, mutant, transcription factor-binding site (TFBS), and cell cycle gene expression data. For each identified cell cycle TF, our method also assigned specific cell cycle phases in which the TF functions and identified the time lag for the TF to exert regulatory effects on its target genes. Moreover, our method can identify novel cell cycle-regulated genes as a by-product.

Importance of Tumour Suppressor Gene Methylation in Sinonasal Carcinomas

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in samples of sinonasal carcinoma by comparison with normal sinonasal tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 64 tissue samples of sinonasal carcinomas with 19 control samples. We also compared the human papilloma virus (HPV) status with DNA methylation. Using a 20% cut-off for methylation, we observed significantly higher methylation in RASSF1, CDH13, ESR1 and TP73 genes in the sinonasal cancer group compared with the control group. HPV positivity was found in 15/64 (23.4 %) of all samples in the carcinoma group and in no sample in the control group. No correlation was found between DNA methylation and HPV status. In conclusion, our study showed that there are significant differences in promoter methylation in the RASSF1, ESR1, TP73 and CDH13 genes between sinonasal carcinoma and normal sinonasal tissue, suggesting the importance of epigenetic changes in these genes in carcinogenesis of the sinonasal area. These findings could be used as prognostic factors and may have implications for future individualised therapies based on epigenetic changes.

Ineffectiveness of Methylation in Rgulation of VHL, ECAD, and RUNX3 Genes in Erythroid Cells Differentiated by Erythropoietin

Background: Vast variety of intermediate factors including cell cycle regulators, growth factors, transcription factors, and signaling pathways are involved in hematopoietic stem cell (HSC) commitment and differentiation into distinct lineages. VHL, Ecad, and RUNX3 are among these. Epigenetics is currently introduced as a potential mechanism to control the gene regulation. The aim of this study is to reveal the correlation between the expression level and methylation pattern of mentioned genes after in vitro differentiation of cord blood HSCs into erythroid lineage mediated by erythropoietin. M aterials and Methods: After isolation and expansion, the CD34+ cord blood stem cells were divided into two parts. The first part was used to extract the DNA and RNA and the second to differentiate into erythroid lineage. Methylation specific PCR (MSP) and Real-time PCR were used to determine the methylation status and expression levels of the genes, respectively. R es ults: Although the significant upregulation observed for VHL and Ecad genes and a down-regulation for RUNX3 gene after differentiation, no remarkable changes were seen in methylation pattern compared with cord blood HSCs by MSP technique. C onclusion: It is appearing that methylation pattern in promoter region has not an effective role in expression of VHL, Ecad, and RUNX3. Moreover, considering the inability of MSP method to detect subtle differences in methylation level a more sensitive method is needed to distinguish the methylation levels of these genes before and after erythroid differentiation.

Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains.

The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains.

Cell cycle transcription factor E2F2 mediates non-stress temperature response of AtHSP70-4 in Arabidopsis

AtHSP70 expression exhibits both stress and non-stress temperature response, however, the molecular mechanisms underlying these temperature signaling pathways remain elusive. Here we performed truncation and deletion assay to investigate the cis-elements within the promoter region of AtHSP70-4 (AT3G12580). And found the region between −1000 and −1100bp from the translation initiation site (TIS) was indispensable for the non-stress temperature response of AtHSP70. Further deletion assay of several candidate motifs within this region suggested that one ‘GCGCCAAA’ sequence played the critical role. This motif was found as the reverse DNA-binding motif of cell cycle transcription factor E2F family. EMSA assay verified one number of Arabidopsis E2F family—E2F2 could bind to AtHSP70-4 promoter via this motif. These results indicated the temperature regulated expression of AtHSP70-4 may be mediated by cell cycle transcription factors and participate in plant acclimations to non-stress temperature changes.

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes.Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.

The transcription factors Tec1 and Ste12 interact with coregulators Msa1 and Msa2 to activate adhesion and multicellular development.

In Saccharomyces cerevisiae and related yeast species, the TEA transcription factor Tec1, together with a second transcription factor, Ste12, controls development, including cell adhesion and filament formation. Tec1-Ste12 complexes control target genes through Tec1 binding sites (TEA consensus sequences [TCSs]) that can be further combined with Ste12 binding sites (pheromone response elements [PREs]) for cooperative DNA binding. The activity of Tec1-Ste12 complexes is known to be under negative control of the Dig1 and Dig2 (Dig1/2) transcriptional corepressors that confer regulation by upstream signaling pathways. Here, we found that Tec1 and Ste12 can associate with the transcriptional coregulators Msa1 and Msa2 (Msa1/2), which were previously found to associate with the cell cycle transcription factor complexes SBF (Swi4/Swi6 cell cycle box binding factor) and MBF (Mbp1/Swi6 cell cycle box binding factor) to control G1-specific transcription. We further show that Tec1-Ste12-Msa1/2 complexes (i) do not contain Swi4 or Mbp1, (ii) assemble at single TCSs or combined TCS-PREs in vitro, and (iii) coregulate genes involved in adhesive and filamentous growth by direct promoter binding in vivo. Finally, we found that, in contrast to Dig proteins, Msa1/2 seem to act as coactivators that enhance the transcriptional activity of Tec1-Ste12. Taken together, our findings add an additional layer of complexity to our understanding of the control mechanisms exerted by the evolutionarily conserved TEA domain and Ste12-like transcription factors.

More of a good thing or less of a bad thing: gene copy number variation in polyploid cells of the placenta.

More of a good thing or less of a bad thing: gene copy number variation in polyploid cells of the placenta.

Effects of TP53 mutational status on gene expression patterns across 10 human cancer types

Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild-type TP53 alleles. Among 113 known p53-up-regulated target genes identified from cell culture assays, 10 were consistently up-regulated in at least eight of 10 cancer types that retain both copies of wild-type TP53. RPS27L, CDKN1A (p21(CIP1)) and ZMAT3 were significantly up-regulated in all 10 cancer types retaining wild-type TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently up-regulated in wild-type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types. Finally, > 93% of tumours with a TP53 mutation exhibited greatly reduced wild-type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild-type TP53 retain some aspects of p53-mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes.

HTLV-1 Tax-activated NF-κB is involved in both cell growth and death

HTLV-1 Tax (Tax1) dysregulates the expression of cellular genes through transcriptional factors such as NF-kB, CREB and AP-1, leading to cell proliferation and immortalization. Conversely, Tax1 is reported to induce growth arrest and apoptosis. The molecular mechanism underlying growth arrest and cell death by Tax1 remains to be elucidated. The interleukin 2 (IL-2)-dependent human cell line Kit 225, in which the resting phase of cell growth is induced by deprivation of IL-2, was used to examine effects of Tax1 in growing and resting Kit 225 cells. Introduction of Tax1 reduced cell growth in growing cells, while Tax1 enhanced cell activity in resting cells. Tax1 activated the cell cycle transcription factor E2F and also increased the expression of cell cycle regulatory genes such as CDK2, CDK4 and cyclin D2 in resting cells. Tax1-induced reduction of cell growth was associated with apoptosis. No reduction in cell growth was observed in growing cells after introduction of Tax2 coded by HTLV-2, which has not been shown to link to malignant diseases. Tax1-induced cell growth reduction and apoptosis were demolished by siRNA against the NF-kB subunit RelA. In contrast, resting Kit 225 cells reduced cell activity by decreasing RelA upon Tax1 introduction. These results indicate that Tax1-activated RelA acts on both cell growth and cell death in a cell cycle-dependent manner. Functional differences between Tax1 and Tax2 may influence HTLV-1 pathogenesis.

Dynamics of the cell-cycle network under genome-rewiring perturbations

The cell-cycle progression is regulated by a specific network enabling its ordered dynamics. Recent experiments supported by computational models have shown that a core of genes ensures this robust cycle dynamics. However, much less is known about the direct interaction of the cell-cycle regulators with genes outside of the cell-cycle network, in particular those of the metabolic system. Following our recent experimental work, we present here a model focusing on the dynamics of the cell-cycle core network under rewiring perturbations. Rewiring is achieved by placing an essential metabolic gene exclusively under the regulation of a cell-cycle's promoter, forcing the cell-cycle network to function under a multitasking challenging condition; operating in parallel the cell-cycle progression and a metabolic essential gene. Our model relies on simple rate equations that capture the dynamics of the relevant protein–DNA and protein–protein interactions, while making a clear distinction between these two different types of processes. In particular, we treat the cell-cycle transcription factors as limited ‘resources’ and focus on the redistribution of resources in the network during its dynamics. This elucidates the sensitivity of its various nodes to rewiring interactions. The basic model produces the correct cycle dynamics for a wide range of parameters. The simplicity of the model enables us to study the interface between the cell-cycle regulation and other cellular processes. Rewiring a promoter of the network to regulate a foreign gene, forces a multitasking regulatory load. The higher the load on the promoter, the longer is the cell-cycle period. Moreover, in agreement with our experimental results, the model shows that different nodes of the network exhibit variable susceptibilities to the rewiring perturbations. Our model suggests that the topology of the cell-cycle core network ensures its plasticity and flexible interface with other cellular processes, without a need for an optimal setting of the kinetic parameters.

Identification of cell cycle–regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors

We identify the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle-regulated genes. FOXM1 is bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle transcription factors (E2F1, E2F4, E2F6, and GABPA) from the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle-regulated genes in a second cancer-derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.

THU0008 Serial Analysis of Gene Expression of Synovial Fibroblasts Discriminates Between Resolving and Persistent Inflammatory Arthritis

BackgroundRheumatoid arthritis synovial fibroblasts (RASF) are key mediators of the persistence of inflammation and joint destruction in RA. Their activated phenotype has been likened to that of cancer associated fibroblasts,...

Methylation analysis of tumor suppressor genes in endometroid carcinoma of endometrium using MS-MLPA

Epigenetic changes are considered to be a frequent event during tumor development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumor suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in endometrial cancer by comparison with normal endometrial tissue. We used MS-MLPA (Methylation-specific Multiplex ligation-dependent probe amplification) to compare the methylation status of 59 tissue samples of endometroid type of endometrial carcinoma with 20control samples of non-neoplastic endometrium. Using 15% cut-off for methylation, we observed significantly higher methylation in the CDH13 gene in endometrial cancer group. We observed significantly higher methylation in both WT1 and GATA5 genes in IB stage of endometroid carcinoma. We also observed significantly higher methylation in GATA5 gene in the group of poorly differentiated endometroid carcinoma. The findings suggest the importance of hypermethylation of CDH13, WT1 and GATA5 genes in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies of endometrial cancer based on epigenetic changes.