MluI Cell Cycle Box Research Articles

FvMbp1-Swi6 complex regulates vegetative growth, stress tolerance, and virulence in Fusarium verticillioides

The mycotoxigenic fungus Fusarium verticillioides is a common pathogen of grain and medicine that contaminates the host with fumonisin B1 (FB1) mycotoxin, poses serious threats to human and animal health. Therefore, it is crucial to unravel the regulatory mechanisms of growth, and pathogenicity of F. verticillioides. Mbp1 is a component of the MluI cell cycle box binding factor complex and acts as an APSES-type transcription factor that regulates cell cycle progression. However, no information is available regarding its role in F. verticillioides. In this study, we demonstrate that FvMbp1 interacts with FvSwi6 that acts as the cell cycle transcription factor, to form the heteromeric transcription factor complexes in F. verticillioides. Our results show that ΔFvMbp1 and ΔFvSwi6 both cause a severe reduction of vegetative growth, conidiation, and increase tolerance to diverse environmental stresses. Moreover, ΔFvMbp1 and ΔFvSwi6 dramatically decrease the virulence of the pathogen on the stalk and ear of maize. Transcriptome profiling show that FvMbp1-Swi6 complex co-regulates the expression of genes associated with multiple stress responses. These results indicate the functional importance of the FvMbp1-Swi6 complex in the filamentous fungi F. verticillioides and reveal a potential target for the effective prevention and control of Fusarium diseases.

Deletion of the MBP1 Gene, Involved in the Cell Cycle, Affects Respiration and Pseudohyphal Differentiation in Saccharomyces cerevisiae.

ABSTRACTMbp1p is a component of MBF (MluI cell cycle box binding factor, Mbp1p-Swi6p) and is well known to regulate the G1–S transition of the cell cycle. However, few studies have provided clues regarding its role in fermentation. This work aimed to recognize the function of the MBP1 gene in ethanol fermentation in a wild-type industrial Saccharomyces cerevisiae strain. MBP1 deletion caused an obvious decrease in the final ethanol concentration under oxygen-limited (without agitation), but not under aerobic, conditions (130 rpm). Furthermore, the mbp1Δ strain showed 84% and 35% decreases in respiration intensity under aerobic and oxygen-limited conditions, respectively. These findings indicate that MBP1 plays an important role in responding to variations in oxygen content and is involved in the regulation of respiration and fermentation. Unexpectedly, mbp1Δ also showed pseudohyphal growth, in which cells elongated and remained connected in a multicellular arrangement on yeast extract-peptone-dextrose (YPD) plates. In addition, mbp1Δ showed an increase in cell volume, associated with a decrease in the fraction of budded cells. These results provide more detailed information about the function of MBP1 and suggest some clues to efficiently improve ethanol production by industrially engineered yeast strains.IMPORTANCE Saccharomyces cerevisiae is an especially favorable organism used for ethanol production. However, inhibitors and high osmolarity conferred by fermentation broth, and high concentrations of ethanol as fermentation runs to completion, affect cell growth and ethanol production. Therefore, yeast strains with high performance, such as rapid growth, high tolerance, and high ethanol productivity, are highly desirable. Great efforts have been made to improve their performance by evolutionary engineering, and industrial strains may be a better start than laboratory ones for industrial-scale ethanol production. The significance of our research is uncovering the function of MBP1 in ethanol fermentation in a wild-type industrial S. cerevisiae strain, which may provide clues to engineer better-performance yeast in producing ethanol. Furthermore, the results that lacking MBP1 caused pseudohyphal growth on YPD plates could shed light on the development of xylose-fermenting S. cerevisiae, as using xylose as the sole carbon source also caused pseudohyphal growth.

Cell Cycle-Regulated Transcription of CENP-A by the MBF Complex Ensures Optimal Level of CENP-A for Centromere Formation.

The centromere plays an essential role in chromosome segregation. In most eukaryotes, centromeres are epigenetically defined by the conserved histone H3 variant CENP-A. Proper centromere assembly is dependent upon the tight regulation of CENP-A level. Cell cycle regulation of CENP-A transcription appears to be a universal feature across eukaryotes, but the molecular mechanism underlying the temporal control of CENP-A transcription and how such regulation contributes to centromere function remains elusive. CENP-A in fission yeast has been shown to be transcribed before S phase. Using various synchronization methods, we confirmed that CENP-A transcription occurs at G1, leading to an almost twofold increase of the protein during S phase. Through a genetic screen, we identified the MBF (MluI box-binding factors) complex as a key regulator of temporal control of CENP-A transcription. The periodic transcription of CENP-A is lost in MBF mutants, resulting in CENP-A mislocalization and chromosome segregation defects. We identified the MCB (MluI cell cycle box) motif in the CENP-A promoter, and further showed that the MBF complex binds to the motif to restrict CENP-A transcription to G1. Mutations of the MCB motif cause constitutive CENP-A expression and deleterious effects on cell survival. Using promoters driving transcription to different cell cycle stages, we found that timing of CENP-A transcription is dispensable for its centromeric localization. Our data instead indicate that cell cycle-regulated CENP-A transcription is a key step to ensure that a proper amount of CENP-A is generated across generations. This study provides mechanistic insights into the regulation of cell cycle-dependent CENP-A transcription, as well as its importance on centromere function.

Nutritional cell cycle reprogramming reveals that inhibition of Cdk1 is required for proper MBF-dependent transcription.

In nature, cells and in particular unicellular microorganisms are exposed to a variety of nutritional environments. Fission yeast cells cultured in nitrogen-rich media grow fast, divide with a large size and show a short G1 and a long G2. However, when cultured in nitrogen-poor media, they exhibit reduced growth rate and cell size and a long G1 and a short G2. In this study, we compared the phenotypes of cells lacking the highly conserved cyclin-dependent kinase (Cdk) inhibitor Rum1 and the anaphase-promoting complex/cyclosome (APC/C) activator Ste9 in nitrogen-rich and nitrogen-poor media. Rum1 and Ste9 are dispensable for cell division in nitrogen-rich medium. However, in nitrogen-poor medium they are essential for generating a proper wave of MluI cell-cycle box binding factor (MBF)-dependent transcription at the end of G1, which is crucial for promoting a successful S phase. Mutants lacking Rum1 and Ste9 showed premature entry into S phase and a reduced wave of MBF-dependent transcription, leading to replication stress, DNA damage and G2 cell cycle arrest. This work demonstrates how reprogramming the cell cycle by changing the nutritional environment may reveal new roles for cell cycle regulators.

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription

SummaryChromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

DNA replication stress induces the transcriptional activation of rhp51 +, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51 + transcription is not understood. The promoter region of rhp51 + contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51 + transcription. The full-length rhp51 + promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51 + promoter abolished the induction of rhp51 + transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51 + transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51 + transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51 + transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51 + transcription. The transcription of rhp51 + and cdc18 +, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51 + similarly to cdc18 +. Collectively, these results suggest that MBF and its regulators mediate rhp51 + transcription in response to DNA replication stress, and underlie rhp51 + transcription at the G1/S transition.

Regulation of Histone Gene Expression in Budding Yeast

We discuss the regulation of the histone genes of the budding yeast Saccharomyces cerevisiae. These include genes encoding the major core histones (H3, H4, H2A, and H2B), histone H1 (HHO1), H2AZ (HTZ1), and centromeric H3 (CSE4). Histone production is regulated during the cell cycle because the cell must replicate both its DNA during S phase and its chromatin. Consequently, the histone genes are activated in late G1 to provide sufficient core histones to assemble the replicated genome into chromatin. The major core histone genes are subject to both positive and negative regulation. The primary control system is positive, mediated by the histone gene-specific transcription activator, Spt10, through the histone upstream activating sequences (UAS) elements, with help from the major G1/S-phase activators, SBF (Swi4 cell cycle box binding factor) and perhaps MBF (MluI cell cycle box binding factor). Spt10 binds specifically to the histone UAS elements and contains a putative histone acetyltransferase domain. The negative system involves negative regulatory elements in the histone promoters, the RSC chromatin-remodeling complex, various histone chaperones [the histone regulatory (HIR) complex, Asf1, and Rtt106], and putative sequence-specific factors. The SWI/SNF chromatin-remodeling complex links the positive and negative systems. We propose that the negative system is a damping system that modulates the amount of transcription activated by Spt10 and SBF. We hypothesize that the negative system mediates negative feedback on the histone genes by histone proteins through the level of saturation of histone chaperones with histone. Thus, the negative system could communicate the degree of nucleosome assembly during DNA replication and the need to shut down the activating system under replication-stress conditions. We also discuss post-transcriptional regulation and dosage compensation of the histone genes.

Abstract 665: Targeting the MluI cell cycle box (MCB) sequence 5’-ACGCGT-3’ in the human Dbf4 promoter using the rationally designed polyamide formamido-imidazole-pyrrole-imidazole (f-IPI)

Abstract Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for the initiation of DNA replication throughout S-phase. The level of Dbf4 is critical for the activation of Cdc7. Human Cdc7/Dbf4 (HuCdc7/Dbf4) kinase activity is essential for cell proliferation by activating DNA replication. High levels of the Cdc7 kinase activity have been implicated with a large number of aggressive solid cancers. The MluI cell cycle box (MCB) sequence 5’-ACGCGT-3’ is the critical site for transcription factor binding that activates the expression of the HuCdc7/Dbf4 core gene in mammalian cells. HuDbf4 promoter activity has previously been shown to be significantly reduced by a point mutation within the MCB. We have previously shown that the rationally designed simple polyamide formamido-imidazole-pyrrole-imidazole (f-IPI) binds to its cognate sequence 5’-ACGCGT-3’ as a stacked dimer in an anti-parallel conformation (2:1 ligand/DNA complex) with exceptionally high affinity and selectivity, superior to the natural product distamycin A binding to its cognate sequence. DNAse I footprinting experiments indicated selective binding of f-IPI to the MCB sequence in the HuDbf4 promoter at 0.1µM. Eletrophoretic mobility shift assays using the MCB-containing sequence from the HuDbf4 promoter showed dose-dependent inhibition of protein binding by f-IPI which was complete at 0.5µM. RT-PCR showed decreased expression of HuDbf4 mRNA in response to incubation of exponentially growing MDA-MB231 cells with 10 μM f-IPI. Selective effects on protein expression, DNA replication and cell cycle are currently underway. These data demonstrate the potential of small molecule polyamides as modulators of gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2011-665

A Cds1-Mediated Checkpoint Protects the MBF Activator Rep2 from Ubiquitination by Anaphase-Promoting Complex/Cyclosome-Ste9 at S-Phase Arrest in Fission Yeast

Transcription of the MluI cell cycle box (MCB) motif-containing genes at G(1) phase is regulated by the MCB-binding factors (MBF) (also called DSC1) in Schizosaccharomyces pombe. Upon S-phase arrest, the MBF transcriptional activity is induced through the accumulation of the MBF activator Rep2. In this study, we show that the turnover of Rep2 is attributable to ubiquitin-mediated proteolysis. Levels of Rep2 oscillate during the cell cycle, with a peak at G(1) phase, coincident with the MBF activity. Furthermore, we show that Rep2 ubiquitination requires the function of the E3 ligase anaphase-promoting complex/cyclosome (APC/C). Ste9 can be phosphorylated by the checkpoint kinase Cds1 in vitro, and its inhibition/phosphorylation at S-phase arrest is dependent on the function of Cds1. Our data indicate that the Cds1-dependent stabilization of Rep2 is achieved through the inhibition/phosphorylation of APC/C-Ste9 at the onset of S-phase arrest. Stabilization of Rep2 is important for stimulating transcription of the MBF-dependent genes to ensure a sufficient supply of proteins essential for cell recovery from S-phase arrest. We propose that oscillation of Rep2 plays a role in regulation of periodic transcription of the MBF-dependent genes during cell cycle progression.

Polyamide Curvature and DNA Sequence Selective Recognition: Use of 4-Aminobenzamide to Adjust Curvature

Imidazole and pyrrole-containing polyamides belong to an important class of compounds that can be designed to target specific DNA sequences, and they are potentially useful in applications of controlling gene expression. The extent of polyamide curvature is an important consideration when studying the ability of such compounds to bind in the minor groove of DNA. The current study investigates the importance of curvature using polyamides of the form f-Im-Phenyl-Im, in which the imidazole heterocycles are placed in ortho-, meta-, and para-configurations of the phenyl moiety. The synthesis and biophysical evaluation of each compound binding to its cognate DNA sequence (5'-ACGCGT-3') and a negative control sequence (5'-AAATTT-3') is reported, along with their comparison to the parent binder, f-Im-Py-Im (3). ACGCGT is a medicinally significant sequence present in the MluI cell-cycle box (MCB) transcriptional element found in the promoter of a gene associated with cell division. The results demonstrated that the para-derivative has the greatest affinity for its cognate sequence, as indicated via thermal denaturation, CD, ITC, SPR analyses, and DNase I footprinting. ITC studies showed that binding of the para-isomer (2c) to ACGCGT was significantly more exothermic than binding to AAATTT. In contrast, no heat change was observed for binding of the meta- (2b) and ortho- (2a) isomers to both DNAs, due to low binding affinities. This is consistent with results from SPR studies, which indicate that the para-derivative binds in a 2:1 fashion to ACGCGT and binds weakly to ACCGGT (K = 1.8 x 10(6) and 4.0 x 10(4) M(-1), respectively). Interestingly, it binds in a 1:1 fashion to AAATTT (K = 5.4 x 10(5) M(-1)). The meta-compound does not bind to any sequence. The para-derivative also was the only compound to show an induced peak via CD at 330 nm, indicative of minor groove binding, and produced a DeltaT(m) value of 5.8 degrees C. Molecular modeling experiments have been performed to determine the shape differences between the three compounds, and the results indicate that the para-derivative 2c has a closest curvature to previously synthesized polyamides. DNase I footprinting studies confirmed earlier observations that only the para-derivative 2c produced a footprint with ACGCGT (1 microM) and no significant footprint was observed at any sites examined for meta-2b and ortho-2a analogs up to 40 microM. The results of these studies suggest that the shape of the ortho- and meta- derivatives is too curved to match the curvature of the DNA minor groove to facilitate binding. The para-derivative gives the highest binding affinity in the series and the results illustrate that 4-aminobenzamide is a reasonable substitute for 4-aminopyrrole-2-carboxylate.

Functional Mapping of the Candida albicans Efg1 Regulator

Efg1p is a key transcriptional regulator in Candida albicans which controls various aspects of morphogenesis and metabolism in this organism. Efg1p contains a central basic helix-loop-helix (bHLH) domain, flanked by sequences highly conserved in fungal APSES proteins, as well as polyglutamine stretches at the N- and C-terminal ends. A systematic deletion approach to specify functional domains of Efg1p revealed that the APSES domain is essential for morphogenesis of the normal yeast and true hyphal cell forms and that bHLH flanking sequences are needed for Efg1p stability. Additional C-terminal sequences were required for hyphal formation on some inducing media, and most Efg1p sequences were needed for chlamydospore morphogenesis. Overexpression of EFG1 led to pseudohypha formation only if a functional APSES domain was present, while a switch from the opaque to the white cell type in addition depended on the presence of certain N- and C-terminal segments. Yeast two-hybrid experiments revealed that binding of Efg1p to its antagonist Czf1p required two regions outside of the APSES domain, which did not coincide with Efg1p sequences needed for its transcriptional repressor activity. Binding of the Flo8 transcription factor to Efg1p did not require the APSES domain but appeared to occur at two or more redundant domains. In contrast, DNA binding of Efg1p to an MluI cell cycle box (MCB) element solely required the APSES domain. Overall, these results suggest that functional domains of Efg1p are spread throughout most of its sequences, including the central APSES domain involved in DNA binding, as well as flanking regions required for various protein interactions and regulatory activities.

Transcription of the Schizosaccharomyces pombe gene cdc18+: roles of MCB elements and the DSC1 complex

In Schizosaccharomyces pombe, commitment to a round of DNA synthesis and entry into the cell cycle are dependent on the function of genes that are transcribed periodically during the cell cycle. Activation of these genes prior to S phase is primarily controlled through cis-acting elements known as MluI Cell-cycle Boxes, or MCBs, and by a family of transcription factors, including Cdc10, Res1, Res2 and Rep2. These transcription factors are also known to be present in a complex, DSC1, that binds to the promoters of pre-S genes. We have demonstrated that within the promoter of cdc18 + , a representative pre-S gene, the orientation and spacing of MCBs are crucial for activation and cell-cycle dependence. To our surprise, electrophoretic mobility shift assays showed a highly active mutant form of the promoter, which alters the spacing of the MCB elements, does not bind DSC1 but does bind a higher mobility complex. The binding of this second complex is not dependent on Cdc10 or the Res/Rep proteins. We conclude that, DSC1 binding does not correlate with cell-cycle dependent transcriptional activation, and the higher mobility species may represent a novel transcriptional activation complex that is also likely to function in pre-S transcription.

High Functional Overlap Between MluI Cell-Cycle Box Binding Factor and Swi4/6 Cell-Cycle Box Binding Factor in the G1/S Transcriptional Program in Saccharomyces cerevisiae

In budding yeast, many genes are induced early in the cell cycle. Induction of these genes has been predominantly attributed to two transcription factors, Swi4-Swi6 (SBF) and Mbp1-Swi6 (MBF). Swi4 and Mbp1 are related DNA-binding proteins with dissimilar target sequences. For most G1/S-regulated genes that we tested in a cdc20 block-release protocol for cell-cycle synchronization, removal of both Swi4 and Mbp1 was necessary and sufficient to essentially eliminate cell-cycle-regulated expression. Detectable SBF or MBF binding sites (SCBs or MCBs) in the promoters or available genome-wide promoter occupancy data do not consistently explain this functional overlap. The overlapping ability of these transcription factors to regulate many promoters with very similar cell-cycle kinetics may provide robustness to the G1/S transcriptional response, but poses a puzzle with respect to promoter-transcription factor specificity. In addition, for some genes, deletion of Mbp1 or Swi4 enhances transcription, suggesting that these factors can also function as transcriptional repressors. Finally, we observe residual G1/S transcriptional regulation in the absence of Swi4 and Mbp1.

Psc3 cohesin of Schizosaccharomyces pombe: cell cycle analysis and identification of three distinct isoforms

Cohesins are a group of proteins that function to mediate correct chromosome segregation, DNA repair and meiotic recombination. This report presents the amino acid sequence for the Schizosaccharomyces pombe cohesin Psc3 based on the translation of the cDNA sequence, showing that the protein is smaller than previously predicted. Interestingly, comparison of the amino acid and DNA coding sequences of Psc3 with fission yeast Rec11 meiotic region-specific recombination activator shows that both intron positioning within the genes and the amino-terminal half of the two proteins are highly conserved. We demonstrate that although the intergenic region upstream of the psc3+ start codon contains a consensus sequence for the cell-cycle regulatory MluI cell-cycle box, psc3+ transcription is not differentially regulated during the mitotic cell cycle. Finally, we demonstrate that an epitope-tagged version of Psc3 undergoes no major changes during the mitotic cell cycle. However, instead we identify at least three distinct isoforms of Psc3, suggesting that post-translational modification of Psc3 contributes to the regulation of cohesion function.

Meiosis-Specific Regulation of the Saccharomyces cerevisiae S-Phase Cyclin CLB5 Is Dependent on MluI Cell Cycle Box (MCB) Elements in Its Promoter but Is Independent of MCB-Binding Factor Activity

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1.

Cell cycle-regulated transcription in fission yeast.

A fundamental process in biology is the mechanism by which cells duplicate and divide to produce two identical daughter cells. The fission yeast, Schizosaccharomyces pombe, has proved to be an excellent model organism to study the role that gene expression plays in this process. The basic paradigm emerging is that a number of groups of genes are expressed in successive waves at different cell cycle times. Transcription of a particular group is controlled by a common DNA motif present in each gene's promoter, bound by a transcription factor complex. Each motif and transcription factor complex is specific to the time in the cell cycle when the group of genes is expressed. Examples of this are the MBF (MCB-binding factor)/MCB (MluI cell cycle box) system controlling gene expression at the start of S-phase, and PBF (PCB-binding factor)/PCB (Pombe cell cycle box) regulation of transcription at the end of mitosis. In some cases, these transcription control systems also operate during the alternative form of cell division, meiosis.

Cell cycle-regulated transcription in fission yeast

A fundamental process in biology is the mechanism by which cells duplicate and divide to produce two identical daughter cells. The fission yeast, Schizosaccharomyces pombe, has proved to be an excellent model organism to study the role that gene expression plays in this process. The basic paradigm emerging is that a number of groups of genes are expressed in successive waves at different cell cycle times. Transcription of a particular group is controlled by a common DNA motif present in each gene's promoter, bound by a transcription factor complex. Each motif and transcription factor complex is specific to the time in the cell cycle when the group of genes is expressed. Examples of this are the MBF (MCB-binding factor)/MCB (MluI cell cycle box) system controlling gene expression at the start of S-phase, and PBF (PCB-binding factor)/PCB (Pombe cell cycle box) regulation of transcription at the end of mitosis. In some cases, these transcription control systems also operate during the alternative form of cell division, meiosis.

Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements.

Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for entry into and traversing through S phase. The level of Dbf4, which is critical for the activation of Cdc7, is regulated by transcription and protein degradation. To gain a better understanding as to how the transcription of human Dbf4 (HuDbf4) is regulated, we have cloned and characterized its promoter. We found that HuDbf4 core promoter is localized within (-)211 to -285 of the translation start-codon. This 75 bp DNA segment contains, among others, a putative MluI Cell-cycle Box (MCB). A point mutation within the MCB dramatically reduced the promoter activity. This is the first example that an MCB element plays an essential role in the activation of a core promoter in mammalian cells. The auxiliary elements required for the full promoter activity are present within 162-bp upstream from the core promoter (i.e., -286/-447). A point mutation within the Sp1 element at -353/-361 resulted in a decrease of promoter activity to the basal level, while the deletion of the putative HES-1 at -326/-331 dramatically increased the promoter activity. Taken together, our data suggests that the MCB element is essential for the core promoter activation, while the Sp1 positive regulator and the HES-1 repressor coordinately determine the efficiency of the HuDbf4 promoter. We have also found: (i) that the major transcription initiations occur at -220, -235 and -245; (ii) that HuDbf4 gene consists of 12 exons, which spread over a 33-kb region.

Genome-Wide analysis of yeast transcription upon calcium shortage

Several regulatory circuits related to important functions, like membrane excitation, immunoresponse, replication, control of the cell cycle and differentiation, among others, cause an increase in intracellular calcium level that finally has a consequence upon transcription of specific genes. The sequencing of the whole genome of eukaryotic cells enables genome-wide analysis of gene expression under many conditions not yet assessed by conventional methods. Using the array technology, the effect of calcium shortage in yeast cells was studied. Correspondence analysis of data showed that there is a response in transcription that is correlated to calcium shortage. The distribution of up-regulated-genes in functional categories suggests a regulatory connection between the cell-cycle progression and the energetic metabolic requirements for growth and division. In silico analysis of promoters reveals the frequent appearance of the Mlu I cell cycle box (MCB) cis element that binds the transcriptional regulatory factor Mcm1.

The cohesin complex: sequence homologies, interaction networks and shared motifs.

Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown. Searches of protein sequence databases have revealed new homologs of cohesin proteins. In mouse, Mmip1 (Mad member interacting protein 1) and Smc3 share 99% sequence identity and are products of the same gene. A phylogenetic tree of SMC homologs reveals five families: Smc1, Smc2, Smc3, Smc4 and an ancestral family that includes the sequences from the Archaea and Eubacteria. This ancestral family also includes sequences from eukaryotes. A cohesion interaction network, comprising 17 proteins, has been constructed using two proteomic databases. Genes encoding six proteins in the cohesion network share a common upstream region that includes the MluI cell-cycle box (MCB) element. Pairs of the proteins in this network share common sequence motifs that could represent common structural features such as binding sites. Scc2 shares a motif with Chk1 (kinase checkpoint protein), that comprises part of the serine/threonine protein kinase motif, including the active-site residue. We have combined genomic and proteomic data into a comprehensive network of information to reach a better understanding of the function of the cohesin complex. We have identified new SMC homologs, created a new SMC phylogeny and identified shared DNA and protein motifs. The potential for Scc2 to function as a kinase - a hypothesis that needs to be verified experimentally - could provide further evidence for the regulation of sister-chromatid cohesion by phosphorylation mechanisms, which are currently poorly understood.