Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle (original) (raw)
Related papers
Genome Research, 1999
Recent developments in genome-wide transcript monitoring have led to a rapid accumulation of data from gene expression studies. Such projects highlight the need for methods to predict the molecular basis of transcriptional coregulation. A microarray project identified the 420 yeast transcripts whose synthesis displays cell cycle-dependent periodicity. We present here a statistical technique we developed to identify the sequence elements that may be responsible for this cell cycle regulation. Because most gene regulatory sites contain a short string of highly conserved nucleotides, any such strings that are involved in gene regulation will occur frequently in the upstream regions of the genes that they regulate, and rarely in the upstream regions of other genes. Our strategy therefore utilizes statistical procedures to identify short oligomers, five or six nucleotides in length, that are over-represented in upstream regions of genes whose expression peaks at the same phase of the cell cycle. We report, with a high level of confidence, that 9 hexamers and 12 pentamers are over-represented in the upstream regions of genes whose expression peaks at the early G 1 , late G 1 , S, G 2 , or M phase of the cell cycle. Some of these sequence elements show a preference for a particular orientation, and others, through a separate statistical test, for a particular position upstream of the ATG start codon. The finding that the majority of the statistically significant sequence elements are located in late G 1 upstream regions correlates with other experiments that identified the late G 1 /early S boundary as a vital cell cycle control point. Our results highlight the importance of MCB, an element implicated previously in late G 1 /early S gene regulation, as most of the late G 1 oligomers contain the MCB sequence or variations thereof. It is striking that most MCB-like sequences localize to a specific region upstream of the ATG start codon. Additional sequences that we have identified may be important for regulation at other phases of the cell cycle.
Comprehensive Identification of Cell Cycle-regulated Genes of the Yeast Saccharomyces cerevisiae by
2000
Biology 3273 tion of 2% (wt/vol). Cells from this culture were harvested every 10 min for 40 min for RNA. The entire control culture was harvested at Cell Cycle-regulated Genes in Yeast Vol. 9, December 1998 3275 Figure 4. The S and M clusters. The transcription profiles are displayed as described in the legend to Figure 1. (A) Histone cluster. The eight genes encoding histones and the yeast histone H1 homologue cluster very tightly and are expressed during S phase of the yeast cell cycle. (B) MET cluster. The expression of many of the members of the methionine pathway peaks just after the histones. (C) CLB2 cluster. A subcluster of genes that are expressed similarly to CLB2 highlights genes that peak during M phase.
Systems analysis of the human cell cycle transcription network
2016
Cell division is one of the most fundamental processes of life whereby one cell replicates itself to produce two. The molecular machinery that drives and regulates this fundamental process has been much studied but much remains unknown. This work describes the use of transcriptomics analyses to identify putative new proteins involved with this process and subsequent attempts to prove their association with this pathway. Using the latest array technology, in Chapter 2 I describe studies that examine the expression of genes regulated during different stages of the human cell cycle. Synchronous populations of neonatal human dermal fibroblasts (NHDFs) were generated by serum starvation and analysed in two separate microarray experiments. For the first set array experiments, samples were taken every 6 hours for 48 hours after serum refeeding, and every 2 hours for 24 hours for the second experiment. Using BioLayout Express 3D , network structure analyses identified four major clusters of gene expression patterns associated with different stages of the cell cycle: G0-, early G1-, late G1-, and S/G2/M-phase. By comparison with datasets of other human cells and tissues, the list of genes in the S/G2/M cluster was refined; genes were only kept in the list if they were found to be co-expressed in cells and tissues with high levels of cell proliferation. 706 genes that were co-expressed during S/G2/M-phase were selected for further analyses. Manual curation showed that 484 are known cell cycle-associated genes, 78 are genes with putative association to the cell cycle, and 75 have known roles in other biological processes, whilst 69 were entirely uncharacterised genes. In order to investigate the 69 genes with unknown function, in Chapter 3 I describe how RNAi was used to screen 42 of these genes to see if their knockdown resulted in an effect on cell proliferation. After extensive assay optimisation, endoribonuclease-prepared siRNA (esiRNA) was delivered to NHDF cells and the effect of knockdown determined using a real time cell analysis (RTCA) system. This system monitors the change in electrical resistance induced by growing cell populations defined as the cell impedance index (CI). Using a Z-scoring cutoff to determine the hits of the RNAi screening, according to the average value of cell impedance growth rate (CIGR i.e. a value from transformed CI), 19 of 42 genes were found to significantly affect the dynamics of cell proliferation, supporting a potential role in cell division. In order to verify that the unknown proteins localise to structures compatible with a role in the cell cycle, in Chapter 4 I describe protein localisation studies on 11 of 19 genes of 'hits' from Chapter 3 (we were unable to obtain clones for the other 8 genes) and other genes of interest. Transfection studies of HEK293T cells with expression clones containing more than
Transcriptional control of the cell cycle
Current Opinion in Cell Biology, 1996
Cell division is a highly coordinated process. In the last decades, many plant cell cycle regulators have been identified. Strikingly, only a few transcriptional regulators are known, although a significant amount of the genome is transcribed in a cell cycle phase-dependent manner. E2F-DP transcription factors and three repeat MYB proteins are responsible for the expression of genes at the G1-to-S and G2-to-M transition, respectively. However, these two mechanisms cannot explain completely the transcriptional regulation seen during the cell cycle. Correspondingly, several new transcriptional regulators have been characterized, stressing the importance of transcriptional control during the cell cycle.
Global control of cell-cycle transcription by coupled CDK and network oscillators
Nature, 2008
A significant fraction of the Saccharomyces cerevisiae genome is transcribed periodically during the cell division cycle 1,2 , 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 3 . However, directed studies interrogating small numbers of genes indicate that their periodic transcription is governed by the activity of cyclin-dependent kinases (CDKs) 4 . 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.
Complex transcriptional circuitry at the G1/S transition in Saccharomyces cerevisiae
Genes & Development, 2002
In the yeast Saccharomyces cerevisiae, SBF (Swi4–Swi6 cell cycle box binding factor) and MBF (MluI binding factor) are the major transcription factors regulating the START of the cell cycle, a time just before DNA replication, bud growth initiation, and spindle pole body (SPB) duplication. These two factors bind to the promoters of 235 genes, but bind less than a quarter of the promoters upstream of genes with peak transcript levels at the G1 phase of the cell cycle. Several functional categories, which are known to be crucial for G1/S events, such as SPB duplication/migration and DNA synthesis, are under-represented in the list of SBF and MBF gene targets. SBF binds the promoters of several other transcription factors, including HCM1, PLM2, POG1, TOS4, TOS8, TYE7, YAP5, YHP1, and YOX1. Here, we demonstrate that these factors are targets of SBF using an independent assay. To further elucidate the transcriptional circuitry that regulates the G1-to-S-phase progression, these factors were epitope-tagged and their binding targets were identified by chIp–chip analysis. These factors bind the promoters of genes with roles in G1/S events including DNA replication, bud growth, and spindle pole complex formation, as well as the general activities of mitochondrial function, transcription, and protein synthesis. Although functional overlap exists between these factors and MBF and SBF, each of these factors has distinct functional roles. Most of these factors bind the promoters of other transcription factors known to be cell cycle regulated or known to be important for cell cycle progression and differentiation processes indicating that a complex network of transcription factors coordinates the diverse activities that initiate a new cell cycle.
Genes & Development, 1996
When yeast cells reach a critical size in late G1 they simultaneously start budding, initiate DNA synthesis, and activate transcription of a set of genes that includes G1 cyclins CLN1, CLN2, and many DNA synthesis genes. Cell cycle-regulated expression of CLN1, CLN2 genes is attributable to the heteromeric transcription factor complex SBF. SBF is composed of Swi4 and Swi6 and binds to the promoters of CLN1 and CLN2. Different cyclin-Cdc28 complexes have different effects on late G1-specific transcription. Activation of transcription at the G1/S boundary requires Cdc28 and one of the G1 cyclins Cln1-Cln3, whereas repression of SBF-regulated genes in G2 requires the association of Cdc28 with G2-specific cyclins Clb1-Clb4. Using in vivo genomic footprinting, we show that SBF (Swi4/Swi6) binding to SCB elements (Swi4/Swi6 cell cycle box) in the CLN2 promoter is cell cycle regulated. SBF binds to the promoter prior to the activation of transcription in late G1, suggesting that Cln/Cdc28 ...