Transcriptional Regulation In Yeast Research Articles

The “glue” that controls transcription

by Mark Ptashne and Alexander Gann Cold Spring Harbor Laboratory Press (2002) 192 pages. ISBN 0-87969-633-8 $39 ![Figure][1] Guano cormorants flying home to the rock on which their baby bird is waiting to be fed face a daunting task. They must distinguish their own child from the

Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells.

Global Transcriptional Regulation in Yeast

Nine transcriptional regulators--MBP1, Swi6, Swi4, Mcm1, Fkh1, Fkh2, Ndd1, Swi5, and Ace2--are responsible for mediating cell cycle-dependent gene expression in yeast. Although much is known about the expression and regulation of each of these transcription factors individually, it would be helpful to know how the transcription factors function together to begin to coordinate complex processes like the cell division cycle. By using epitope-tagged transcription factors, DNA immunoprecipitation, and ligation-mediated polymerase chain reaction, Simon et al. arrayed the expression of hundreds of cell cycle-regulated genes and determined, with high probabilty, which transcription factors were required for the expression of those genes. Additionally, Simon et al. have begun to map out the profiles of transcription factors required for the expression of subsequent transcription factors during the cell cycle. Thus, this work helps to collate molecular observations into the better understanding of cellwide control networks.I. Simon, J. Barnett, N. Hannett, C. T. Harbison, N. J. Rinaldi, T. L. Volkert, J. J. Wyrick, J. Zeitlinger, D. K. Gifford, T. S. Jaakkola, R. A. Young, Serial regulation of transcriptional regulators in the yeast cell cycle. Cell 106, 697-708 (2001). [Online Journal]

Global Transcriptional Regulation in Yeast

Nine transcriptional regulators--MBP1, Swi6, Swi4, Mcm1, Fkh1, Fkh2, Ndd1, Swi5, and Ace2--are responsible for mediating cell cycle-dependent gene expression in yeast. Although much is known about the expression and regulation of each of these transcription factors individually, it would be helpful to know how the transcription factors function together to begin to coordinate complex processes like the cell division cycle. By using epitope-tagged transcription factors, DNA immunoprecipitation, and ligation-mediated polymerase chain reaction, Simon et al. arrayed the expression of hundreds of cell cycle-regulated genes and determined, with high probabilty, which transcription factors were required for the expression of those genes. Additionally, Simon et al. have begun to map out the profiles of transcription factors required for the expression of subsequent transcription factors during the cell cycle. Thus, this work helps to collate molecular observations into the better understanding of cellwide control networks. I. Simon, J. Barnett, N. Hannett, C. T. Harbison, N. J. Rinaldi, T. L. Volkert, J. J. Wyrick, J. Zeitlinger, D. K. Gifford, T. S. Jaakkola, R. A. Young, Serial regulation of transcriptional regulators in the yeast cell cycle. Cell 106 , 697-708 (2001). [Online Journal]

The Human Homologue of the Yeast DNA Repair and TFIIH Regulator MMS19 Is an AF-1-specific Coactivator of Estrogen Receptor

Steroid/nuclear hormone receptors are ligand-dependent transcriptional regulators that control gene expression in a wide array of biological processes. The transcriptional activity of the receptors is mediated by an N-terminal ligand-independent transcriptional activation function AF-1 and a C-terminal ligand-dependent transcriptional activation function AF-2. The nuclear receptor coactivator RAC3 (also known as AIB1/ACTR/pCIP/TRAM-1/SRC-3) is amplified in breast cancer cells, where it forms a complex with estrogen receptor (ER) and enhances AF-2 activity of the receptor. Here, we identify a putative human homologue of the yeast DNA repair and transcriptional regulator MMS19 as a RAC3-interacting protein. The human MMS19 interacts with the N-terminal PAS-A/B domain of RAC3 in vivo and in vitro through a conserved C-terminal domain. Interestingly, the human MMS19 also interacts with estrogen receptors in a ligand-independent manner but not with retinoic acid receptor or thyroid hormone receptor. Overexpression of the interacting domain of hMMS19 strongly inhibits ER-mediated transcriptional activation, indicating a dominant negative activity. In contrast, over expression of the full-length hMMS19 enhances ER-mediated transcriptional activation. We find that hMMS19 stimulates the AF-1 activity of ERalpha, but not the AF-2 activity, suggesting that hMMS19 may be an AF-1-specific transcriptional coactivator of estrogen receptor.

High Affinity Interaction of Yeast Transcriptional Regulator, Mot1, with TATA Box-binding Protein (TBP)

Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y = T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y=T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

Yap1 and Skn7 Control Two Specialized Oxidative Stress Response Regulons in Yeast

Yap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals. Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response. The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis. Yap1 controls a large oxidative stress response regulon of at least 32 proteins. Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2. Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro. The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets. Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH. Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance.

Genomic Organization and Chromosomal Localization of the Human Histone Deacetylase 3 Gene

Reversible acetylation of histone proteins plays a critical role in transcriptional regulation, cell cycle progression, and developmental events. The steady state of histone acetylation is controlled by the enzymatic activities of multiple histone acetyltransferases and histone deacetylases (HDACs). Three distinct human HDACs are homologous to RPD3, a yeast transcriptional regulator. We have isolated and sequenced a genomic clone for the humanHDAC3gene. This is a single-copy gene spanning a region of at least 13 kb. Determination of the intron–exon splice junctions established that the gene is encoded by 15 exons ranging in size from 56 to 657 bp. Fluorescencein situhybridization studies localized this gene to 5q31. Double-target experiments in which bothHDAC3and the early-growth response 1 gene (EGR1), which is localized in the 5q31.2 region, were used as probes showed that theHDAC3gene lies in region 5q31.3, immediately distal toEGR1with respect to the centromere.

Being at the right place at the right time: the role of nuclear transport in dynamic transcriptional regulation in yeast.

As a consequence of compartmentalization in eukaryotic cells, the transcriptional response to extracellular stimuli requires signals to be transmitted from the cytoplasm to the nucleus. Thus, nuclear import and export of signal transduction and transcription factors provide an important level of transcriptional control in eukaryotes. Recent investigations have illustrated the importance of this control for dynamic gene expression, as will be discussed on transcriptional activators and repressors (Mac1p, Mig1p, Msn2p/4p, Swi5p) and protein kinases (Hog1p, Sty1/Spc1, protein kinase A).

A Linker Region of the Yeast Zinc Cluster Protein Leu3p Specifies Binding to Everted Repeat DNA

Yeast zinc cluster proteins form a major class of yeast transcriptional regulators. They usually bind as homodimers to target DNA sequences, with each monomer recognizing a CGG triplet. Orientation and spacing between the CGG triplet specifies the recognition sequence for a given zinc cluster protein. For instance, Gal4p binds to inverted CGG triplets spaced by 11 base pairs whereas Ppr1p recognizes a similar motif but with a spacing of 6 base pairs. Hap1p, another member of this family, binds to a direct repeat consisting of two CGG triplets. Other members of this family, such as Leu3p, also recognize CGG triplets but when oriented in opposite directions, an everted repeat. This implies that the two zinc clusters of Leu3p bound to an everted repeat must be oriented in opposite directions to those of Gal4p or Ppr1p bound to inverted repeats. In order to map the domain responsible for proper orientation of the zinc clusters of Leu3p, we constructed chimeric proteins between Leu3p and Ppr1p and tested their binding to a Leu3p and a Ppr1p site. Our results show that the linker region, which bridges the zinc cluster to the dimerization domain, specifies binding of Leu3p to an everted repeat. We propose that the Leu3p linker projects the two zinc clusters of a Leu3p homodimer in opposite directions allowing binding to everted repeats.

Evidence of an overlap between the two half-sites of UAS1-B/CYC1--a new model for Cyp1p (Hap1p) DNA binding.

Cyp1p (Hap1p) is a yeast transcriptional regulator belonging to the zinc-cluster family. CGGNNNTANCGG was identified by PCR selection as the DNA sequence allowing its optimal binding. Nevertheless, this sequence is not a consensus sequence, the simultaneous presence of the two CGGs and the TA generally not being found in the known natural Cyp1p targets. In fact, our previous studies showed that the mechanism of Cyp1p DNA binding was target dependent. Data concerning the binding of Cyp1p to the UAS1-B/CYC1 are presented here. This target, containing the CGGGGTTTACGG sequence, was found to present the particular ability of stabilizing the binding of only one molecule of some monomeric Cyp1p fragments. This property was used to investigate the actual contribution of the TT and CGG sequences in the binding of Cyp1p. Our results indicate that each CGG belongs to a different half-site and, in contrast to a previous hypothesis, that the T nucleotide located four bases downstream from the left CGG is essential for the binding of one monomer to each half-site. The two half-sites of the UAS1-B/CYC1 thus overlap.

Genetics of transcriptional regulation in yeast: connections to the RNA polymerase II CTD.

Transcriptional regulation is important in all eukaryotic organisms for cell growth, development, and responses to environmental change. Saccharomyces cerevisiae, or bakers' yeast, has provided a powerful system for genetic analysis of transcriptional regulation, and findings from the study of this model system have proven broadly applicable to higher organisms. Transcriptional regulation requires the interactions of regulatory proteins with various components of the transcription machinery. Recently, genetic analysis of a diverse set of transcriptional regulatory responses has converged with studies of the function of the RNA polymerase II carboxy-terminal domain (CTD) to reveal regulatory roles for proteins associated with the CTD. These proteins, designated Srb/mediator proteins, are broadly involved in both positive and negative regulatory responses in vivo. This review focuses on the connections between genetic analysis of transcriptional regulation and the functions of the Srb/mediator proteins associated with the RNA polymerase II CTD.

Signaling activation and repression of RNA polymerase II transcription in yeast.

Activators of RNA polymerase II transcription possess distinct and separable DNA-binding and transcriptional activation domains. They are thought to function by binding to specific sites on DNA and interacting with proteins (transcription factors) binding near to the transcriptional start site of a gene. The ability of these proteins to activate transcription is a highly regulated process, with activation only occurring under specific conditions to ensure proper timing and levels of target gene expression. Such regulation modulates the ability of transcription factors either to bind DNA or to interact with the transcriptional machinery. Here we discuss recent advances in our understanding of these mechanisms of transcriptional regulation in yeast.

Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure.

Transcriptional regulation in yeast involves a number of general trans-acting factors affecting chromatin structure. The Swi-Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro. The Ssn6p-Tup1p repressor complex may be involved in chromatin organization through the interaction with pathway-specific DNA-binding proteins. To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild-type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations. We have mapped nucleosome positioning of the repressed gene in wild-type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state. In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p-Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter. Our data indicate the involvement of chromatin remodeling factors distinct from the Swi-Snf complex in SUC2 transcriptional regulation and suggest that Swi-Snf may antagonize Ssn6p-Tup1p by controlling remodeling activity. We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes.

Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities.

CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA-binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild-type CYP1 target sites. In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1-(1-200)-peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding. (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1-(1-200)-peptide is usually a monomer in solution but binds DNA as a dimer. Finally, we found evidence for the presence of two half-sites with different measured affinities in the asymmetric sequences of some CYP1 targets.

Modified Steroid Receptors and Steroid-Inducible Promoters as Genetic Switches for Gene Therapy

Steroids or steroid-like molecules are already in use in clinical protocols, and are therefore potential reagents for gene therapy. To be practical, steroid receptors must be converted into exquisitely specific signal transducers, a transcription factors with unique DNA-binding specificities that are activated by an otherwise physiologically inactive signal. Many of the required experimental precedents for the production of an appropriately modified steroid-inducible expression system are already in place. Steroid receptors can act as powerful and specific signal transducers by activation of synthetic promoters composed solely of hormone response elements and a TATA region in stably transfected cells. These “minimal” promoters are responsive to only one signal, and their degree of induction can be modulated by changing the number of hormone response elements in the promoter and/ or the concentration of hormone in the growth medium. There are several potential options available for modification, or indeed replacement, of the DNA-binding domains of steroid receptors. Many structure-function studies have been performed with chimeric nuclear receptors in which the receptor DNA-binding domain has been replaced by that of the yeast transactivator GAL4. GAL4 is a member of a large family of yeast transcriptional regulators that possess DNA-binding domains containing so-called zinc cluster motifs. It is well established that chimeric steroid receptors with altered DNA-binding specificities can activate genes in a ligand-dependent manner. The combination of all these results, along with the use of a receptor bearing a modified ligand-binding domain, may result in the engineering of a protein that acts as an exquisitely specific signal transducer.

A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p.

Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity matrix was used to isolate two nuclear proteins that copurified with histone deacetylase activity. Both proteins were identified by peptide microsequencing, and a complementary DNA encoding the histone deacetylase catalytic subunit (HD1) was cloned from a human Jurkat T cell library. As the predicted protein is very similar to the yeast transcriptional regulator Rpd3p, these results support a role for histone deacetylase as a key regulator of eukaryotic transcription.

酵母における転写制御機構の研究 高等真核生物のモデル系として

酵母における転写制御機構の研究 高等真核生物のモデル系として