Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.
Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae. There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified. We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter. One of the high affinity Pho2 sites largely overlaps the Pho4 binding site at UASp1. Cooperative DNA binding of the two proteins to their overlapping sites, resulting in a high-affinity ternary complex, was demonstrated. Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site. Finally, Pho2 facilitates binding of Pho4 to a third, cryptic Pho4 binding site which binds Pho4 with lower affinity than UASp1 or UASp2. These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene.
All ribosomal protein (rp) gene promoters from Saccharomyces cerevisiae studied so far contain either (usually two) binding sites for the global gene regulator Rap1p or one binding site for another global factor, Abf1p. Previous analysis of the rpS33 and rpL45 gene promoters suggested that apart from the Abf1 binding site, additional cis-acting elements play a part in transcription activation of these genes. We designed a promoter reconstruction system based on the beta-glucuronidase reporter gene to examine the role of the Abf1 binding site and other putative cis-acting elements in promoting transcription. An isolated Abf1 binding site turned out to be a weak activating element. A T-rich sequence derived from the rpS33 proximal promoter was found to be stronger, but full transcription activation was only achieved by a combination of these elements. Both in the natural rpL45 promoter and in the reconstituted promoter, a Rap1 binding site could functionally replace the Abf1 binding site. Characteristic rp gene nutritional control of transcription, evoked by a carbon source upshift or by nitrogen re-feeding to nitrogen starved cells, could only be mediated by the combined Abf1 (or Rap1) binding site and T-rich element and not by the individual elements. These results demonstrate that Abf1p and Rap1p do not activate rp genes in a prototypical fashion, but rather may serve to potentiate transcription activation through the T-rich element.
The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection.
We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background) mutation rate, the selection coefficient, and the effective population size.
The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.
Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose. Rgt1 contains a Zn2Cys6 binuclear cluster responsible for DNA binding. Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG. However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence. We identified the Rgt1 consensus binding site sequence 5′-CGGANNA-3′, multiple copies of which are present in all HXT promoters regulated by Rgt1. Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription. We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression. This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability. Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions. These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation.
The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.
The short length and high degeneracy of sites recognized by DNA-binding transcription factors limit the amount of information they can carry, and individual sites are rarely sufficient to mediate the regulation of specific targets. Computational analysis of microbial genomes has suggested that many factors function optimally when in a particular orientation and position with respect to their target promoters. To investigate this further, we developed and trained spatial models of binding site positioning and applied them to the genome of the yeast Saccharomyces cerevisiae. We found evidence of non-random organization of sites within promoters, differences in binding site density, or both for thirty-eight transcription factors. We show that these signatures allow transcription factors with substantial differences in binding site specificity to share similar promoter specificities. We illustrate how spatial information dictating the positioning and density of binding sites can in principle increase the information available to the organism for differentiating a transcription factor’s true targets, and we indicate how this information could potentially be leveraged for the same purpose in bioinformatic analyses.
Global genome repair (GG-NER) removes DNA damage from non-transcribing DNA. In Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for GG-NER. We reported that autonomously replicating sequence-binding factor 1 (A BF1) protein forms a stable complex with Rad7 and Rad16 proteins. ABF1 functions in transcription, replication, gene silencing and NER in yeast. We show that binding of ABF1 to its DNA recognition sequence found at multiple genomic locations promotes efficient GG-NER in yeast. Mutation of the I silencer ABF1 binding site at the HMLα locus causes loss of ABF1 binding, which results in a domain of reduced GG-NER efficiency on one side of the ABF1 binding site. During GG-NER, nucleosome positioning at this site is not altered, and this correlates with an inability of the GG-NER complex to reposition nucleosomes in vitro. We discuss how the GG-NER complex might facilitate GG-NER, whilst preventing unregulated gene transcription during this process.
Transcription factors (TF) regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS), DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription.
We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO) classification) against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8000 human (and 23,000 mouse) genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1000 bp upstream the TSS) for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally.
We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central importance for regulation of transcription in human and mouse, just as it is in bacteria and yeast.
The large tumor antigen (TAg) of simian virus 40 regulates transcription of the viral genes. The early promoter is repressed when TAg binds to the origin and DNA replication begins, whereas the late promoter is activated by TAg through both replication-dependent and -independent mechanisms. Previously it was shown that activation is diminished when a site in the viral enhancer to which the factor TEF-1 binds is disrupted. We show here that the NH2-terminal region of TAg binds to the TEA domain of TEF-1, a DNA binding domain also found in the Drosophila scalloped and the Saccharomyces cerevisiae TEC1 proteins. The interaction inhibits DNA binding by TEF-1 and activates transcription in vitro from a subset of naturally occurring late start sites. These sites are also activated by mutations in the DNA motifs to which TEF-1 binds. Therefore, TEF-1 appears to function as a repressor of late transcription, and its involvement in the early-to-late shift in viral transcription is discussed. The mutation of Ser-189 in TAg, which reduces transformation efficiency in certain assays, disrupts the interaction with TEF-1. Thus, TEF-1 might also regulate genes involved in growth control.
This study presents the Yeast Promoter Atlas (YPA, http://ypa.ee.ncku.edu.tw/ or http://ypa.csbb.ntu.edu.tw/) database, which aims to collect comprehensive promoter features in Saccharomyces cerevisiae. YPA integrates nine kinds of promoter features including promoter sequences, genes’ transcription boundaries—transcription start sites (TSSs), five prime untranslated regions (5′-UTRs) and three prime untranslated regions (3′UTRs), TATA boxes, transcription factor binding sites (TFBSs), nucleosome occupancy, DNA bendability, transcription factor (TF) binding, TF knockout expression and TF–TF physical interaction. YPA is designed to present data in a unified manner as many important observations are revealed only when these promoter features are considered altogether. For example, DNA rigidity can prevent nucleosome packaging, thereby making TFBSs in the rigid DNA regions more accessible to TFs. Integrating nucleosome occupancy, DNA bendability, TF binding, TF knockout expression and TFBS data helps to identify which TFBS is actually functional. In YPA, various promoter features can be accessed in a centralized and organized platform. Researchers can easily view if the TFBSs in an interested promoter are occupied by nucleosomes or located in a rigid DNA segment and know if the expression of the downstream gene responds to the knockout of the corresponding TFs. Compared to other established yeast promoter databases, YPA collects not only TFBSs but also many other promoter features to help biologists study transcriptional regulation.
Originating from COMPEL, the TRANSCompel® database emphasizes the key role of specific interactions between transcription factors binding to their target sites providing specific features of gene regulation in a particular cellular content. Composite regulatory elements contain two closely situated binding sites for distinct transcription factors and represent minimal functional units providing combinatorial transcriptional regulation. Both specific factor–DNA and factor–factor interactions contribute to the function of composite elements (CEs). Information about the structure of known CEs and specific gene regulation achieved through such CEs appears to be extremely useful for promoter prediction, for gene function prediction and for applied gene engineering as well. Each database entry corresponds to an individual CE within a particular gene and contains information about two binding sites, two corresponding transcription factors and experiments confirming cooperative action between transcription factors. The COMPEL database, equipped with the search and browse tools, is available at http://www.gene-regulation.com/pub/databases.html#transcompel. Moreover, we have developed the program CATCH™ for searching potential CEs in DNA sequences. It is freely available as CompelPatternSearch at http://compel.bionet.nsc.ru/FunSite/CompelPatternSearch.html.
It is widely suspected that gene regulatory networks are highly plastic. The rapid turnover of transcription factor binding sites has been predicted on theoretical grounds and has been experimentally demonstrated in closely related species. We combined experimental approaches with comparative genomics to focus on the role of combinatorial control in the evolution of a large transcriptional circuit in the fungal lineage. Our study centers on Mcm1, a transcriptional regulator that, in combination with five cofactors, binds roughly 4% of the genes in Saccharomyces cerevisiae and regulates processes ranging from the cell-cycle to mating. In Kluyveromyces lactis and Candida albicans, two other hemiascomycetes, we find that the Mcm1 combinatorial circuits are substantially different. This massive rewiring of the Mcm1 circuitry has involved both substantial gain and loss of targets in ancient combinatorial circuits as well as the formation of new combinatorial interactions. We have dissected the gains and losses on the global level into subsets of functionally and temporally related changes. One particularly dramatic change is the acquisition of Mcm1 binding sites in close proximity to Rap1 binding sites at 70 ribosomal protein genes in the K. lactis lineage. Another intriguing and very recent gain occurs in the C. albicans lineage, where Mcm1 is found to bind in combination with the regulator Wor1 at many genes that function in processes associated with adaptation to the human host, including the white-opaque epigenetic switch. The large turnover of Mcm1 binding sites and the evolution of new Mcm1–cofactor interactions illuminate in sharp detail the rapid evolution of combinatorial transcription networks.
In explaining the diversity of organisms on Earth, it is increasingly evident that evolutionary changes in when and where genes are expressed provide a crucial source of variation. By using genome-wide transcription factor localization experiments in S. cerevisiae, K. lactis, and C. albicans, combined with comparative genomics across many more yeast species, we examined how a large combinatorial transcription circuit evolves over the course of hundreds of millions of years. Combinatorial regulation is pervasive in eukaryotic organisms and is thought to allow for increased specificity and integration of multiple signals in the control of gene expression. Our studies focused on one prolific combinatorial regulator, Mcm1, which, in combination with five cofactors, binds and regulates genes functioning in a diverse range of cellular processes in S. cerevisiae. We found evidence of massive network rewiring, including high rates of gain and loss of Mcm1 binding sites and the formation of new Mcm1–cofactor combinations and the breaking of old ones. We propose that the multiple protein–protein and protein–DNA interactions that specify transcription in combinatorial circuits allow for a richness of compensatory mutations and thereby provide ample opportunity for both adaptive and neutral evolution.
Experimental approaches combined with comparative genomics show how a large combinatorial transcription circuit has rewired over evolutionary time scales.
To know the map between transcription factors (TFs) and their binding sites is essential to reverse engineer the regulation process. Only about 10%–20% of the transcription factor binding motifs (TFBMs) have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory “DNA words.” From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%—far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of “DNA words,” newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.
For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.
Defects in human DNA mismatch repair have been reported to underlie a variety of hereditary and sporadic cancer cases. We characterized the structure of the MSH6 promoter region to examine the mechanisms of transcriptional regulation of the MSH6 gene. The 5′-flanking region of the MSH6 gene was found to contain seven functional Sp1 transcription factor binding sites that each bind Sp1 and Sp3 and contribute to promoter activity. Transcription did not appear to require a TATA box and resulted in multiple start sites, including two major start sites and at least nine minor start sites. Three common polymorphisms were identified in the promoter region (−557 T→G, −448 G→A, and −159 C→T): the latter two were always associated, and each of these functionally inactivated a different Sp1 site. The polymorphic allele −448 A −159 T was demonstrated to be a common Caucasian polymorphism found in 16% of Caucasians and resulted in a five-Sp1-site promoter that had 50% less promoter activity and was more sensitive to inactivation by DNA methylation than the more common seven Sp1 site promoter allele, which was only partially inactivated by DNA methylation. In cell lines, this five-Sp1-site polymorphism resulted in reduced MSH6 expression at both the mRNA and protein level. An additional 2% of Caucasians contained another polymorphism, −210 C→T, which inactivated a single Sp1 site that also contributes to promoter activity.
The mouse thymidylate synthase (TS) promoter lacks a TATAA box and directs transcriptional initiation at multiple sites over a 60 nucleotide region. All of the sequences that are important for transcription are located within close proximity to the first initiation site. Gel mobility shift and footprinting analyses with various sequences from this region of the TS promoter identified three major protein-DNA interactions. One of these corresponds to Sp1 interacting with a nonconsensus binding site downstream of the first transcriptional initiation site. Inactivation of the binding site by site-directed mutagenesis led to a 3-fold reduction in gene expression as well as a significant change in distribution of transcriptional start sites. The proteins responsible for the other two complexes (CII and CIII) do not appear to correspond to any of the common transcription factors that have been studied previously. The CII protein binds very close to the first initiation site. Inactivation of the binding site by site-directed mutagenesis had little effect on expression. The CIII protein binds immediately upstream of CII. Inactivation of this site led to a 12-fold reduction in expression, indicating that it is important for expression of the TS gene.
Combinatorial regulation by transcription factor complexes is an important feature of eukaryotic gene regulation. Here, we propose a new method for identification of interactions between transcription factors (TFs) that relies on the relationship of their binding sites, and we test it using Saccharomyces cerevisiae as a model system. The algorithm predicts interacting TF pairs based on the co-occurrence of their binding motifs and the distance between the motifs in promoter sequences. This allows investigation of interactions between TFs without known binding motifs or expression data. With this approach, 300 significant interactions involving 77 TFs were identified. These included more than 70% of the known protein–protein interactions. Approximately half of the detected interacting motif pairs showed strong preferences for particular distances and orientations in the promoter sequences. These one dimensional features may reflect constraints on allowable spatial arrangements for protein–protein interactions. Evidence for biological relevance of the observed characteristic distances is provided by the finding that target genes with the same characteristic distances show significantly higher co-expression than those without preferred distances. Furthermore, the observed interactions were dynamic: most of the TF pairs were not constitutively active, but rather showed variable activity depending on the physiological condition of the cells. Interestingly, some TF pairs active in multiple conditions showed preferences for different distances and orientations depending on the condition. Our prediction and characterization of TF interactions may help to understand the transcriptional regulatory networks in eukaryotic systems.
Divergence of transcription factor binding sites is considered to be an important source of regulatory evolution. The associations between transcription factor binding sites and phenotypic diversity have been investigated in many model organisms. However, the understanding of other factors that contribute to it is still limited. Recent studies have elucidated the effect of chromatin structure on molecular evolution of genomic DNA. Though the profound impact of nucleosome positions on gene regulation has been reported, their influence on transcriptional evolution is still less explored. With the availability of genome-wide nucleosome map in yeast species, it is thus desirable to investigate their impact on transcription factor binding site evolution. Here, we present a comprehensive analysis of the role of nucleosome positioning in the evolution of transcription factor binding sites.
We compared the transcription factor binding site frequency in nucleosome occupied regions and nucleosome depleted regions in promoters of old (orthologs among Saccharomycetaceae) and young (Saccharomyces specific) genes; and in duplicate gene pairs. We demonstrated that nucleosome occupied regions accommodate greater binding site variations than nucleosome depleted regions in young genes and in duplicate genes. This finding was confirmed by measuring the difference in evolutionary rates of binding sites in sensu stricto yeasts at nucleosome occupied regions and nucleosome depleted regions. The binding sites at nucleosome occupied regions exhibited a consistently higher evolution rate than those at nucleosome depleted regions, corroborating the difference in the selection constraints at the two regions. Finally, through site-directed mutagenesis experiment, we found that binding site gain or loss events at nucleosome depleted regions may cause more expression differences than those in nucleosome occupied regions.
Our study indicates the existence of different selection constraint on binding sites at nucleosome occupied regions than at the nucleosome depleted regions. We found that the binding sites have a different rate of evolution at nucleosome occupied and depleted regions. Finally, using transcription factor binding site-directed mutagenesis experiment, we confirmed the difference in the impact of binding site changes on expression at these regions. Thus, our work demonstrates the importance of composite analysis of chromatin and transcriptional evolution.
The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.
The spatial organization of transcription factor binding sites in regulatory DNA, and the composition of intersite sequences, influences the assembly of the multiprotein complexes that regulate RNA polymerase recruitment and thereby affects transcription. We have developed a genetic approach to investigate how reporter gene transcription is affected by varying the spacing between transcription factor binding sites. We characterized the components of promoter architecture that govern the yeast transcription factors Cbf1 and Met31/32, which bind independently, but collaboratively recruit the coactivator Met4.
A Cbf1 binding site was required upstream of a Met31/32 binding site for full reporter gene expression. Distance constraints on coactivator recruitment were more flexible than those for cooperatively binding transcription factors. Distances from 18 to 50 bp between binding sites support efficient recruitment of Met4, with only slight modulation by helical phasing. Intriguingly, we found that certain sequences located between the binding sites abolished gene expression.
These results yield insight to the influence of both binding site architecture and local DNA flexibility on gene expression, and can be used to refine computational predictions of gene expression from promoter sequences. In addition, our approach can be applied to survey promoter architecture requirements for arbitrary combinations of transcription factor binding sites.
MicroRNAs of the miR-302 cluster are involved in early embryonic development and somatic cell reprogramming. Expression of the miR-302 gene is regulated by the binding of the pluripotency factors Oct4, Sox2 and Nanog to the miR-302 promoter. The specific expression pattern of the miR-302 gene suggested that additional transcription factors might be involved in its regulation. Here, we show that the miR-302 promoter is a direct target of the Wnt/β-catenin signaling pathway. We found that the miR-302 promoter contains three different functional Tcf/Lef binding sites. Two of the three sites were located within the cluster of Oct4/Sox2/Nanog binding sites and were essential for Wnt/β-catenin-mediated regulation of the miR-302 gene. Tcf3, the only Tcf/Lef factor that bound to the miR-302 promoter, acted as a repressor of miR-302 transcription. Interestingly, mutations in the two Tcf/Lef binding sites and the Oct4/Nanog binding sites abolished miR-302 promoter responsiveness to Wnt signaling, suggesting that the Tcf/Lef and the Oct4/Nanog sites interact genetically.
The CCAAT-binding factor is an evolutionarily conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites within the promoters of numerous eukaryotic genes. The CCAAT-binding factor from Saccharomyces cerevisiae is a heterotetramer that contains the subunits Hap2p, Hap3p, Hap4p, and Hap5p and that functions in the activation of genes involved in respiratory metabolism. Here we describe the isolation of the cDNA encoding the Schizosaccharomyces pombe homolog of Hap5p, designated php5+. We have shown that Php5p is a subunit of the CCAAT-binding factor in fission yeast and is required for transcription of the S. pombe cyc1+ gene. Analysis of the evolutionarily conserved regions of Hap5p, Php5p, and the mammalian homolog CBF-C revealed two essential domains within Hap5p that are required for DNA binding and transcriptional activation. One is an 87-amino-acid core domain that is conserved among Hap5p, Php5p, and CBF-C and that is required for the assembly of the Hap2p-Hap3p-Hap5p heterotrimer both in vitro and in vivo. A second domain that is essential for the recruitment of Hap4p into the CCAAT-binding complex was identified in Hap5p and Php5p.
The human and murine MOK2 proteins are factors able to recognize both DNA and RNA through their zinc finger motifs. This dual affinity of MOK2 suggests that MOK2 might be involved in transcription and post-transcriptional regulation of MOK2 target genes. The IRBP gene contains two MOK2-binding elements, a complete 18 bp MOK2-binding site located in intron 2 and the essential core MOK2-binding site (8 bp of conserved 3′-half-site) located in the IRBP promoter. We have demonstrated that MOK2 can bind to the 8 bp present in the IRBP promoter and repress transcription from this promoter by competing with the CRX activator for DNA binding. In this study, we identify a novel interaction between lamin A/C and hsMOK2 by using the yeast two-hybrid system. The interaction, which was confirmed by GST pull-down assays and co-immunolocalization studies in vivo, requires the N-terminal acidic domain of hsMOK2 and the coiled 2 domain of lamin A/C. Furthermore, we show that a fraction of hsMOK2 protein is associated with the nuclear matrix. We therefore suggest that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription.
Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.