Biomedical subjects
Publications and source records attributed to B Franklin Pugh.
There are many regulated steps in the assembly of a transcription preinitiation complex (PIC). In this issue of Molecular Cell, Black et. al. (2006) reveal a catalytic switch mechanism in which autoacetylation of the HAT p300 triggers its dissociation from a promoter in a manner that is coupled to TFIID association.
DNA sequence has long been recognized as an important contributor to nucleosome positioning, which has the potential to regulate access to genes. The extent to which the nucleosomal architecture at promoters is delineated by the underlying sequence is now being worked out. Here we use comparative genomics to report a genome-wide map of nucleosome positioning sequences (NPSs) located in the vicinity of all Saccharomyces cerevisiae genes. We find that the underlying DNA sequence provides a very good predictor of nucleosome locations that have been experimentally mapped to a small fraction of the genome. Notably, distinct classes of genes possess characteristic arrangements of NPSs that may be important for their regulation. In particular, genes that have a relatively compact NPS arrangement over the promoter region tend to have a TATA box buried in an NPS and tend to be highly regulated by chromatin modifying and remodeling factors.
Eukaryotic genes are controlled by sequence-specific DNA-binding proteins, chromatin regulators, general transcription factors, and elongation factors. Here we examine the genome-wide location of representative members of these groups and their redistribution when the Saccharomyces cerevisiae genome is reprogrammed by heat shock. As expected, assembly of active transcription complexes is coupled to eviction of H2A.Z nucleosomes, and disassembly is coupled to the return of nucleosomes. Remarkably, a large number of promoters assemble into partial preinitiation complexes (partial PICs), containing TFIIA, TFIID (and/or SAGA), TFIIB, TFIIE, and TFIIF. However, RNA polymerase II and TFIIH are generally not recruited, and nucleosomes are not displaced. These promoters may be preparing for additional stress that naturally accompany heat stress. For example, we find that oxidative stress, which often occurs with prolonged exposure of cells to high temperature, converts partial PICs into full PICs. Partial PICs therefore represent novel regulated intermediates that assemble at promoters in the midst of chromatin.
Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise approximately 90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes.
Histone acetylation regulates gene expression, yet the functional contributions of the numerous histone acetyltransferases (HATs) to gene expression and their relationships with each other remain largely unexplored. The central role of the putative HAT-containing TAF1 subunit of TFIID in gene expression raises the fundamental question as to what extent, if any, TAF1 contributes to acetylation in vivo and to what extent it is redundant with other HATs. Our findings herein do not support the basic tenet that TAF1 is a major HAT in Saccharomyces cerevisiae, nor do we find that TAF1 is functionally redundant with other HATs, including Gcn5, Elp3, Hat1, Hpa2, Sas3, and Esa1, which is in contrast to previous conclusions regarding Gcn5. Our findings do reveal that of these HATs, only Gcn5 and Esa1 contribute substantially to gene expression genome wide. Interestingly, histone acetylation at promoter regions throughout the genome does not require TAF1 or RNA polymerase II, indicating that most acetylation is likely to precede transcription and not depend upon it. TAF1 function has been linked to Bdf1, which binds TFIID and acetylated histone H4 tails, but no linkage between TAF1 and the H4 HAT Esa1 has been established. Here, we present evidence for such a linkage through Bdf1.
Organisms respond to heat stress by reprogramming gene expression. In Saccharomyces cerevisiae, heat-induced genes tend to be regulated by factors that belong to the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription regulatory pathway, whereas heat-repressed genes tend to be regulated by a parallel pathway involving transcription factor IID (TFIID). Here, we examine whether heat stress affects the occupancy of representative factors of each pathway at promoter regions throughout the yeast genome. Representatives of the SAGA pathway include the TATA binding protein, Spt3, and Mot1. Representatives of the TFIID pathway include the TATA binding protein, TAF1, and Bdf1. We find that heat stress causes disassembly of the TFIID pathway at genes that are inhibited by stress. In contrast, heat induces assembly of the SAGA pathway at stress-induced genes, although many also assemble along the TFIID pathway. Other genes were found to assemble almost exclusively along the TFIID pathway. Strikingly, these genes are lowly transcribed and are generally not induced. Thus, heat stress leads to factor assembly along each pathway but with distinct transcriptional outcomes. Further investigation of these pathways reveals that Bdf1 and Mot1 negatively regulate the SAGA pathway in different ways. The findings suggest that Bdf1 blocks assembly, whereas Mot1 promotes disassembly of the transcription machinery.
The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III. When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization. Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers. Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate. The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1. A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.
The TATA-binding protein (TBP) plays a central role in assembling eukaryotic transcription complexes and is subjected to extensive regulation including auto-inhibition of its DNA binding activity through dimerization. Previously, we have shown that mutations that disrupt TBP dimers in vitro have three detectable phenotypes in vivo, including decreased steady-state levels of the mutants, transcriptional derepression, and toxicity toward cell growth. In an effort to more precisely define the multimeric structure of TBP in vivo, the crystallographic dimer structure was used to design mutations that might enhance dimer stability. These mutations were found to enhance dimer stability in vitro and significantly suppress in vivo phenotypes arising from a dimer-destabilizing mutation. Although it is conceivable that phenotypes associated with dimer-destabilizing mutants could arise through defective interactions with other cellular factors, intragenic suppression of these phenotypes by mutations designed to stabilize dimers provides compelling evidence for a crystallographic dimer configuration in vivo.
Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions.
TFIID and SAGA share a common set of TAFs, regulate chromatin, and deliver TBP to promoters. Here we examine their relationship within the context of the Saccharomyces cerevisiae genome-wide regulatory network. We find that while TFIID and SAGA make overlapping contributions to the expression of all genes, TFIID function predominates at approximately 90% and SAGA at approximately 10% of the measurable genome. Strikingly, SAGA-dominated genes are largely stress induced and TAF independent, and are downregulated by the coordinate action of a variety of chromatin, TBP, and RNA polymerase II regulators. In contrast, the TFIID-dominated class is less regulated, but is highly dependent upon TAFs, including those shared between TFIID and SAGA. These two distinct modes of transcription regulation might reflect the need to balance inducible stress responses with the steady output of housekeeping genes.
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The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF(II)145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.
The TATA binding protein (TBP) is required for the expression of nearly all genes and is highly regulated both positively and negatively. Here, we use DNA microarrays to explore the genome-wide interplay of several TBP-interacting inhibitors in the yeast Saccharomyces cerevisiae. Our findings suggest the following: The NC2 inhibitor turns down, but not off, highly active genes. Autoinhibition of TBP through dimerization contributes to transcriptional repression, even at repressive subtelomeric regions. The TAND domain of TAF1 plays a primary inhibitory role at very few genes, but its function becomes widespread when other TBP interactions are compromised. These findings reveal that transcriptional output is limited in part by a collaboration of different combinations of TBP inhibitory mechanisms.