A TATA binding protein regulatory network that governs transcription complex assembly

Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Genome biology (Impact Factor: 10.81). 02/2007; 8(4):R46. DOI: 10.1186/gb-2007-8-4-r46
Source: PubMed


Eukaryotic genes are controlled by proteins that assemble stepwise into a transcription complex. How the individual biochemically defined assembly steps are coordinated and applied throughout a genome is largely unknown. Here, we model and experimentally test a portion of the assembly process involving the regulation of the TATA binding protein (TBP) throughout the yeast genome.
Biochemical knowledge was used to formulate a series of coupled TBP regulatory reactions involving TFIID, SAGA, NC2, Mot1, and promoter DNA. The reactions were then linked to basic segments of the transcription cycle and modeled computationally. A single framework was employed, allowing the contribution of specific steps to vary from gene to gene. Promoter binding and transcriptional output were measured genome-wide using ChIP-chip and expression microarray assays. Mutagenesis was used to test the framework by shutting down specific parts of the network.
The model accounts for the regulation of TBP at most transcriptionally active promoters and provides a conceptual tool for interpreting genome-wide data sets. The findings further demonstrate the interconnections of TBP regulation on a genome-wide scale.

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    • "GCN5 has been extensively characterized as a global regulator of gene expression [36], [47], [50], [51]. Is it then possible that all effects we have observed are indirect via deregulated gene expression? "
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    ABSTRACT: GCN5 encodes one of the non-essential Histone Acetyl Transferases in Saccharomyces cerevisiae. Extensive evidence has indicated that GCN5 is a key regulator of gene expression and could also be involved in transcriptional elongation, DNA repair and centromere maintenance. Here we show that the deletion of GCN5 decreases the stability of mini-chromosomes; that the tethering of Gcn5p to a crippled origin of replication stimulates its activity; that high dosage of GCN5 suppresses conditional phenotypes caused by mutant alleles of bona fide replication factors, orc2-1, orc5-1 and mcm5-461. Furthermore, Gcn5p physically associates with origins of DNA replication, while its deletion leads to localized condensation of chromatin at origins. Finally, Deltagcn5 cells display a deficiency in the assembly of pre-replicative complexes. We propose that GCN5 acts as a positive regulator of DNA replication by counteracting the inhibitory effect of Histone Deacetylases.
    PLoS ONE 01/2010; 5(1):e8964. DOI:10.1371/journal.pone.0008964 · 3.23 Impact Factor
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    • "This is somewhat paradoxical because the expression of RPGs is strongly dependent on TFIID but only modestly dependent on SAGA (9,12,13). However, genome-wide ChIP (chromatin immunoprecipitation)-chip analysis combined with computer modeling has revealed that RPGs exhibit the highest occupancy levels of Spt3, the TBP-delivering subunit of SAGA (18–20). Furthermore, it has also been shown that heat stress can induce transcription of many genes through recruitment of both TFIID and SAGA (18). "
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    ABSTRACT: In Saccharomyces cerevisiae, TFIID and SAGA principally mediate transcription of constitutive housekeeping genes and stress-inducible genes, respectively, by delivering TBP to the core promoter. Both are multi-protein complexes composed of 15 and 20 subunits, respectively, five of which are common and which may constitute a core sub-module in each complex. Although genome-wide gene expression studies have been conducted extensively in several TFIID and/or SAGA mutants, there are only a limited number of studies investigating genome-wide localization of the components of these two complexes. Specifically, there are no previous reports on localization of a complete set of Tafs and the effects of taf mutations on localization. Here, we examine the localization profiles of a complete set of Tafs, Gcn5, Bur6/Ncb2, Sua7, Tfa2, Tfg1, Tfb3 and Rpb1, on chromosomes III, IV and V by chromatin immunoprecipitation (ChIP)-chip analysis in wild-type and taf1-T657K mutant strains. In addition, we conducted conventional and sequential ChIP analysis of several ribosomal protein genes (RPGs) and non-RPGs. Intriguingly, the results revealed a novel relationship between TFIIB and NC2, simultaneous co-localization of SAGA and TFIID on RPG promoters, specific effects of taf1 mutation on Taf2 occupancy, and an indirect evidence for the existence of different TFIID conformations.
    Nucleic Acids Research 12/2009; 38(6):1805-20. DOI:10.1093/nar/gkp1172 · 9.11 Impact Factor
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    • "The dominant role of NC2 on TFIIB in vivo appears to be a positive role, and this seems to be determined by the promoters of the target genes, as we determined for one such gene, RPS14b. In particular, TFIIB association with all strongly expressed genes that we looked at requires NC2, in accordance with recent reports suggesting that NC2 is generally activating ribosomal protein (RP) genes (24), but NC2 also has a positive effect on TFIIB recruitment to some lowly expressed genes such as HSP26 or DAN1. Factors which assemble after TFIIB in the PIC, namely TFIIE, TFIIH as well as RNA polymerase II, generally also decrease in NC2 mutants. "
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    ABSTRACT: Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP.
    Nucleic Acids Research 03/2008; 36(2):539-49. DOI:10.1093/nar/gkm1078 · 9.11 Impact Factor
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