Mutations In TATA-binding Protein Research Articles

Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism.

Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96 h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism.

Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome

The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP–BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells.

TATA-binding protein mutants that are lethal in the absence of the Nhp6 high-mobility-group protein.

The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p.

Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex.

The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes. In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant. In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

Role of the TATA binding protein-transcription factor IIB interaction in supporting basal and activated transcription in plant cells.

The TATA binding protein (TBP) and transcription factor IIB (TFIIB) play crucial roles in transcription of class II genes. The requirement for TBP-TFIIB interactions was evaluated in maize cells by introducing mutations into the Arabidopsis TBP (AtTBP2) within the C-terminal stirrup. Protein binding experiments indicated that amino acid residues E-144 and E-146 of AtTBP2 are both essential for TFIIB binding in vitro. Activation domains derived from herpes simplex viral protein VP16, the Drosophila fushi tarazu glutamine-rich domain (ftzQ), and yeast Gal4 were tested in transient assays. TBP-TFIIB interactions were dispensable for basal transcription but were required for activated transcription. In general, activated transcription was more severely inhibited by TBP mutation E-146R than by mutation E-144R. However, these TBP mutations had little effect on activity of the full-length cauliflower mosaic virus 35S and maize ubiquitin promoters, thus demonstrating that strong TBP-TFIIB contacts are not always required for transcription driven by complex promoters.

Architecture of protein and DNA contacts within the TFIIIB-DNA complex.

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.

An RNA polymerase III-defective mutation in TATA-binding protein disrupts its interaction with a transcription factor IIIB subunit in drosophila cells.

A subunit of the Drosophila RNA polymerase III transcription factor IIIB (TFIIIB) complex has been identified using antibodies directed against the analogous human protein, hIIIB90. This protein has an apparent molecular mass of 105 kDa and has been designated dTAFIII105. Drosophila S-2 cell extracts that were immunodepleted of dTAFIII105 were substantially reduced in their capacity to support tRNA gene transcription. A protein (far Western) blot analysis revealed that dTAFIII105, present in a TFIIIB fraction, directly interacts with TATA-binding protein (TBP). Coimmunoprecipitation assays demonstrated that this protein associates with TBP in S-2 cell extracts. Our previous studies have identified a mutation at position 332 within Drosophila TBP that changes a highly conserved arginine residue to a histidine residue, which renders it specifically defective in its ability to support RNA polymerase III transcription in S-2 cells (Trivedi, A., Vilalta, A., Gopalan, S., and Johnson, D. L. (1996) Mol. Cell. Biol. 16, 6909-6916). We further demonstrate that extracts prepared from a stable cell line expressing epitope-tagged wild-type TBP exhibit an increase in tRNA gene transcription, whereas extracts derived from cells expressing the mutant TBP protein do not. Coimmunoprecipitation assays and far Western blot analysis demonstrate that this mutation in TBP abolishes its ability to stably interact with dTAFIII105. Thus, we have identified both a Drosophila protein that is directly associated with TBP in the TFIIIB complex, dTAFIII105, and an amino acid residue within the highly conserved carboxyl-terminal region of TBP that is critical for dTAFIII105-TBP interactions.

A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB

The TDS4 gene of S. cerevisiae was isolated as an allele-specific high copy suppressor of mutations within the basic region of the TATA-binding protein (TBP). The gene is essential for viability and encodes a 596 aa protein. The first 300 aa of the TDS4 protein exhibit significant sequence similarity to the RNA polymerase II transcription factor TFIIB. However, TDS4 is required for RNA polymerase III transcription in vivo and in vitro. Antibodies specific for TDS4 or TBP react with the TFIIIB complex, indicating that both proteins are components of the RNA polymerase III initiation complex. These findings suggest that the RNA polymerase II and III initiation mechanisms are extremely similar, and they explain how the TATA-binding protein can function in both systems.