Molecular Genetic Dissection of <i>TAF25</i>, an Essential Yeast Gene Encoding a Subunit Shared by TFIID and SAGA Multiprotein Transcription Factors

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The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF II s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF II -containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF II 25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF II 25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF II 25 or chimeras with the human homologue TAF II 30 arrested cell growth at either the G 1 or G 2 /M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF II 25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF II 25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF II 25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF II mutant allele reflects the full range of its normal functions.Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) requires the assembly of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) on promoters to form a preinitiation complex (31). Preinitiation complex assembly on both TATA-containing and TATA-less promoters is often nucleated by transcription factor TFIID, which comprises the TATA-binding protein (TBP) and 14 TBP-associated factors (TAF II s) (4,23,68). To date, most of the TFIID components from Saccharomyces cerevisiae, Drosophila, and humans have been identified, partially characterized, and shown to be well conserved throughout evolution (18,19,68).A large number of recent studies have provided a direct molecular link between histone acetylation and transcriptional activation (reviewed in references 11 and 40). It has been shown that several previously identified coactivators/adapters of transcription possess intrinsic histone acetyltransferase (HAT) activity or are associated in large multiprotein complexes with HATs (71). One of these, the 1.8-to 2-MDa S. cerevisiae SAGA (ySAGA) complex (26), comprises at least four distinct classes of gene products: (i) the Ada proteins (yAda1, yAda2, yAda3, yGcn5 [yAda4], and yAda5 [ySpt20]), which were isolated in a genetic screen for mutations that relieve the toxicity of the strong VP16 activation domain when overexpressed as a fusion with the Gal4 DNA-binding domain in yeast cells (5); (ii) Spt proteins (ySpt3, ySpt7, ySpt8, and ySpt20), initially identified as suppressors of transcription initiation defects caused by promoter insertions ...

Section: Discussionsupporting

confidence: 83%

Distinct Mutations in Yeast TAF_II25 Differentially Affect the Composition of TFIID and SAGA Complexes as Well as Global Gene Expression Patterns

Kirschner

Baur

Thibault

et al. 2002

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“…8B), further suggesting that only TAF9-or TAF9b-regulated genes exist and that the two proteins regulate only partially overlapping transcription programs. In agreement with several previous studies (11,20,27,28,32,53), our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up-and down-regulation of gene expression.…”

supporting

confidence: 82%

“…Our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up-and down-regulation of gene expression, further suggesting that TAFs can act as both positive and negative regulatory factors. This finding together with other TAF knockdown studies carried out mainly in yeast (11,20,27,28,32,53) raise an interesting hypothesis that TAFs, when binding to promoters in the different TAF-containing complexes (TFIID and TFTC type), would be involved at certain promoters as repressors and at others as positive cofactors of transcription.…”

Section: Discussionmentioning

confidence: 85%

TAF9b (Formerly TAF9L) Is a Bona Fide TAF That Has Unique and Overlapping Roles with TAF9

Frontini¹,

Soutoglou²,

Argentini

et al. 2005

TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process.Transcription initiation of protein-encoding genes by RNA polymerase II (Pol II) requires the transcription factor TFIID that is comprised of the TATA binding protein (TBP) and series of TBP-associated factors (TAFs) (1, 6, 57). In human HeLa cells, we showed that different human TFIID complexes containing or lacking TAF10 which exhibit functionally distinct properties exist (6,7,25). Cell type-specific TFIID complexes have been found to be composed of core TAFs and cell typespecific TAFs. TAF4b was found to be enriched in differentiated human B lymphocytes, and a unique TAF4b-containing TFIID was isolated from these cells (13). Moreover, during spermatogenesis, TAF7L-containing TFIID complexes have been found (48). Another set of human transcriptional regulatory multiprotein complexes containing TAFs are called TFTC, STAGA, or PCAF/GCN5 (6a, 36, 63). These complexes are functional homologues of the Saccharomyces cerevisiae SAGA complex, and all contain human homologues of the yeast histone acetyltransferase Gcn5 as well as a subset of SPT and ADA proteins, the 400-kDa TRRAP protein, and a number of TAFs (shared TAFs) also found in TFIID (36).TAF9 was first identified as a TFIID subunit from multiple organisms: human ( (44), and human TFTC-type complexes (10,36,67). Drosophila TAF9 has also been described in the Polycomb group complex and the e(y)2 protein-containing complex (17, 50).TAF9 was shown to interact directly with the tumor suppressor protein p53, the herpes simplex virus activator VP16, and the basal transcription factor TFIIB, and these interactions were suggested to be ...

“…The relevant background strains used in our studies are BY4741 (MATa ura3 leu2 his3 met15), BY4742 (MAT␣ ura3 leu2 his3 lys2), BY4743 (MATa/␣ ura3/ura3 leu2/leu2 his3/his3 lys2/LYS2 met15/MET15), BY4716 (MAT␣ lys2) (8), YSLS14 [MAT␣ ura3 ade2 trp1 his3 leu2 tbp(spt15)⌬::TRP1 pDP15-HA 1 -TBP(SPT15)], YSLS18 (MAT␣ ura3 ade2 trp1 his3 leu2 taf130⌬::TRP1 pRS313-HA 1 -TAF130) (61), and 21R (MATa ura3 leu2 ade1) (31). S. cerevisiae cell growth, manipulation, and epitope tagging were all performed as described before (33,61).…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of Saccharomyces cerevisiae TFIID

Sanders

Garbett

Weil

2002

Self Cite

100

127

We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.The general transcription factor TFIID plays a central role in the initiation of mRNA gene transcription. TFIID is the only general transcription factor with specific TATA box binding activity, and the expression of many RNA polymerase IItranscribed genes is dependent on TFIID function (1, 26). Saccharomyces cerevisiae TFIID is composed of 15 subunits, TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs) (61). The TBP subunit is responsible for the TATA box binding activity of TFIID, and TBP sequence and structure are both highly conserved throughout eukaryotic species. Like TBP, the TAF subunits of TFIID have also been highly conserved during eukaryotic evolution (6,22,66), and in aggregate, TAFs appear to serve multiple functions within the TFIID holocomplex. Early models, based on in vitro studies with individual recombinant subunits or subcomplexes of TFIID subunits, argued that TAFs functioned either as core promoter selectivity factors or as general coactivators, integrating signals between gene-specific trans-activating factors and the general transcription machinery (1). Although initially questioned, recent in vivo studies with S. cerevisiae and metazoans support both the coactivator and core promoter functions of TAFs within TFIID (1, 42, 59).Studies with numerous eukaryotic systems have yielded invaluable insights into the role of TFIID in the regulation of mRNA gene transcription. However, despite this large body of information regarding its functional properties, many fund...