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

Abstract

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 ...

Functional Analysis of the TFIID-specific Yeast TAF4 (yTAFII48) Reveals an Unexpected Organization of Its Histone-fold Domain

“…The presence of an extended L2 loop within the HFD of yTAF10 and yTAF11 has previously been noted (30,33,40). However, in contrast to yTAF4, these loops are not present in the metazoan orthologues and can be deleted in the yeast proteins without loss of function.…”

New Insights into TAFs as Regulators of Cell Cycle and Signalling Pathways

“…Indeed, different TS mutants of a given TAF can elicit distinct effects on gene expression and arrest at either of these checkpoints. 22,23 Genetic studies in vertebrate cells also point to an essential role of TAFs in cell cycle progression. This was first revealed when it was discovered that the ccg1 gene encodes TAF1.…”

Genome-wide Transcriptional Dependence on TAF1 Functional Domains

“…Inasmuch as different TFIID subunits and different portions of TAF5 and TAF10 have been ascribed a gene-specific function (10,11,13,14), we sought to examine, using microarray analysis, whether the four functional domains make gene-specific contributions to gene expression on a genome-wide scale. To minimize potential indirect effects where the impact on the expression of certain genes like transcriptional regulators alters the expression of a large number of other genes, we sought a means of rapidly replacing the functional copy of TAF1.…”

“…This suggests that distinct parts of TFIID might play important promoter-specific roles. Similarly, a variety of temperature-sensitive alleles located throughout TAF10 reveal potential promoter-selective roles for distinct regions of a single TAF (11).…”