Mark J. Swanson

The Yeast TAF145 Inhibitory Domain and TFIIA Competitively Bind to TATA-Binding Protein

ABSTRACT The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-binding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperature-sensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory domain is involved in TFIIA function.

A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p

ABSTRACT The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Δ cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Δ mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Δ strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator.

Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

Recruitment of SWI/SNF by Gcn4p Does Not Require Snf2p or Gcn5p but Depends Strongly on SWI/SNF Integrity, SRB Mediator, and SAGA

ABSTRACT The nucleosome remodeling complex SWI/SNF is a coactivator for yeast transcriptional activator Gcn4p. We provide strong evidence that Gcn4p recruits the entire SWI/SNF complex to its target genes ARG1 and SNZ1 but that SWI/SNF is dispensable for Gcn4p binding to these promoters. It was shown previously that Snf2p/Swi2p, Snf5p, and Swi1p interact directly with Gcn4p in vitro. However, we found that Snf2p is not required for recruitment of SWI/SNF by Gcn4p nor can Snf2p be recruited independently of other SWI/SNF subunits in vivo. Snf5p was not recruited as an isolated subunit but was required with Snf6p and Swi3p for optimal recruitment of other SWI/SNF subunits. The results suggest that Snf2p, Snf5p, and Swi1p are recruited only as subunits of intact SWI/SNF, a model consistent with the idea that Gcn4p makes multiple contacts with SWI/SNF in vivo. Interestingly, Swp73p is necessary for efficient SWI/SNF recruitment at SNZ1 but not at ARG1, indicating distinct subunit requirements for SWI/SNF recruitment at different genes. Optimal recruitment of SWI/SNF by Gcn4p also requires specific subunits of SRB mediator (Gal11p, Med2p, and Rox3p) and SAGA (Ada1p and Ada5p) but is independent of the histone acetyltransferase in SAGA, Gcn5p. We suggest that SWI/SNF recruitment is enhanced by cooperative interactions with subunits of SRB mediator and SAGA recruited by Gcn4p to the same promoter but is insensitive to histone H3 acetylation by Gcn5p.

Activator Gcn4p and Cyc8p/Tup1p Are Interdependent for Promoter Occupancy at ARG1 In Vivo

ABSTRACT The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

Disrupting Vesicular Trafficking at the Endosome Attenuates Transcriptional Activation by Gcn4

ABSTRACT The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that decreasing cargo proteins in the MVB through impaired delivery or enhanced degradation, and not merely the failure to transport cargo properly to the vacuole or downregulate plasma membrane proteins by endocytosis, is required to attenuate substantially transcriptional activation by Gcn4.

Telomere protein RAP1 levels are affected by cellular aging and oxidative stress

Telomeres are important for maintaining the integrity of the genome through the action of the shelterin complex. Previous studies indicted that the length of the telomere did not have an effect on the amount of the shelterin subunits; however, those experiments were performed using immortalized cells with stable telomere lengths. The interest of the present study was to observe how decreasing telomere lengths over successive generations would affect the shelterin subunits. As neonatal human dermal fibroblasts aged and their telomeres became shorter, the levels of the telomere-binding protein telomeric repeat factor 2 (TRF2) decreased significantly. By contrast, the levels of one of its binding partners, repressor/activator protein 1 (RAP1), decreased to a lesser extent than would be expected from the decrease in TRF2. Other subunits, TERF1-interacting nuclear factor 2 and protection of telomeres protein 1, remained stable. The decrease in RAP1 in the older cells occurred in the nuclear and cytoplasmic fractions. Hydrogen peroxide (H2O2) stress was used as an artificial means of aging in the cells, and this resulted in RAP1 levels decreasing, but the effect was only observed in the nuclear portion. Similar results were obtained using U251 glioblastoma cells treated with H2O2 or grown in serum-depleted medium. The present findings indicate that TRF2 and RAP1 levels decrease as fibroblasts naturally age. RAP1 remains more stable compared to TRF2. RAP1 also responds to oxidative stress, but the response is different to that observed in aging.

Identification of Genes in Saccharomyces cerevisiae that Are Haploinsufficient for Overcoming Amino Acid Starvation

The yeast Saccharomyces cerevisiae responds to amino acid deprivation by activating a pathway conserved in eukaryotes to overcome the starvation stress. We have screened the entire yeast heterozygous deletion collection to identify strains haploinsufficient for growth in the presence of sulfometuron methyl, which causes starvation for isoleucine and valine. We have discovered that cells devoid of MET15 are sensitive to sulfometuron methyl, and loss of heterozygosity at the MET15 locus can complicate screening the heterozygous deletion collection. We identified 138 cases of loss of heterozygosity in this screen. After eliminating the issues of the MET15 loss of heterozygosity, strains isolated from the collection were retested on sulfometuron methyl. To determine the general effect of the mutations for a starvation response, SMM-sensitive strains were tested for the ability to grow in the presence of canavanine, which induces arginine starvation, and strains that were MET15 were also tested for growth in the presence of ethionine, which causes methionine starvation. Many of the genes identified in our study were not previously identified as starvation-responsive genes, including a number of essential genes that are not easily screened in a systematic way. The genes identified span a broad range of biological functions, including many involved in some level of gene expression. Several unnamed proteins have also been identified, giving a clue as to possible functions of the encoded proteins.

Abstract 2261: Natural redistribution of end-protection proteins in aging cells as telomeres shorten

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Telomeres are the nucleoprotein structures at the ends of linear chromosomes. In mammalian cells, a six-member protein complex, called shelterin, protects telomeres from being recognized as DNA double-strand breaks and from nucleolytic degradation. It has been speculated that as telomeres become progressively shorter with each round of replication, the number of binding sites for the components of shelterin would decrease as well. In an attempt to determine whether there is a relationship between the length of telomeres and shelterin binding, primary human dermal fibroblasts from neonatal foreskin (HDFn) were grown in culture, and the levels of a number of shelterin components were measured over time. Unlike other previous studies where artificially constructed telomeres were used to address this question, we are following the natural progression of aging of a population of cultured cells. Comparisons were made between young cells at a population doubling (PDL) between 3 and 6 and aged cells at a PDL of 28 to 38. Southern blotting using a telomeric probe showed that the aged cells possessed significantly shorter telomeres than their younger counterparts, supporting the notion that there will be fewer shelterin binding sites. Using immunoblotting to compare the levels of several shelterin subunits (TRF2, hRAP1, and POT1), we found that TRF2, the DNA binding subunit that targets hRAP1 to the telomeres, and POT1 decrease in level as would be expected when fewer binding sites are present for the complex. Interestingly, the levels of hRAP1 did not decrease proportionally and remained relatively high compared to other subunits. These data suggest that as telomeres decrease in length and shelterin binding sites become less numerous, most shelterin subunits are degraded, but hRAP1 most likely plays an additional role. We have stressed young HDFn cells with hydrogen peroxide to age them prematurely. Upon treatment, the young cells (PDL of 8) became similar in appearance to the untreated, aged cells at PDL greater than 25. In the treated, young cells, hRAP1 levels in the nucleus decreased in a manner that was unaffected by inhibition of the proteasome, suggesting that during the oxidative stress, hRAP1 translocates from the nucleus to the cytoplasm. Taken together, our data suggest that RAP1 plays a role in stress signaling or response to cellular aging. Citation Format: Michelle E. Baribault, Mark J. Swanson, Nancy S. Bae. Natural redistribution of end-protection proteins in aging cells as telomeres shorten. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2261. doi:10.1158/1538-7445.AM2014-2261