Differential Requirement of SAGA Components for Recruitment of TATA-Box-Binding Protein to Promoters In Vivo

Abstract

The multisubunit Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is required to activate transcription of a subset of RNA polymerase II-dependent genes. However, the contribution of each SAGA component to transcription activation is relatively unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay, we have systematically analyzed the role of SAGA components in the recruitment of TATA-box binding protein (TBP) to SAGA-dependent promoters. We show that recruitment of TBP is diminished at a number of SAGA-dependent promoters in ada1⌬, spt7⌬, and spt20⌬ null mutants, consistent with previous biochemical data suggesting that these components maintain the integrity of the SAGA complex. We also find that Spt3p is generally required for TBP binding to SAGAdependent promoters, consistent with biochemical and genetic experiments, suggesting that Spt3p interacts with and recruits TBP to the core promoter. By contrast, Spt8p, which has been proposed to be required for the interaction between Spt3p and TBP, is required for TBP binding at only a subset of SAGA-dependent promoters. Ada2p and Ada3p are both required for TBP recruitment to Gcn5p-dependent promoters, supporting previous biochemical data that Ada2p and Ada3p are required for the histone acetyltransferase activity of Gcn5p. Finally, our results suggest that TBP-associated-factor components of SAGA are differentially required for TBP binding to SAGA-dependent promoters. In summary, we show that SAGA-dependent promoters require different combinations of SAGA components for TBP recruitment, revealing a complex combinatorial network for transcription activation in vivo.

Actively Transcribed GAL Genes Can Be Physically Linked to the Nuclear Pore by the SAGA Chromatin Modifying Complex

“…Standard deviations in the data are indicated by the error bars. not required to link actively transcribed genes to the nuclear pore, which is consistent with previous reports that Gcn5 is not required for expression of the GAL genes (23,24), integrity of the SAGA complex (43), or visual detection of the GAL genes at the nuclear periphery (9).…”

“…To disrupt the SAGA complex, we deleted the SPT7 gene, which encodes a protein required for the structural integrity of the SAGA complex (37). However, disruption of the SAGA complex due to deletion of the SPT7 gene also results in a severe slow-growth phenotype when galactose is the sole carbon source (37) and decreased recruitment of TBP to the GAL1 promoter (23), suggesting that transcription of the GAL loci might be decreased in spt7⌬ cells. Thus a decreased association between the GAL loci and the Mlp proteins in spt7⌬ cells could be due to either the physical absence of the SAGA complex at the GAL UAS or to a decrease in transcription of the GAL genes.…”

“…The SAGA complex then facilitates recruitment of the general transcription factors and RNA polymerase II (27). As the SAGA complex regulates transcription of the GAL genes (23)(24)(25), and the GAL genes relocalize to the nuclear periphery upon transcriptional activation (5), we hypothesized that a physical interaction between Mlp proteins and the SAGA complex might mediate the recruitment of the GAL genes to the NPC. We verified the interaction between the Mlp proteins and SAGA components, and we found that both Mlp1 and Mlp2 interact with the GAL UAS, the region of the GAL genes that interacts with the SAGA complex.…”

“…With this approach, we identified three components of the evolutionarily conserved SAGA complex, a histone acetyltransferase complex that regulates transcription of ϳ10% of the yeast genome (21,22), including the GAL genes (23)(24)(25). SAGA interacts with genespecific transcriptional activators that recruit SAGA and additional transcription machinery to the promoters of target genes (26).…”

The 19 S Proteasome Subcomplex Establishes a Specific Protein Interaction Network at the Promoter for Stimulated Transcriptional Initiation in Vivo

“…To compare the PCR signal arising from the immunoprecipitated DNA with that of the input DNA, 1 l of input DNA was used in the PCR analysis. Serial dilutions of the input and immunoprecipitated (IP) DNAs were used to assess the linear range of PCR amplification as described previously (66). The PCR data presented in this article are within the linear range of PCR analysis.…”