Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jun;25(12):4863-72.
doi: 10.1128/MCB.25.12.4863-4872.2005.

Differential requirement of SAGA subunits for Mot1p and Taf1p recruitment in gene activation

Chris J C van Oevelen  1 , Hetty A A M van Teeffelen, H T Marc Timmers
Affiliations

Differential requirement of SAGA subunits for Mot1p and Taf1p recruitment in gene activation

Chris J C van Oevelen et al. Mol Cell Biol. 2005 Jun.

Abstract

Transcription activation in yeast (Saccharomyces cerevisiae) involves ordered recruitment of transcription factor complexes, such as TFIID, SAGA, and Mot1p. Previously, we showed that both Mot1p and Taf1p are recruited to the HXT2 and HXT4 genes, which encode hexose transporter proteins. Here, we show that SAGA also binds to the HXT2 and HXT4 promoters and plays a pivotal role in the recruitment of Mot1p and Taf1p. The deletion of either SPT3 or SPT8 reduces Mot1p binding to HXT2 and HXT4. Surprisingly, the deletion of GCN5 reduces Taf1p binding to both promoters. When GCN5 is deleted in spt3Delta or spt8Delta strains, neither Mot1p nor Taf1p binds, and this results in a diminished recruitment of TATA binding protein and polymerase II to the HXT4 but not the HXT2 promoter. This is reflected by the SAGA-dependent expression of HXT4. In contrast, SAGA-independent induction of HXT2 suggests a functional redundancy with other factors. A functional interplay of different SAGA subunits with Mot1p and Taf1p was supported by phenotypic analysis of MOT1 SAGA or TAF1/SAGA double mutant strains, which revealed novel genetic interactions between MOT1 and SPT8 and between TAF1 and GCN5. In conclusion, our data demonstrate functional links between SAGA, Mot1p, and TFIID in HXT gene regulation.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Transcription of HXT4 but not HXT2 is affected in SAGA mutant strains. (A) Kinetics of mRNA expression of HXT genes in wild-type, spt7Δ, and spt20Δ strains upon a shift to low glucose. Cells were grown in 4% glucose and shifted to 0.1% glucose for 0, 2, 5, 10, 20, and 30 min before collection (see Materials and Methods). RNA samples from wild-type, spt7Δ, and spt20Δ strains were processed in parallel and analyzed on a single Northern blot to ensure direct comparison of hybridization signals. HXT mRNAs were detected using oligonucleotide probes described previously (1, 17). ACT1 probes were used us an internal loading control. Data shown are representative of at least two independent experiments. (B and C) Kinetics of mRNA expression of the HXT2 gene (B) or the HXT4 gene (C) in various SAGA mutant strains. RNA samples from wild-type, ada2Δ, spt3Δ, gcn5Δ, spt8Δ, spt3Δ gcn5Δ, spt8Δ gcn5Δ, and spt3Δ spt8Δ strains were analyzed on single Northern blots as in panel A. Reprobing of the blots with the ACT1 probe verified equal mRNA loading (data not shown). (D) Quantification of HXT2 and HXT4 mRNAs. HXT hybridization signals were quantified by a PhosphorImager and are expressed relative to ACT1 mRNA signals.
FIG. 2.
FIG. 2.
SAGA specifically binds to the upstream region of both HXT2 and HXT4 genes. (A) HXT promoter structure and location of primers used to analyze Spt20p and Spt3 binding. Boxes indicate bind-ing sites for Mig1p (vertical stripes), Rgt1p (horizontal stripes), putative UAS (filled), and TBP (open) (39). Lines represent the DNA fragment, which was amplified in a multiplex PCR analysis. Numbers represent the exact location of HXT2 and HXT4 primers relative to the start site of the coding region. (B and C) Cells expressing a TAP-tagged version of Spt20p (B) or Spt3p (C) were subjected to a glucose shift, and cross-linking was initiated by addition of formaldehyde at the indicated time points. Input and immunoprecipitated DNA was analyzed by multiplex PCR with primers spanning the TATA box (TATA), putative UAS, or further upstream (UPS1) regions of the HXT genes as indicated. (D) Quantification of ChIP signals over the HXT2 and HXT4 genes. Radioactive signals of the indicated PCR fragments were quantified with a PhosphorImager. The signals are expressed relative to the POL1 signal, which was included as a negative control in the multiplex PCR analysis.
FIG. 3.
FIG. 3.
Binding kinetics of SAGA, Mot1p, Taf1p, TBP, and Pol II to HXT genes in SAGA and MOT1 mutant strains after a shift to low glucose. (A) Representative PCR and PhosphorImager quantification of Mot1p binding to HXT genes in wild-type (Wt), gcn5Δ, spt3Δ, and spt8Δ cells. The top panel shows multiplex PCR analysis in which POL1 primers have been included as a normalization control. Primers amplifying HXT2 and HXT4 are specific for the core promoter region as indicated for Fig. 2A. Each sample was analyzed at least in duplo. The lower panel displays the quantification. Factor binding is expressed as HXT/POL1 ratios, as described previously (1). (B) Analysis of Spt20p binding to HXT genes in a mot1-1 mutant strain after a shift to low glucose. (C) Taf1p binding to HXT in Wt, gcn5Δ, spt3Δ, and spt8Δ cells. (D) TBP and Pol II recruitment to HXT genes in Wt, gcn5Δ, spt3Δ, and spt8Δ strains.
FIG. 4.
FIG. 4.
Binding kinetics of Mot1p, Taf1p, TBP, and Pol II to HXT genes in SAGA double deletion strains. (A) Recruitment of Mot1p, Taf1p, TBP, and Pol II in gcn5Δ spt3Δ and gcn5Δ spt8Δ cells after a shift to low glucose. Multiplex PCR signals of immunoprecipitated DNA as indicated above the lanes are displayed for the TATA-containing fragments of HXT2 and HXT4 as described for Fig. 2A. (B) PhosphorImager quantification of Mot1p, Taf1p, TBP, and Pol II binding to the indicated HXT promoters.
FIG. 5.
FIG. 5.
Genetic interactions between SAGA, MOT1, and TAF1 during conditions of stress. (A) Wild-type (FHY58) and SAGA/TAF1 or (B) wild-type (W303) and SAGA/MOT1 mutant strains (as indicated) were serially diluted (10-fold steps) and grown on SC plus 2% glucose or SC plus 0.5% glucose. Cells were grown for 4 days at 33°C.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andrau, J.-C., C. J. C. van Oevelen, H. A. A. M. van Teeffelen, P. A. Weil, F. C. P. Holstege, and H. T. M. Timmers. 2002. Mot1 is essential for TBP recruitment to selected promoters during in vivo gene activation. EMBO J. 21:5173-5183. - PMC - PubMed
    1. Auble, D. T., K. E. Hansen, C. G. F. Mueller, W. S. Lane, J. Thorner, and S. Hahn. 1994. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8:1920-1934. - PubMed
    1. Bai, Y., G. M. Perez, J. M. Beechem, and P. A. Weil. 1997. Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130. Mol. Cell. Biol. 17:3081-3093. - PMC - PubMed
    1. Barbaric, S., H. Reinke, and W. Horz. 2003. Multiple mechanistically distinct functions of SAGA at the PHO5 promoter. Mol. Cell. Biol. 23:3468-3476. - PMC - PubMed
    1. Basehoar, A. D., S. J. Zanton, and B. F. Pugh. 2004. Identification and distinct regulation of yeast TATA box-containing genes. Cell 116:699-709. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources