1. Volume 9, Issue 6, Pages 1297-1305 (June 2002)
Cti6, a PHD Domain Protein, Bridges the Cyc8-Tup1 Corepressor and the SAGA Coactivator to Overcome Repression at GAL1 
Manolis Papamichos-Chronakis, Theodoros Petrakis, Eleni Ktistaki, Irini Topalidou, Dimitris Tzamarias 
Molecular Cell 
Volume 9, Issue 6, Pages (June 2002)
DOI: /S (02)

2. Figure 1 Cyc8-Tup1 Is Permanently Tethered at Target Promoters In Vivo
(A) Upper panel: HA-Cyc8 (left), HA-Tup1 (middle), and Tup1 (right) occupancy of GAL1URS and GAL1ORF in the presence of glucose (Glu) or galactose (Gal). Chromatin-IP was performed in parallel with RNA analysis in the wild-type strain (for a-Tup1) and in the yeast mutants (cyc8Δ or tup1Δ) expressing functional HA-tagged Cyc8 or Tup1 proteins, respectively. Cells were grown in 2% glucose or 2% galactose for 3 hr. HA-specific antibody, rabbit anti-Tup1 polyclonal antiserum, and primers specific for GAL1URS and GAL1ORF were used. Lower panel: HA-Cyc8, HA-Tup1, and Tup1 occupancy of SUC2URS and SUC2ORF in the presence of 2% glucose or in low glucose (0.05%, indicated “L-Gl”) for 3 hr. Numbers indicate the ratios between the immunoprecipitated and the input PCR products (see Experimental Procedures).
(B) Immunoblotting analysis of HA-tagged Cyc8 or Tup1 protein expressed in cyc8Δ or tup1Δ cells, respectively, in the presence of glucose or galactose. Total protein amounts were normalized by ponceau staining. The proteolytic product of Cyc8 is present in most yeast protein preparations.
(C) Upper panel: HA-Tup1 occupancy of the ANB1 promoter in cells grown aerobically (+O2) or anaerobically (−O2) for 4 hr for induction of ANB1. Lower panel: HA-Tup1 occupancy of HSP26 and GAL1 in cells grown at 30°C or 38°C for 30 min for HSP26 induction. Chromatin-IP was performed as in (A) with primers specific for ANB1, HSP26, GAL1 URS, and POL1 ORF as a background control.
(D) Serial dilutions of DNA obtained from chromatin-IP served as template for GAL1URS amplification, demonstrating the linearity of the PCR assay.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2
The PHD Domain Protein Cti6 Is Required for Activation of GAL1 Transcription
(A) The Cti6 protein sequence (506 amino acid long) is indicated along with regions comprising the PHD Zn finger and the Cyc8-interacting domain (CTI). Conserved PHD residues are in bold.
(B) Activation defect of GAL1 transcription in cti6Δ mutants. Wild-type and cti6Δ cells were grown in glucose (Gluc.) or in galactose for 1, 2, and 4 hr. Total RNA was extracted, transferred to nylon membrane, and hybridized with probes specific for GAL1 and actin sequences.
(C) Wild-type and cti6Δ cells were grown in normal (NO2) or low (LO2) oxygen for 3 hr, and total RNA was hybridized with a ANB1-specific probe. The nonregulated transcript 1 (Tr1) cross-hybridizes with ANB1 and serves as a loading control.
(D) cyc8Δ and gal80Δ single mutants, as well as cyc8Δ,gal80Δ double mutants, were grown in YP-glucose medium and harvested in early exponential phase (OD ≅ 0.6). Total RNA was extracted and hybridized with probes specific for GAL1 and actin sequences.
(E) GAL1 mRNA analysis in cyc8Δ single and cyc8Δ,cti6Δ double mutants, as described in (A).
(F) GAL1 mRNA analysis in gal80Δ single and gal80Δ,cti6Δ double mutants performed as in (A) and (D), except that cell cultures were harvested 1 and 2 hr following galactose induction.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3 Cti6 Is Physically Present at the GAL1 Promoter
(A) Cti6 occupancy of the GAL1 promoter in wild-type and cyc8Δ mutant cells. Chromatin-IP was performed in wild-type and cyc8Δ yeast strains expressing HA-Cti6 protein using HA-specific antibody and primers specific for GAL1 UAS or ORF sequences. Cells were grown in glucose (Glu) or galactose (Gal). Numbers indicate the ratios between IP and input PCR products.
(B) Cyc8-Cti6 interaction in vivo. Immunoblotting of total input (In) and sepharose-bound fraction (B) with HA-specific antibody and protein extracts obtained from yeast cells expressing HA-Cti6 along with GST or GST-Cyc8 (G-Cyc8) hybrid. Input lane contains the 1:150th of the protein loaded at the sepharose beads. The amount of GST present in yeast extracts was significantly higher than that of GST-Cyc8 (data not shown).
(C) Cyc8-Cti6 interaction in vitro. Western blotting with anti-His antibody of histidine-tagged Cyc8 (His-Cyc8) bound to glutathione sepharose beads containing either GST or GST-Cti6 (G-Cti6) hybrid proteins. Lane labeled “input” contains 20% of the protein amount incubated with the sepharose beads.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4
Gcn5-Dependent Histone Acetylation, TBP Recruitment, and Gal4 Binding at the GAL1 Promoter
(A) Gal4 binding at the GAL1 promoter. Chromatin-IP was conducted in wild-type or cti6Δ mutants with antibodies specific for the Gal4 DNA binding domain and primers for the UASG DNA sequence. Yeast cells were grown in YP-glucose and washed and cultured in YP-galactose for 2 hr. Primers specific for an internal region of POL1 were used to amplify the same DNA samples as a negative control. Numbers indicate the ratios between IP and input PCR products.
(B) Gcn5 occupancy of the GAL1 promoter. Upper panel: chromatin-IP was performed in wild-type and cti6Δ cells expressing HA-tagged Gcn5, using HA-specific antibody and UASG or POL1ORF specific primers. Cells were treated as in (A). Lower panel: chromatin-IP was performed in the strains described above with primers specific for TRP3 UAS and ORF sequences. Cells were grown in SC media to induce TRP3 transcription.
(C) H3 histone acetylation in cti6Δ mutants. Chromatin-IP was conducted in wild-type, cti6Δ, and gcn5Δ yeast strains grown in YP-galactose or SC media to induce transcription of GAL1 or TRP3, respectively. Antibodies specific for the acetylated forms of lysine residues 9 and 14 of histone H3 and primers specific for UASG or TRP3 UAS were used.
(D) TBP binding at GAL1. Chromatin-IP in wild-type and cti6Δ mutants expressing a triple HA epitope-tagged TBP protein and primers specific for the GAL1 promoter, POL1 coding sequence, or the ADH1 promoter, served as a positive control.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5 Cti6 Links the Cyc8-Tup1 and SAGA Complexes
(A) Cti6 interaction with HA-Gcn5, HA-Ada2, and Ada3. Immunoblotting of total input (In) and glutathione sepharose-bound fraction (B) with HA-specific or anti-Ada3 antibodies in wild-type or spt20 mutant strains are shown. Protein extracts were obtained from cells expressing GST or GST-Cti6 along with HA-Gcn5 or HA-Ada2. Extracts obtained from cells expressing GST or GST-Cti6 along with HA-Med3 or HA-TBP were used as negative controls. Input proteins were equal to 1:150th of the total extract incubated with the sepharose beads.
(B) Cti6-Spt3 interaction. Protein extracts obtained from cells expressing HA-Cti6 and myc-tagged Spt3 were immunoprecipitated using preimmune serum (−HA) or anti-HA antibody (+HA). Precipitated proteins were analyzed by immunobloting using anti-myc antiserum.
(C) Cyc8-Tup1 interacts with Gcn5 via Cti6. Immunoblotting of total input (In) and sepharose-bound fraction (B) with extracts obtained from wild-type or cti6Δ cells expressing HA-Cti6 along with GST-Cyc8 hybrid protein.
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6
TUP1 or CYC8 Deletion Overcomes the Requirement of Gcn5 and Spt3 for GAL1 Transcription
(A) RNA blotting analysis of the wild-type, tup1Δ, cti6Δ, gcn5Δ, spt3Δ, and spt20Δ single mutants, as well as the double mutants tup1Δ,cti6Δ; tup1Δ,gcn5Δ; tup1Δ,spt3Δ; and tup1Δ,spt20Δ. Yeast cells grown in glucose were washed and transferred in galactose for 2 hr. Total RNA was extracted and hybridized with probes specific for GAL1 and actin as internal control.
(B) RNA blotting analysis as in (A) in cyc8Δ single and cyc8Δ,gcn5Δ; cyc8Δ,spt3Δ; and cyc8Δ,spt20Δ double mutants.
Molecular Cell 2002 9, DOI: ( /S (02) )