Figure 8

CBP interacts with multiple sites in the MOZ portion of MOZ-TIF2. A. The MOZ portion (amino acids 1–759) of MOZ-TIF2 interacts with the C-terminus of CBP. As described in Experimental Procedures, a ³⁵S-methionine labeled CBP fragment (amino acids 1680–2441) was incubated with the GST-MOZ-TIF2 fragment (amino acids 1–759) (MT2 1–759) and the reaction mixture absorbed to Sepharose 4B-GSH beads. After extensive washing the beads were treated with sample buffer, absorbed proteins separated on SDS-PAGE, and CBP fragments detected with radioautography. GST, the pull-down by GST tag peptide alone mixed with MT2 1–759. CBP 1680–2441, 10 % of the CBP 1680–2441 peptide input into the reaction. MT2 1–759, CBP absorbed to Sepharose-GSH beads through GST-MT2 1–759 peptides. B. The MOZ portion of MOZ-TIF2 interacts with the CH3 domain of CBP. Three peptides-MOZ-TIF2 1–759, MOZ-TIF2 760–1644 (amino acids 760–1644), and MOZ-TIF2 760–1644 with deleted CID domain (deleted amino acids 1183–1311)-were labeled with ³⁵S-methionine by synthesis by in vitro translation. GST-CBP peptides (amino acids 1680–1892) were expressed in E. coli and purified by Glutathione Sepharose™ 4B beads. MOZ-TIF2 peptides and GST-CBP peptides were allowed to interact as described in Experimental Procedures and the proteins absorbed to GST-CBP beads were analyzed as described. Input was 10% of ³⁵S-methionine labeled polypeptides used in the binding reaction. GST, the pull-down by GST peptide alone mixed with MOZ-TIF2 peptides. CBP 1680–1892, pull-down by GST-CBP peptides (amino acids 1680–1892) incubated with ³⁵S-methionine labeled MOZ-TIF2 1–759, MOZ-TIF2 760–1644, and MOZ-TIF2 760–1644 with deleted CID domain. Input-MT2 1–759, Input-MT2 760–1644, and Input-MT2 D 760–1644 are 10 % input of ³⁵S-methionine labeled peptides of MOZ-TIF2 1–759, MOZ-TIF2 760–1644, and MOZ-TIF2 760–1644 with deleted CID domain used in the reaction. C. The PHD and MYST domains of the MOZ moiety in the fusion protein interact with the CH3 domain of CBP. The ³⁵S-methionine labeled CH3 domain peptides of CBP were incubated with GST-MOZ peptides, absorbed to Glutathione Sepharose™ beads, the absorbed proteins separated by SDS-PAGE, and subjected to radioautography. The input represents 10% of ³⁵S-methionine labeled peptides used in the binding reaction. GST and input CBP 1680–1892 have the same meanings as described in A above. MT2 1–235, MT2 1–311, and MT2 523–759, peptides of the MOZ partner from amino acids 1–235, 1–311, and 523–759 respectively were used in the incubation with the CH3 domain of CBP. D. GST pull-down analysis of CBP binding sites in MOZ partner of MOZ-TIF2. GST tagged C terminal of CBP(amino acids 1680–2441) were expressed in E. coli and purified by Glutathione Sepharose™ 4B beads. The expression plasmid with Xpress-tagged MOZ-TIF2 fragments was transfected into HEK 293 cells. After 36 hours whole cell extract was prepared and incubated with GST-tagged C terminal of CBP absorbed onto Glutathione-Sepharose beads. The Xpress-tagged MOZ-TIF2 fragments pulled down by GST-tagged C-terminal of CBP were detected with anti-Xpress antibody and are seen in lane 3. Lane 1 represents 5% of the input of expressed Xpress-tagged MOZ-TIF2 fragment. Lane 2, incubation of Xpress-tagged MOZ-TIF2 fragment with GST peptide alone. E. Deletion of CBP binding sites in MOZ portion decreases the inhibition of the transcriptional activation of ER by MOZ-TIF2. As described in Figure 3, an estrogen response element-driven luciferase reporter plasmid was co-transfected into CV-1 cells with an estrogen receptor expression vector and pCDNA3.1 plasmid inserted with MOZ-TIF2 fragments or vector alone. The relative luciferase activity in light units is shown after correction of transfection efficiency by measurement of beta galactosidase activity. Double stars represents a significant difference at P < 0.01 level respectively by two tail student T- test compared to the transfection with MOZ-TIF2 in the ligand added condition. Open bar, 50 nM estradiol. Dark bar, no added estradiol. The numbers on top of the open bars indicate the ratio of light units in the presence and absence of ligand as described in Figure 3.