Hop recruits HDAC activity to repress transcription. (a) Cos cells were transfected with a luciferase reporter plasmid including regulatory elements from the SM22α promoter with binding sites for SRF. Myocardin cotransfection induces dramatic activation assigned a value of 100% maximal activation (M). Cotransfection of myocardin and Hop (H) results in inhibition of myocardin-induced activation. The ability of Hop to inhibit myocardin-induced activation is attenuated in a dose-dependent fashion by TSA. (b) Hop inhibitory activity is also blocked by Scriptaid but not by Nullscript. (c) Immunoprecipitation of Hop from transfected Cos cells results in precipitation of HDAC activity. Cells were transfected with vector alone (pcDNA3) or myc-tagged control (pSecTag-PSA), Hop, or HopH2, followed by immunoprecipitation with anti-myc Ab. HDAC activity in the pellet was assayed and reported as arbitrary units (see Methods). Western blot analysis with anti-myc Ab of input material used for immunoprecipitation is shown below. (d) Chromatin immunoprecipitation reveals decrease in acetylated histones after Hop expression. The 10T1/2 cells were transfected as in a, followed by chromatin immunoprecipitation with Ab’s to acetylated histone H4 or H3 and detection of SM22α promoter fragments by PCR in precipitate. (e) Coimmunoprecipitation of HDAC2 and Hop. The 293T cells were transfected with control vector (pcDNA3), Hop, or HopH2, followed by immunoprecipitation and Western blot analysis for HDAC2. A Western blot using anti-HDAC2 Ab of the cell extracts used in each case is shown (Input).