Figure 6.

HSF1 binding to HDAC 1 and HDAC2 in heat-shocked cells correlates with increased HDAC activities. (A) Increased HDAC1 and 2 activities after heat shock. Immunoprecipitation of transiently transfected Flag-tagged HDAC1 and 2 or Flag tag alone from non-heat-shocked (NHS) or heat-shocked (HS) HeLa cells were performed with anti-Flag antibodies. The same amounts of immunoprecipitates were used in a deacetylase assay to quantify the HDAC activity in each sample. Increased HDAC1 and 2 activities were both observed in heat-shocked cells. Each sample was analyzed in duplicates in two independent experiments; error bars, SEM. (B) Endogenous HSF1 coimmunoprecipitates with Flag-HDAC1 and HDAC2 but not with HDAC3 in heat-shocked cells. Flag immunoprecipitations were performed as in A on cellular extracts from unstressed and heat-shocked cells transiently transfected with Flag-HDAC1, -HDAC2, or -HDAC3. Western blot analysis of the immunoprecipitates were performed with anti-Flag (top panel) and anti-HSF1 (bottom panel) antibodies. (C) Increased HDAC activity in HSF1 immunoprecipitates after heat shock. Cos cells were transiently cotransfected with Myc-tagged HSF1 and a mixture of flag-tagged HDAC1/2. Immunoprecipitations from non-heat-shocked (NHS) or heat-shocked (HS) cells were performed with anti-Myc or anti-Flag antibodies as indicated. The level of HSF1 and HDAC1/2 in NHS and HS cells and the level of HSF1 and of HDAC1/2 in the corresponding immunoprecipitates are analyzed by Western blot using the anti-Flag and anti-Myc antibodies. Each immunoprecipitate was incubated ON with an acetylated H4 synthetic peptide. The percentage of deacetylated and acetylated peptides in the reaction was determined by MALDI-TOF mass spectrometry. The peak at 1655 Da (left peak) corresponds to the acetylated H4 peptide and the peak at 1613 Da (right peak) to the deacetylated H4 peptide. The percentages of deacetylated H4 peptides is indicated on each spectrum.