HSP70 Transcription and Protein Aggregation Figure 3. Role of PKAca in binding of HSF1, p300 Ac-histone H4, Pol II, phospho-S2-Pol II, phospho-S5-Pol II and CDK9 to the hsp70.1 promoter at 426C. Cells were heat shocked at 42uC/15 min before ChIP. (A) Schematic diagram of hsp70.1 gene with 59 residue of each real time PCR fragment shown underneath (approximately to scale). (B–I) Antibodies used for immunoprecipitation are indicated on each panel. The x axes show each PCR fragment along hsp70.1 gene and the y-axes represent the percentage of input. (Mean value +/2 SEM of three experiments is shown), except for D where arbitrary units are used for the ratio of ChIPed acetylated H4 against total H4. doi:10.1371/journal.pone.0028950.g003 (Figure S4E, F). Untreated cells contained abundant CFP- containing granules/inclusions, which were reduced by heat shock at 42uC for 30 min (Figure S4E). PKAca knockdown with siRNA reduced the effects of heat shock and CFP-containing granules were again apparent (Figure S4F). The findings that similar effects are seen when using either GFP or CFP as a reporter suggest that inclusion formation and resolution are properties of the aggrega- tion–prone poly-Q tract rather than the fluorescent proteins themselves (Fig. 4A–G, Figure S4E, F). The extent of PKAca knockdown in SKNSH cells is indicated in the adjacent panel showing lack of immunofluorescence staining for PKAca (Figure S4F). We next examined the effects of HSF1 perturbation on the relative levels of soluble poly-Q-associated reporter proteins and poly-Q-containing structures that sediment at 4006g in cell extracts as described by Meriin et al. We showed first that in HeLa cells expressing poly-94Q-CFP (Htt 94Q-CFP), most of the CFP is detected in the pellet fraction (Fig. 4H) while after heat shock at 42uC most of the CFP was soluble and did not sediment at 4006g (Fig. 4H). CFP fused to a shorter pol-Q tract (poly-25Q or Htt 94Q-CFP) was found largely in the soluble fraction of HeLa cells (Fig. 4H). Likewise, in PC12 cells expressing poly-103Q-GFP, GFP was enriched in the pellet fraction in non-heat shocked cells expressing a scrambled RNA, while 42uC heat shock caused a shift to the soluble fraction (Fig. 4H). However after PKAca knockdown with siRNA, poly-103Q-GFP was found largely in the pellet fraction (Fig. 4 H). Likewise with HSF1 knockdown, poly-103Q-GFP was found largely in the pellet fraction of control cells, compared to heat shocked cells expressing the scrambled RNA in which it was more abundant in the soluble fraction (Fig. 4H). HSF1 knockdown reversed this distribution and poly- 103Q-GFP was again enriched in the insoluble fraction (Fig. 4H). Discussion HSF1 activity in neurons is diminished compared to other tissues and declines with aging through mechanisms not fully elucidated [3]. Our studies indicate a pathway involving PKA- dependent activation of HSF1 and recruitment of a p300/p-TEFB complex that permits transcriptional activation (Figs. 1, 3). The transcriptional activation mechanism involving HSF1 and PKAca may be a general one in mammalian genes responding to heat shock and we observed PKA-dependent co-localization of HSF1, p300, CDK9 and RNA Pol II phospho-S2 in both mRNA PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28950