HSP70 Transcription and Protein Aggregation Figure 2. HSF1 regulation of hsp70.1 mRNA expression by p-TEFb, PKAca and histone acetylation. (A) Time-course of hsp70.1 mRNA synthesis at 42uC. (B) Effects of p-TEFb inhibitor FP (500 nM/18 hr) and CTD kinase inhibitor DRB (100 mM/18 hr) on hsp70.1 mRNA synthesis at 42uC/ 45 min. (C) Effects of CDK9 knockdown with siRNA on hsp70.1 mRNA synthesis at 42uC. (D) Effects of PKAca knockdown and TSA treatment at 37uC for 18 hr on hsp70.1 mRNA synthesis at 37uC (no heat shock) or 42uC (heat shock) (E) PKAca mRNA expression in control and PKAca knock-down cells. (F) Stress (42uC/1 hr) -induced induction of pHuR98ncRNA was inhibited by PKA kd as in D. Each experiment was repeated reproducibly at least 3 times and data are plotted as mean +/2 SEM. doi:10.1371/journal.pone.0028950.g002 utilized siRNA molecule targeting CDK9 (Fig. 1C). While heat shock activated Hsp70.1 mRNA expression in cells exposed to control RNA (lane 2), the CDK9-targeted siRNA markedly reduced Hsp70.1 expression (lane 4) (Fig. 2C). As active HSF1 can be found complexed with p300 and PKAca (Fig. 1) [4] we next asked if hsp70.1 induction by stress requires PKAca expression and involves HAT activity using PKAca shRNA and histone deacetylase (HDAC) inhibitor TSA (Fig. 2D). Exposure to TSA caused a mild increase in hsp70.1 expression while induction was decreased by PKAca shRNA (Fig. 2D, lanes 2, 3). TSA exposure was additive with heat shock and caused a strong induction of hsp70.1 expression (Fig. 2D, lane 5). As shown previously PKAca reduction by RNA interference reduces hsp70.1 expression (lane 7) and this decrease was partially compensated by TSA exposure (lane 8) (Fig. 2D). Fig. 2E shows relative levels of PKAca mRNA in control cells and after PKAca shRNA. We were also interested in the behavior of genes expressed in the NsB and examined expression of a non-coding RNA transcribed at the pHuR98nc locus (Fig. 2F). In humans, HSF1 granules have been previously observed localizing to the 9q11–q12 heterochromatic region [18]. Within this locus, pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. We have performed both real time reverse transcription qPCR and ChIP- qPCR to show that HSF1 directly binds and regulates pHuR 98 (Figure S2). Heat shock induced a large increase in expression of pHuR98ncRNA (Fig. 2F). As with the hsp70.1 mRNA, stress- induced induction of pHuR98ncRNA was inhibited by PKA knockdown (Fig. 2F). (3) PKAca is required for association of HSF1, p300 and p- TEFb, histone H4 acetylation and RNA Pol II with hsp70.1 on chromatin We next examined the role of PKAca in association of HSF1 and other factors with the hsp70.1 promoter and downstream sequences on the gene, using the standard cell fixation/ChIP approach. We carried out the experiments using 15 min 42uC as Pol II was enriched on the gene at this time while levels returned to baseline levels at 45 and 60 min when RNA ceased to accumulate further (Data not shown). We scanned four regions of the hsp70.1 gene by ChIP including an HSE-containing promoter motif (2233), two promoter proximal zones including the 263 and +28 regions and a sequence in the open reading frame (+752) (Fig. 3A). As shown previously, stress induces strong binding of HSF1 to the hsp70.1 promoter but not the downstream regions of hsp70.1 and PKAca knockdown inhibits HSF1 promoter associ- ation (Fig. 3 B). The p300 was enriched on the downstream and promoter regions of HSF1 after heat shock and this effect was again strongly inhibited by PKAca knockdown (Fig. 3C). We next examined acetylation of histones on the gene concentrating on histone H4, a known p300 substrate [19] (Fig. 3D). The analysis was complicated by the finding that total histone H4 levels were depleted by 15 min of heat shock in accordance with earlier findings in yeast and Drosophila showing rapid nucleosome clearance after heat stress [20,21] (Fig. 3E). Levels of acetylated histone H4 were increased in the +28 region but not elevated in other parts of the gene. RNA Pol II was bound to the hsp70.1 gene and these levels were increased by heat shock in each region of the gene, particularly the promoter proximal 263 region (Fig. 3F). PKAca depletion markedly reduced Poll II association with the gene (Fig. 3F) in keeping with its effects on Hsp70 mRNA synthesis (Fig. 2). RNA Pol II phospho-S2 levels were also markedly increased on the gene after heat shock, consistent with abundant transcriptional elongation (Fig. 3G). As with total Pol II, decrease in PKAca inhibited accumulation of Pol II phospho-S2 (Fig. 3G). Likewise Pol II phospho-S5 levels increased in heat shocked cells although PKAca knockdown had a minimal effect on the accumulation of this phospho-form of RNA polymerase II on hsp70.1 (Fig. 3H). Pol II S5 is phosphorylated by a kinase activity in TFIIH and is involved with transcriptional initiation, the early stages of elongation [22]. Interestingly, CDK9 levels were increased in the one promoter proximal region, +28 (Fig. 3I) in which acetylated histone H4 appears to accumulate, and this effect was reduced by PKAca knockdown (Fig. 3C). The p-TEFb recruit- ment to chromatin has been shown previously to be regulated by histone H4 acetylation [23]. (4) Role of PKAca and HSF1-mediated transcription in protein aggregation We next examined the potential role of this cascade response in cellular homeostasis using neuronal cells expressing an extended polyglutamine repeat sequence fused to GFP (poly-Q103-GFP) [24]. Forced expression of poly-103Q-GFP in PC-12 pheochro- mocytoma cells led to formation of large intracellular GFP- containing inclusion bodies, detectable by confocal microscopy, as shown previously [24] (Fig. 4A). The number of these structures was however markedly reduced after a heat shock of 42uC for 1 hr that leads to HSF1 activation and HSP synthesis (Fig. 2) and we observed a more even distribution of poly-Q-GFP within the cytoplasm of such cells (Fig. 4B). Heat shock at 42uC may promote resolution of protein inclusions, as HSP mRNAs are translated at this temperature, while at higher temperatures translation is profoundly inhibited and heat shock may exacerbate protein insolubilization [25]. A more complete time course for the heat treatments is shown in Figure S3 indicating that the number of poly-103Q-GFP structures begins to decline in cells by 20 min. This corresponds to the time at which Hsp70 begins to accumulate in and co-localize with the poly-103Q-GFP containing structures in some cells (Figure S3). Hsp70 has been shown to modulate aggregation of poly-Q proteins in yeast [26]. Treatment with an RNAi species targeting PKAca almost completely reversed the effects of heat shock and poly-103Q-GFP inclusions were seen in both un-heat shocked and heat shocked cells (Fig. 4C, D). Similar results were seen with CDK9 knockdown which caused partial inhibition of the effects of heat shock (Fig. 4F). Numbers of cells containing inclusions/aggregate bodies in the different treatments are quantitated in Fig. 4G. In addition, knockdown of HSF1 by siRNA reduced the occurrence of inclusion bodies containing Poly-103Q-GFP in PC12 cells to a similar degree compared to PKAca knockdown (Fig. 4G). We next carried out similar experiments in human neuronal cells (SKNSH) expressing a different polyglutamate containing fusion protein (poly-94Q-CFP) PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28950