Plasmids PC12 cells stably expressing Poly Q (103Q)-GFP were a gift from Michael Sherman (Boston University, MA). Plasmids encoding shRNA for PKAca and scrambled shRNA were from Open Biosystems as in [4]. The CDK9 siRNA construct was from Santa Cruz Antibodies and PKA siRNA from Cell Signaling. HSF1 siRNA was from Qiagen. The Htt94Q-CFP was purchased from Addgene. Cells and Culture Lentivirus production and transduction was performed as described [4]. PC12 cells were grown in DMEM with 10% horse serum, penicillin-streptomycin (1000 u/ml) and 5% FBS [13]. PC12 cells stably expressing Poly-Q (103Q) GFP [13] were grown in DMEM with 10% horse serum, penicillin-streptomycin (1000 u/ml) and 5% FBS, G418 (200 mg/ml) (Sigma) and zeocin (100 mg/ml). Ponasterone A (2.5 mM) was then incubated with the cells 4 days for 103Q-GFP induction. HeLa cells (ATCC) were grown in DMEM with 10% FBS and antibiotic mix. SKNSH cells (ATCC) grew in DMEM with 10% FBS. FBS was replaced with Tet-system-approved FBS (Clontech) and cells transfected with Htt94Q-CFP and then treated with doxycyline (500 ng/ml)/24 hr for poly-Q induction. RNA isolation and quantitative Real-Time PCR (qRT-PCR) Total RNA was isolated by RNeasy Mini kit (Qiagen) including on- column DNase digestion to eliminate DNA (Rnase-Free DNase Set, Qiagen). RNA quantification was then performed using the QuantiTect Reverse Transcription –PCR Kit (QIAGEN) on the ABI 7300 real time PCR system according to the manufacturer’s protocol and the -fold increase in ChIP-PCR products by 22DDCT compared with an internal control (b-actin) was plotted for the expression of hsp70.1. All experiments were performed 3 times for each sample and the primers were designed using Primer3 online software. Chromatin Immunoprecipitation ChIP assays for HSF1 were performed as described previously [4]. ChIP was carried out by precipitating with anti-HSF1, p300, RNA pol II, pCDK9, phospho-ser-2-RNA pol II, phospho-ser5- RNA pol II, acetylated histone H4 or total histone H4 antibodies. ChIP data were normalized to 1% of starting input of genomic DNA as a positive control and pre-immune IgG mock ChIP as negative control. Amplified PCR products were first analyzed by size on agarose gel, then independently quantitated using the ABI 7300 real time PCR system and enrichment of ChIP-PCR products by 22DDCT compared with the input was plotted for the respective regions of hsp70.1. ChIP–qPCR experiments were performed 3 times for each sample. Immunoprecipitation and immunoblot Procedures were performed as described [4]. Confocal Microscopy GFP and fluorescently labeled secondary antibodies were visualized using Zeiss 510 confocal microscopy using the right wavelength and filter sets as described [4]. Results (1) PKAca-dependent association of HSF1 with p300 and p-TEFb after stress We first examined the potential role of PKAca in HSF1 binding to p-TEFb and p300. HSF1 has also been shown to associate with HSP70 Transcription and Protein Aggregation nuclear stress bodies (nSB) after heat shock and regulate transcription of multiple copies of satellite III DNAs which encode non-coding RNA Pol II-dependent RNAs [14]. Associated HSF1 can thus be visualized by light microscopy in these regions. We confirmed, using confocal immunofluorescence analysis that HSF1 localizes to bright-stained nuclear bodies resembling nSB after heat shock (Fig. 1A) and was co-localized with p300 and the p- TEFb subunit CDK9, in a PKAca-dependent manner. Co- localization of the three factors in the nSB was abrogated by PKAca knockdown with shRNA, using conditions described previously (Fig. 1B). Previous studies have shown the enhancement of histone acetylation in nSB after stress, consistent with of p300 association with the granules [15]. PKAca knockdown reduced the levels of nuclear HSF1 and HSF1 was observed in the cytoplasm of heat shocked cells with depleted PKAca (Figure S1). One of the primary targets of p-TEFb in transcriptional elongation is the CTD of RNA Pol II [9]. The CTD contains a series of heptad repeats with amino acid consensus YSPTSPS, in which S2, S5 and S7 are subject to modification according to transcriptional status of the gene [8]. Transcriptional elongation and mRNA processing are triggered by the phosphorylation of Pol II at the S2 site by p- TEFb [8]. (We found no evidence in the literature for direct phosphorylation of CDK9 by PKA). We observed marked enrichment of RNA Pol II phospho-S2 in NsB after heat shock, in association with p300 and HSF1 (Fig. 1C), an effect that was reduced by PKAca knockdown (Fig. 1D). Relative levels of stress granules containing HSF1, p300 and CDK9 in cells without and with PKAca knockdown are quantitated in Fig. 1E. The relative incidence of granules containing HSF1, p300 and RNA Pol II phospho-S2 are shown in Fig. 1F. We also detected complexes containing p300, CDK9 and HSF1 in nuclear extracts from heat shocked HeLa cells after immuno- precipitation with anti-p300 antibodies indicating a direct interaction (Fig. 1G). Association of the three factors in the immunoprecipitates was inhibited by PKAca knockdown and minimal in unstressed cells (Fig. 1G). As PKAca mediates the function of HSF1 through a mechanism that involves phosphor- ylation on serine 320 [4], we examined the ability of HSF1 in which S320 was replaced by either aspartate (S320D) or alanine (S320A) (Fig. 1H) to bind p300. Experiments were carried out in cells co-transfected with HA-p300 and GFP-HSF1 constructs. The electrophoretic mobility of GFP-HSF1 is indicated in the 7th lane. The p300 was effectively co-precipitated with HSF1-S320D but only minimally with HSF1-S320A – compare to WT, suggesting involvement of negatively charged residues at S320 for complex formation. In the immunoblots of HSF1-GFP precipitated from cells we observed a lower band not observed in the rHSF1-GFP control lane. This band was observed in control immunoprecip- itations with IgG suggesting that it is not specific (Fig. 1H). (2) Gene activation by stress involves PKAca, histone acetylases and p-TEFb To further study the role of PKAca, p300 and p-TEFb in HSF1 activation, we investigated the heat inducible hsp70.1 gene (Fig. 2). Hsp70.1 is synthesized at basal levels in HeLa cells and is induced by heat shock (42uC) as shown previously [16] (Fig. 2A). The mRNA levels peaked at 45 min of heat shock and were not increased by 60 min. Stress activation was partially inhibited by the p-TEFb/CDK9 inhibitor flavopiridol (FP) suggesting a functional role for p-TEFb in Hsp70.1 expression (Fig. 2B). Transcriptional inhibitor 5, 6-dichloro-1-b-D-ribofuranosylbenz- midazole (DRB) which inhibits TFII kinase and Pol II hyperpho- sphorylation [17] completely blocked stress-induced mRNA production (Fig. 1 B). To further probe the role of p-TEFb, we PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28950