Supplementary Information: Materials and Methods. Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered

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1 Supplementary Information: Materials and Methods Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered saline (PBS) and lysed in TNN lysis buffer (50mM Tris at ph 8.0, 120mM NaCl and 0.5% NP-40) containing protease inhibitor cocktail (Calbiochem), 4mM dithiothreitol (DTT) and 0.2mM phenylmethylsulphonyl fluoride (PMSF) at 4 C for 30min. Immunoblot and immunoprecipitation were carried out as described previously (Shikama et al. 1999). Equal protein aliquots were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane and probed with the indicated antibodies: anti-p300 monoclonal antibody (Upstate), anti-hsf1, anti-hsp70 inducible (Stressgen) and anti-pcna antibody (Santa Cruz). Anti-Strap peptide 510 antibody was as described previously against a peptide representing residues 211 to 234 (Demonacos et al. 2001). For immunoprecipitation, cells were lysed in high salt TNN buffer (240mM NaCl). Cell extracts were incubated with Protein-G agarose beads (25μl) at 4 C for 1h and centrifuged at 13,000g for 3min to remove the beads. The supernatant was collected and incubated with protein A agarose beads (25μl) and the indicated antibodies overnight at 4 C. After overnight incubation, the beads were washed three times in TNN buffer and once in PBS buffer. Proteins bound to the beads were eluted in SDS loading buffer. The eluted complex was resolved on SDS-PAGE gel, transferred to nitrocellulose membranes and analysed by using the appropriate antibodies as previously described. Protein purification. Fusion proteins were expressed in E.coli BL21 (DE3) plyss cells and purified respectively by histidine affinity chromatography or glutathione

2 affinity chromatography as described (Shikama et al. 1999). Eluted proteins were dialyzed in 20mM Tris, 1% glycerol, 5mM EDTA and 100nM PMSF; the purity of each protein was greater than 80%. For the recombinant Strap protein used in the in vitro HAT assay, the dialysed Strap protein was further purified on a Mono-Q column using a HPLC system. All Strap fractions were tested for purity; the final protein purity was greater than 95%. Reporter assays and reverse transcription PCR. HA-Strap and β-galactosidase expression vectors were co-transfected with either the pgl3 vector containing hsp70b promoter or a control pgl3 vector into U2OS cells. After 24h of transfection, cells were harvested and lysed in 1 lysis buffer (Promega). The activities of luciferase and β-galactosidase were measured as previously described (Demonacos et al. 2001). All assays were performed in triplicate. Total RNA from U2OS cells was extracted using RNAeasy columns (Qiagen). RNA levels were normalized and cdna templates were generated using the First Strand Synthesis kit (InVitrogen). Primers targeting the hsp70b gene encoding sequence (Stressgen) were used to amplify a 234bp fragment by PCR. GAPDH primers (Stratagene) were used as an internal control. In vitro and in vivo p300 acetylation assays. Flag-p300 was expressed in baculovirus and purified as described previously (Shikama et al. 1999). The indicated input proteins were incubated at 30 C for 1h in 40µl containing 5 HAT assay buffer (250mM Tris ph 8.0, 25% glycerol, 0.5mM EDTA, 10mM sodium butyrate and 250mM KCl), 5μM 3 Η acetyl-coa (2-10μCi/mM, Amersham Life Science). Core histones (Upstate) were used as the substrate and each reaction was washed and then

3 spotted onto 2cm Whatman (p81) circles, washed, and the activity measured in a scintillation counter (Beckman). HAT assays were performed on material purified from the following cell lines: Tet-On U2OS parental cell line (par), inducible Strap stable cell line in the absence (wt) and presence of doxycyclin (wt+dox). Cell lysates were immunoprecipitated with anti-p300 or control antibodies and the HAT activity was measured as described above. Chromatin immunoprecipitation (ChIP). U2OS cells were plated and treated with heat shock (1h at 43 C) before harvesting. To crosslink chromatin, formaldehyde was added to the culture medium at a final concentration of 1% and incubated at room temperature for 10 min. Cells were resuspended in lysis buffer I (5mM Tris-HCl ph 8.0, 85mM KCl and 0.5% NP-40) and incubated on ice for 20 min. After centrifugation, cells were resuspended in lysis buffer II (RIPA buffer: 10mM Tris- HCl ph 7.5, 150mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS and 1mM EDTA) and incubated on ice for 10min. Cell lysates were sonicated and the chromatin suspension incubated with BSA pre-blocked ProA/G agarose beads (Sigma). Chromatin was then incubated with the indicated antibodies at 4 C overnight. Immunocomplexes were harvested by incubating with ProA/G agarose and washed in buffer I (20mM Tris-HCl ph 8.0, 150mM NaCl, 2mM EDTA, 0.1% SDS and 1% Triton X-100) twice, wash buffer II (20mM Tris-HCl ph 8.0, 500mM NaCl, 2mM EDTA, 0.1% SDS and 1% Triton X-100) for 3 times and wash buffer III (0.25M LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA and 10mM Tris-HCl ph 8.0) for 4 times and TE buffer once. Immunocomplexes were eluted in 250µl elution buffer (1% SDS, 0.1 M NaHCO 3 ). To reverse the crosslink, 0.4mg/ml proteinase K and 0.2mg/ml RNAse diluted in buffer containing 2M NaCl, 100mM EDTA and 400mM Tris-HCl ph 6.5

4 was added to the elutes and then incubated at 55 C for 3h following by 65 C overnight. DNA samples were purified by PCR purification kit (Qiagen) and dissolved in 50µl TE buffer. For the sequential ChIP assay, the primary immunocomplexes were eluted with 50µl 10mM DTT with agitation at 37 C for 30min.The elutes were diluted 1:50 in 20mM Tris-HCl ph 8.0, 150mM NaCl, 2mM EDTA and 1% Triton X-100 and the second antibody added to the immunoprecipitates, followed by overnight incubation. The immunocomplexes were rinsed in wash buffer I, II, III and TE once each. The procedures for reversing the crosslink were as described above. The following primers were used to amplify the genomic sequences on the promoters region of hsp70b and hsp90β. The albumin primers were as previously described (Zhu et al. 2004). The PCR products were electrophoresed on 1% agarose gel and visualized on a gel image station. The sequences of the primers designed were: hsp70b promoter F 5 -GGAAGGTGCGGGAAGGTTCG-3 R 5 -TTCTTGTCGGATGCTGGA-3 hsp90β promoter F 5 -GCGTCTCTCGGACAGGTCAG-3 R 5 -TCTCGCAGGAGTAGAGGAAGG-3 Quantitation by real time PCR ChIP experiments were quantified by real-time PCR in the MX3005P system (Stratagene) using Brilliant II SYBR Green QPCR Master Mix (Stratagene). The primers used were as follows: Hsp70B forward: 5 - GGAAGGTGCGGGAAGGTTCG-3 ; Hsp70B reverse 5 -

5 TTCTTGTCGGATGCTGGA-3 ; Hsp90β forward 5 -CTCCCCAACCCTCCATCC - 3, Hsp90β reverse 5 -ATCCCGACTCTCGCAGGA-3. Samples were checked for specific amplification using dissociation curve analysis included with the manufacturer s software. Data analysis was performed on the MXPro QPCR software and relative quantities were obtained by plotting the observed Ct values (threshold values) against serial dilutions of positive control samples. The relative quantities were normalized to input DNA (DNA before the immunoprecipitation step). The data represent the mean values from three reactions and are shown as fold change, above control ChIP, relative to input DNA, with error bars indicating standard deviation according to the formula: SD = (SD IP - m IP ) + (SD Input - m Input ) m IP / m IN sirna. sirna for lamin A/C was purchased from Dharmacon, and the sirna Strap sequence was CCAGCTGGGTGAGGTGTAC. In general, 100nM sirna Strap or control sirna lamin was transfected into U2OS cells using the oligofectamine transfection reagent (InVitrogen) according to the manufacturer s protocol. After 72h of transfection, cells were harvested for further analysis. Flow cytometry. U2OS cells seeded in 6cm dish were transfected with sirna oligos Strap or lamin. After 72h, cells were treated with lethal heat shock at 45ºC for 90min (Shamovsky et al. 2006; Westerheide et al. 2006) and recovered at 37ºC for 16h. Cells were harvested, fixed by 60% ethanol in PBS, stained by propidium iodide and analysed by flow cytometry. About 2x10 4 events were collected for each sample.

6 SI Figure 1 a) U2OS cells were treated with or without lamin or Strap sirna (100nM) for 48h followed by heat shock (43ºC) for 1h. Cell extracts were prepared and subjected to immunoblotting as indicated. b) Extracts prepared from either normal or heat shocked (43ºC for 1h) U2OS cells were immunoprecipitated with anti-p300 or control antibodies, and subsequently immunoblotted with anti-p300, anti-hsf1 or anti-strap antibodies. c) Chromatin prepared from heat shocked (43ºC for 1h) or normal cells transfected with or without HA-Strap expression vector (2µg) was immunoprecipitated with anti-hsf1 or control antibodies followed by PCR reactions against the promoters of hsp70b and albumin genes.

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