PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription

Lew, Qiao Jing; Chu, Kai Ling; Lee, Jialing; Koh, Poh Ling; Rajasegaran, Vikneswari; Teo, Jin Yuan; Chao, Sheng-Hao

Chromatin immunoprecipitation (ChIP) assays were carried out using EZ ChIP kit (Millipore) according to the manufacturer's protocol with some modifications. HEK293 cells were treated with Tm (10 mg/ml) or Tg (300 nM) for 16 h prior to cross-linking. DNA fragments at around 200-1000 bp were achieved by sonication with Microson Ultrasonic Cell Disruptor (Misonix). For the IP, the indicated antibodies (i.e. anti-XBP-1 or anti-PCAF antibodies) were added to the sheared chromatin individually and incubated at 4 C overnight. The DNA/protein/antibody complex was then pulled down by protein G agarose and the DNA in the complex was purified using QIAquick PCR purification kit (Qiagen). Quantitative-PCR was performed to determine the relative amount of DNA that was immunoprecipitated by anti-XBP-1 or anti-PCAF antibodies in the presence of Tm or Tg. The primer pairs used to amplify the promoter regions of BiP and CHOP genes include: BiP (5 0 -GATGGGGCGGATGTTATCTA-3 0 and 5 0 -CTCT CACACTCGCGAAACAC-3 0 ) and CHOP (5 0 -GACA CTACGTCGACCCCCTA-3 0 and 5 0 -GGTTCCAGCTC TGATTTTGG-3 0 ). Cells treated with DMSO were served as a negative control. For the overexpression study, MCF7 cells were co-transfected with a PCAF expression vector and one of the XBP-1 plasmids (XBP-1S or XBP-1U plasmids) 2 days prior to cross-linking. Cells co-transfected with a PCAF plasmid and an empty vector served as a negative control.