Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor.

The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.

, related to "Experimental Procedures" Table S1. List of peaks found after ChIP -Seq analysis in LNCaP   Table S2. List of peaks found after ChIP-Seq analysis in LNsip53   Table S3. Motif enrichment analysis of ChIP-Seq data prepared by CEAS

Supplemental experimental procedures p53 knockdown preparation
The expression of endogenous p53 was inhibited by infection with recombinant lentivirus constructs pLSL-puro-expressing siRNA hairpin under control of the RNA H1 promoter (Budanov et al., 2004).
The structure of the 19 bp siRNA complementary to human p53 mRNA was as follows: 5′-GACTCCAGTGGTAATCTAC-3′. The structure of the control siRNA derived from the HPV18 E6 gene was as follows: 5′-CTAACACTGGGTTATACAA-3′. LNCaP was infected with lentivirus with siE6 or si-p53 followed by puromycin selection. The effect of siRNA expression was verified by assessing p53 protein expression by Western blot analyses.

Caspase activity in cell lysates
Caspase activity in cell lysates was measured as described previously (Rokhlin et al., 2001). Briefly, cell lysates were prepared in 1% Triton X-100 buffer, pH 7.2, containing protease inhibitors. Protein lysate (40 μg) was incubated for 60 min in assay buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS and 10% sucrose) with 40 μM of fluorescent substrates Ac-DEVD-AMC or Ac-DEHC -AMC (Bio-Mol, Plymouth Meeting, PA, USA) and substrate hydrolysis was monitored using a fluorescence reading system set to 360 nm for excitation and 460 nm for emission.

ChIP
Sampels for Solexa ChiP sequencing experiment-high throughout sequencing (ChiP-Seq) were prepared using ChiP IT Express Magnetic Chromatin Immunoprecipitation Kit. (Active Motif, CA cat # 53008) following manufactures instruction with minor modifications. Briefly, cells were crosslinked with 1 % formaldehyde for 10 min at room temperature and the cross linking was inactivated by 0.125 M glycine for 3 min at room temperature. Cells were washed with cold PBS twice. Cells were harvested in cell lysis buffer (5mM PIPES pH 8.0, 5mM KCl, 0.5% NP40, PMSF and protease inhibitors cocktail) using scraper. After centrifugation, pellets were resuspended in 1ml of ice cold Lysis Buffer from ChiP IT kit and homogenized on ice with dounce homogenizer. After 10 minutes of centrifucation nuclei were resuspended in Shearing Buffer (ChiP IT kit). DNA was sheared by sonication on Sonics Vibracell sonicator at 55% power in volume 350 l with 3mm stepped microtip. Twelve pulses of 30 second each, with a 40-second rest on ice between each pulse. 50 l of each sample were reverse cross linked, treated with RNAs A and proteinase K according to protocol and DNA were cleaned up using QI Aquick MINelute kit (Qiagene, Valencia, CA, USA, No 28104) and separated in 1% agarose gel. 10 g of DNA were taken for each IP probe with 2 g of anti-AR mAbs overnight at 4C. DNA samples were cleaned up using QI Aquick MINelute kit (QIAGEN, Valencia, CA, USA) and DNA concentrations were detected using NanoDrop 2000 (Thermo Scientific NanoDrop products Wilmington, DE, USA).

CHIP-Sequence data analysis
Fastq files were acquired from ISU aligned with ELAND extended. The fastq files where first converted to aln files using a simple script to rearrange the columns of data. These files were then analyzed using cisGenome with the following settings; width equal to 100, step size equal to 25 and a cutoff of 5. The .bar files output from this process where converted to .txt files using the cisGenome supplied program affy_bar2txt. These txt files where then converted to .wig files for uploading to the UCSC genome browser for manual viewing. The .cod files output from the same process as the .bar files were converted to .bed files using the cisGenome supplied program cod2bed. These .bed files where uploaded to the cisregulatory element annotation system (CEAS) for processing to find enriched regions and motifs (Ji et al., 2006), (Web Server issue):W551-4. CEAS: cis-regulatory element annotation system). An additional script was developed to extend the data from CEAS adding additional sequence information as well as adjacent gene information based on the motif files from CEAS. This script queried UCSC genome database for gene information and used the alignments reported from CEAS to obtain adjacent sequence information.