Bolton_et_al._Supplement SUPPLEMENTAL RESULTS AND DISCUSSION Some HPr-1AR ARE-containing Genes Are Unresponsive to Androgen Intracellular receptors specify complex patterns of gene expression that are cell and gene specific. For example, among the 62 androgen non-responsive genes in HPr-1AR that nevertheless bear AR-occupied intragenic AREs in those cells, seven are androgen regulated in LNCaP cells (DePrimo et al. 2002; Nelson et al. 2002). In general, it seems likely that many of these AR binding sites may confer androgen responses in different cellular contexts, reflecting, for example, requirements for additional coregulators to enable hormonal control. Thus, for this subset of genes, AR occupancy is not the primary determinant for AR regulation. Alternatively, some of these AR-occupied genes may be strongly expressed prior to androgen treatment, rendering their induction difficult to measure. However, our qPCR expression data do not support this alternative. ARBRs Function as AREs in Androgen-Mediated Transcriptional Regulation Interestingly, four of the 500-bp ARBRs identified near repressed ARGs produced activation rather than repression of luciferase expression when tested in reporter contexts, whereas three failed to confer androgen regulation in either direction (ECB and KRY, unpublished). Although we do not yet understand this regulatory “polarity reversal”, our result is similar to earlier findings in which “negative” glucocortoicoid response elements direct transcriptional activation in simple contexts (M. Cronin and KRY, unpublished); thus, the mechanisms of transcriptional repression may prove to be more complex than induction, perhaps requiring components of chromatin structure or additional regulatory factors that fail to function in the reporter context. 1 Bolton_et_al._Supplement SUPPLEMENTAL MATERIALS AND METHODS Expression Microarray Analysis We identified androgen-regulated transcripts by converting RNA from vehicle or androgen- treated HPr-1AR cells into double-stranded cDNA, and hybridizing to spotted cDNA microarrays comprised of at least 10,865 predicted protein-coding genes and 3,035 expressed sequence tags. Total RNA was isolated from HPr-1AR cells with QIAshredder and RNeasy Mini kits (Qiagen). Microarray analysis was performed as described (Fox et al. 2003). In brief, RNA was linearly amplified through two rounds of in vitro transcription (Baugh et al. 2001) and coupled to N-hydroxysuccunimidyl esters of Cy3 or Cy5 (Amersham) (Hughes et al. 2001). For printing the arrays, DNA was prepared by colony-PCR (Bohlander et al. 1992) of the sequence verified Research Genetics cDNA library sets containing 21,632 clones. Primary data were analyzed using GENEPIX 3.0 software (Axon Instruments) and normalized by NOMAD (http://ucsf-nomad.sourceforge.net). The ratio-of-medians values were log-transformed and differentially expressed genes were identified using Significance Analysis of Microarrays (SAM) analysis with a q-value cutoff < 4.2% (Tusher et al. 2001). DNase I Hypersensitive Site Mapping The procedure was adapted from previous protocols (Zaret and Yamamoto 1984; Cleutjens et al. 1997). Briefly, HPr-1AR cells treated with vehicle or R1881 for 4 hr were cooled to 4°C, harvested by scraping in DNase I buffer (20 mM HEPES-KOH, pH 7.4, 3 mM MgCl2, 0.5 mM CaCl2, 5% glycerol, 0.2 mM spermine, 0.2 mM spermidine, supplemented with protease inhibitors) and disrupted in a Dounce homogenizer. Ten A260 units of nuclei in DNase I buffer 2 Bolton_et_al._Supplement were treated with 20 Kunitz units/ml of DNAse I (Qiagen) for 0, 0.5, 1,2,3,4,6 or 8 min at room temperature. The DNase digestion was stopped with an equal volume of 2x Stop buffer (50 mM Tris-HCl, pH 8; 5 mM EDTA; 200 mM NaCl and 200 µg/ml Proteinase K) and incubated for 1 hr at 55°C. The DNA fragments were purified using QIAquick PCR Purification kit (Qiagen) and digested with Xba I. DNA fragments were fractionated by agarose gel electrophoresis, transferred to Hybond-N+ filters (Amersham) and fixed by UV crosslinking. Membrane-bound DNAs were hybridized to an SGK promoter-specific DNA probe (-375 to +544 relative to the transcription start site) that was internally labeled and purified over G25 Sephadex spin columns. Hybridization was measured using a Molecular Dynamics PhosphorImager. Computational Analysis Similar motifs were clustered using CAST (Ben-Dor et al. 1999; Li et al. 2002; Patil et al. 2004). The P values of enrichment were calculated from the number of occurrences of each motif within the 524 ARBRs (Nobs) and the expected number of occurrences (Nexp). Nexp was calculated from its frequency of occurrence within background sequences (200 randomly sampled 100-kb ChIP-chip regions). The Poisson distribution was then used to calculate the probability of observing the number of occurrences ≥ Nobs by chance given the expected number of occurrences Nexp. These P values were then Bonferroni corrected by the number of tests (Patil et al. 2004). 3 Bolton_et_al._Supplement SUPPLEMENTAL TABLES Supplemental Table 1. The putative AR binding peaks detected by Mpeak for duplicate independent experiments, resulting in 524 AR binding regions (ARBRs) for HPr-1AR Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr1:6205301-6305301 chr1:6253985-6254035 3.1267 chr1:6253985-6254035 2.8205 6254010 1.01 chr1:6277021-6277071 1.3378 chr1:6288888-6288938 2.7649 chr1:6289485-6289535 2.6236 chr1:6289538-6289588 2.6882 6289537 1.02 chr1:6547195-6647195 chr1:6582471-6582521 1.2674 chr1:6585714-6585764 2.3387 chr1:6585661-6585711 1.6361 6585713 1.03 chr1:6594809-6594859 1.7126 chr1:6615966-6616016 1.5021 chr1:16177870-16277870 chr1:23581591-23681591 chr1:23607642-23607692 3.1094 chr1:23680719-23680769 1.7228 chr1:23680878-23680928 1.5661 23680824 1.04 chr1:29278977-29378977 chr1:32850360-32950360 chr1:32914864-32914914 1.3549 chr1:41569390-41669390 chr1:41600899-41600949 2.5646 chr1:41600899-41600949 2.7303 41600924 1.05 chr1:41623117-41623167 1.4250 chr1:41633414-41633464 1.9657 chr1:41633361-41633411 1.9050 41633413 1.06 chr1:41633838-41633888 1.7977 chr1:41635408-41635458 2.5735 chr1:41635779-41635829 3.2329 chr1:41635779-41635829 2.8095 41635804 1.07 chr1:46468409-46568409 chr1:46518804-46518854 1.8402 chr1:46519607-46519657 2.3492 chr1:46926761-47026761 chr1:46927693-46927743 2.4370 chr1:46927693-46927743 3.0594 46927718 1.08 chr1:46935128-46935178 1.7578 chr1:46935499-46935549 1.7719 chr1:46984587-46984637 3.2702 chr1:46984481-46984531 3.5516 46984559 1.09 chr1:46985070-46985120 1.9752 chr1:46985229-46985279 1.8889 46985175 1.10 chr1:46997825-46997875 1.3461 chr1:55014926-55114926 chr1:55052978-55053028 2.4988 chr1:55052978-55053028 2.1344 Yes 55053003 1.11 chr1:55061339-55061389 1.5554 chr1:55061339-55061389 2.2130 Yes 55061364 1.12 chr1:55105456-55105506 2.0910 chr1:58911806-59011806 chr1:58946248-58946298 2.2190 chr1:58959343-58959393 1.9244 chr1:58963477-58963527 1.7674 chr1:58963477-58963527 2.3196 58963502 1.13 chr1:59002804-59002854 1.7687 chr1:65920884-66020884 chr1:78045828-78145828 chr1:78052676-78052726 2.1833 chr1:78052623-78052673 2.2000 78052675 1.14 chr1:84818201-84918201 chr1:84866414-84866464 3.7892 chr1:84866467-84866517 3.7788 Yes 84866466 1.15 chr1:85708480-85808480 chr1:85715753-85715803 1.6689 chr1:85806989-85807039 1.5336 chr1:85806936-85806986 1.4157 85806988 1.16 chr1:92013676-92113676 chr1:94669336-94769336 chr1:94721989-94722039 1.7732 chr1:94729539-94729589 3.9766 chr1:94729698-94729748 3.9257 94729644 1.17 chr1:94730074-94730124 3.1268 chr1:94731038-94731088 2.3926 chr1:113160847-113260847 chr1:116577877-116677877 chr1:116584318-116584368 2.0176 chr1:116584159-116584209 1.9252 116584264 1.18 chr1:116585013-116585063 3.5620 chr1:116585066-116585116 3.3979 116585065 1.19 chr1:116589226-116589276 1.5016 chr1:116629803-116629853 1.7711 chr1:116631205-116631255 4.0244 chr1:116631292-116631342 3.2328 116631274 1.20 chr1:116632881-116632931 2.6574 chr1:116635630-116635680 1.6490 chr1:116642858-116642908 2.4223 chr1:116642975-116643025 3.0361 116642942 1.21 chr1:142380067-142615067 chr1:142472089-142472139 3.1179 chr1:142472407-142472457 2.8853 142472273 1.22 chr1:142586124-142586174 3.1253 chr1:147795123-147895123 chr1:147841007-147841057 1.9187 chr1:151140741-151254741 chr1:151200154-151200204 2.5446 chr1:151200207-151200257 1.9806 Yes 151200206 1.23 chr1:151203890-151203940 1.9899 chr1:151203890-151203940 2.1120 Yes 151203915 1.24 chr1:151671562-152041562 chr1:151697622-151697672 2.8655 chr1:151723198-151723248 2.8218 chr1:151723145-151723195 3.0027 Yes 151723197 1.25 chr1:151729172-151729222 4.3135 chr1:151756822-151756872 2.0705 chr1:151756928-151756978 2.7010 Yes 151756900 1.26 chr1:151791274-151791324 1.3867 chr1:151802784-151802834 1.8998 chr1:151808602-151808652 3.6921 chr1:151808496-151808546 2.9280 Yes 151808574 1.27 chr1:151820339-151820389 3.8045 chr1:151820233-151820283 3.4778 Yes 151820311 1.28 chr1:151824453-151824503 4.4061 chr1:151824559-151824609 3.3467 Yes 151824531 1.29 chr1:151835344-151835394 2.1675 chr1:151854236-151854286 2.0203 chr1:151854236-151854286 3.0808 Yes 151854261 1.30 chr1:151863719-151863769 3.1661 chr1:151863772-151863822 2.7226 Yes 151863771 1.31 chr1:151865137-151865187 1.3468 chr1:151870669-151870719 2.5251 chr1:151870760-151870810 2.9078 Yes 151870740 1.32 chr1:151916996-151917046 3.3198 chr1:151916996-151917046 3.1002 Yes 151917021 1.33 chr1:151976759-151976809 2.1210 chr1:151981350-151981400 0.8543 chr1:151993365-151993415 2.2438 chr1:151995885-151995935