Intracellular receptors specify complex patterns of gene expression that are cell and gene

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1 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when tested in reporter contexts, whereas three failed to confer androgen regulation in either direction (ECB and KRY, unpublished).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negative glucocortoicoid response elements direct transcriptional activation in simple contexts (M. Cronin and KRY, unpublished); thus, the mechanisms of transcriptional repression may prove to be more complex than induction, perhaps requiring components of chromatin structure or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

2 SUPPLEMENTAL MATERIALS AND METHODS Expression Microarray Analysis We identified androgen-regulated transcripts by converting RNA from vehicle or androgentreated 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 ( 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 MgCl 2, 0.5 mm CaCl 2, 5% glycerol, 0.2 mm spermine, 0.2 mm spermidine, supplemented with protease inhibitors) and disrupted in a Dounce homogenizer. Ten A 260 units of nuclei in DNase I buffer 2

3 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 (N obs ) and the expected number of occurrences (N exp ). N exp 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 N obs by chance given the expected number of occurrences N exp. These P values were then Bonferroni corrected by the number of tests (Patil et al. 2004). 3

4 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: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: chr1: Yes chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: Yes chr1:

5 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr1: chr1: chr1: Yes chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: Yes chr1: chr1: chr1: chr1: No chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr1: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: No chr2: chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: chr2: Yes chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2:

6 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: Yes chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: chr2: chr2: Yes chr2: chr2: Yes chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: chr2: Yes chr2: chr2: chr2: Yes chr2: chr2: Yes chr2: chr2: chr2: chr2: chr2: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: Yes chr3: chr3: Yes chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: Yes chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: Yes chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: Yes chr3:

7 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr3: chr3: chr3: chr3: Yes chr3: Yes chr3: chr3: Yes chr3: chr3: Yes chr3: chr3: Yes chr3: chr3: Yes chr3: Yes chr3: chr3: Yes chr3: chr3: Yes chr3: chr3: Yes chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr3: chr4: chr4: chr4: chr4: Yes chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: Yes chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: Yes chr4: Yes chr4: chr4: chr4: Yes chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: Yes chr4: Yes chr4: chr4: Yes chr4: Yes chr4: chr4: chr4: chr4: Yes chr4: chr4: chr4: Yes chr4: chr4: chr4: chr4: Yes chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: chr4: Yes chr4: chr4: chr5: chr5: chr5: Yes chr5: chr5: chr5:

8 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: Yes chr5: chr5: Yes chr5: chr5: chr5: chr5: Yes chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: chr5: Yes chr5: chr5: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6:

9 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: Yes chr6: chr6: chr6: Yes chr6: chr6: Yes chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: Yes chr6: chr6: Yes chr6: chr6: Yes chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr6: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7:

10 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr7: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: Yes chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr8: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: Yes chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9:

11 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr9: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: Yes chr10: chr10: chr10: Yes chr10: chr10: Yes chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10: chr10:

12 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr10: chr10: chr10: chr10: chr10: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr11: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: Yes chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12:

13 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr12: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr13: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr14: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15:

14 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr15: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr16: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17:

15 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr17: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr18: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19: chr19:

16 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chr19: chr19: chr19: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr20: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr21: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chr22: chrx: chrx: chrx: chrx: X.01 chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: X.02 16

17 Chromosomal_region_tiled Mpeak_peak#1 Mpeak_Value#1 Mpeak_peak#2 Mpeak_Value#2 Validated Mpeak_peaks_mean ARBR chrx: chrx: X.03 chrx: chrx: chrx: chrx: chrx: chrx: chrx: chrx: X.04 chrx: chrx: chrx: X.05 chrx: chrx: chrx: chrx: chrx: X.06 chrx: chrx: X.07 chrx: chrx: chrx: chrx: chrx: chrx: chrx: X.08 chrx: chrx: chrx: chrx: X.09 chrx: chrx: X.10 chrx: chrx: chrx: X.11 17

18 Supplemental Table 2. Primers used for PCR cloning and mutating wild-type ARBRs Nearest ARG Reporter Activity ARBR FORWARD PRIMER REVERSE PRIMER Wild-type ARBRs 1.25 TAGGTACCGATTGCTTTTCTAGCCGCAG ATCTCGAGGGCGTTGATGAGTGCAG Induced Induced 1.41 GCTGCAGGTACCGTCACACGACGTGAACTGGGGCGGT TGCAGCCTCGAGCGGTTCTGCTGTTCAGTGACCAGCTT Induced Unresponsive 1.48 GCTGCAGGTACCAATGCCGAGCACAACCACCCCAAGCC TGCACTCGAGCTCATCACCCTGCTGTCCAGAGAAC Induced Induced 2.16 GCTGCAGGTACCTGCAGGGGTCCTCTAGGTC TGCACTCGAGCACAGTGTAAAAAGGGCCAAGTAC Induced Induced 2.32 GCTGCAGGTACCGTGAGGGTGTAGGATGAGGCCCGCT TGCAGCCTCGAGGGACACCCGGTGTTCTGCAACCCAT Induced Induced 2.33 GCTGCAGGTACCGGTCAAATGCTGGGTGCGTTTCAAG TGCAGCCTCGAGACTGGGGTGTGAAGAAGCACAGCTT Induced Induced 4.02 TAGGTACCTTCATCTTGAAGGCTGCC ATCTCGAGCCAAGGCCCAGGGTGTG Induced Induced 4.29 TAGGTACCAGCCAACTTGGGCAGATAG ATCTCGAGGAAAGTGCCTGCATAGC Induced Induced 5.18 GCTGCAGGTACCTTATTTGAGAAGGGAGGATATCACT TGCAGCCTCGAGTTGTGTCAGTGCTCGGTTCCTAAGC Induced Induced 6.14 GCTGCAGGTACCTTGGTAGAGAAAGAAATAAACAAGTT TGCACTCGAGAAATAGACACTTACCAGAGCTAATGTC Induced Induced 6.17 GCTGCAGGTACCGTTTAGAACCTAAAGACGGGGAGTGGG TGCAGCCTCGAGCTGGGCTGCTGGGGCTGTCTGCACCC Induced Induced 6.18 GCTGCAGGTACCTCATTTCAATACACCCAATATTTATTG TGCAGCCTCGAGGAACAAAGCATTTATATCGCACGTTTACC Induced Induced 6.35 GTACCCGGACAAAATGTTCTC TCGAGAGAACATTTTGTCCGG Induced Induced 7.07 GCTGCAGGTACCTATTTTTTCAAACTTTCCCATCAGTTTG TGCAGCCTCGAGATAAAGCCCCTGGCCTCAGTTTT Induced Unresponsive 7.08 GCTGCAGGTACCGCCACCCTGGGTCCCCCACCAGTCT TGCAGCCTCGAGCAGAGAATCCGCAGGAAATCCCTGT Induced Induced 7.11 GCTGCAGGTACCTCCTGTAACCTCTCCAGGAGGGAAAAGC TGCAGCCTCGAGCTGCCCCCTGCCTGGGTCCACCCCTA Induced Induced 9.06 GCTGCAGGTACCTTTGTCTCCAGCCCTGGGCAGACAT TGCAGCCTCGAGCACCCATGGGGGACCTGTGCCTCGTGCC Induced Induced TAGGTACCTCTCCAGAGGGATTTTCC ATCTCGAGATCAGTGACTTGTGCG Induced Induced TAGGTACCTGAGTTCAAGATCTTCTCC ATCTCGAGAAAGTTGCCTGAGCCAG Induced Induced GCTGCAGGTACCAACTAGGAAAGGAAGGGGCGGGGCA TGCAGCCTCGAGTCAATACTCCCATTCTAGGCATTTT Induced Induced TAGGTACCATCAGTTCGTCCTCTCTC ATCTCGAGAGGTGACCAATATTCGGTG Induced Induced X.11 TAGGTACCAGCTATCCTAGCAAAGTC ATCTCGAGGCCAATAAGACAATCTCAGTG Induced Induced 1.23 TAGGTACCAGTTCTGTGGAGGGAATG ATCTCGAGGAACAGCTTCCCCAGTC Repressed Induced 1.24 GCTGCAGGTACCACATGGCCTTAGATATCTTTTCCTC TGCAGCCTCGAGCTCGGCCACTGTCCAGGAAGAGGCA Repressed Unresponsive 3.03 GCTGCAGGTACCAACCATATGCATCAGAGCTGGGGCT TGCAGCCTCGAGGTCCCCATCTCACAGACATCGTCTC Repressed Induced 3.11 TAGGTACCTGTCAAGATGCCCTGTAC ATCTCGAGGGAAGTTGCTATGCAGC Repressed Induced 4.16 GCTGCAGGTACCCTCAGAGATACCAGTAACCGCCTG ATCTCGAGGTTCCAAGATTTCAAAGAGAGAAAGG Repressed Unresponsive GCTGCAGGTACCCCTAAAGGTCTCAGAAGCTCAATTT TGCAGCCTCGAGGACTCAGGTTCAAGGAAGAGCATGA Repressed Induced Mutated ARBRs m1.48 CCCAGACTTCTATGTCCTGACCAGCTGCCAATCACCTTG GACATAGAAGTCTGGGCAACTCCCAGCCCTCCCCTGCC Induced Unresponsive m2.33 CTAGATTGCATTTTTTCCTCAGGTCTCTGAAATATCAGG CCTGATATTTCAGAGACCTGAGGAAAAAATGCAATCTAG Induced Unresponsive m5.18 CAGAGAGCTTGAGCTTAACGTGAAGCTACCGGTGCTGTTGGG CCCAACAGCACCGGTAGCTTCACGTTAAGCTCAAGCTCTCTG Induced Unresponsive m6.17 CATTCCGGTCCCAGCGTATTAAACTAGACCAGCCTTTTTGAG CTCAAAAAGGCTGGTCTAGTTTAATACGCTGGGACCGGAATG Induced Unresponsive m6.18 TACCTTTCTCACGGCGGAGCGTTCGGCCTGTCTCATATTC TACCTTTCTCACGGCGGAGCGTTCGGCCTGTCTCATATTC Induced Unresponsive m6.35 GTACCCGGGTAAAATTCTCTC TCGAGAGAGAATTTTACCCGG Induced Unresponsive m7.08#1 CGTCCTGGATACAGTAGAGGTTTCCCAGATGTTTAC GTAAACATCTGGGAAACCTCTACTGTATCCAGGACG Induced Modestly Induced m7.08#2 CTGGACTGCTTGCACGTCCATACCTGTGCCATTCTTGG CCAAGAATGGCACAGGTATGGACGTGCAAGCAGTCCAG Induced Modestly Induced m7.08 combination of m7.08#1 and m7.08#2 Induced Unresponsive m7.11 CCTTGAGATACAGGTCCGACGTGTCCTGTCTACGCA TGCGTAGACAGGACACGTCGGACCTGTATCTCAAGG Induced Unresponsive m9.06 CGTGAACTGGGAAAGCACCAAACGCTCTACTCAAAAGCC GGCTTTTGAGTAGAGCGTTTGGTGCTTTCCCAGTTCACG Induced Unresponsive m19.01 CACTGGTTTCGATGTGCCATTACATAGTCCTAACAACC CATCGAAACCAGTGACTGGCCTCTACCGCCCCCAGC Repressed Unresponsive 18

19 Supplemental Table 3. Real-time quantitative PCR (qpcr) primers for androgen-responsive gene (ARG) validation and expression profiling HUGO Gene Symbol ENTREZ GeneID Forward Primer Reverse Primer Response to R1881 in HPr-1AR RPL ATGTATCACAGCCTGTACCTG TTCTTGGTCTCTTCCTCCTTG Control, Unresponsive gene SNAI AGCTACCCAATGGCCTCTCT GGGTCTGAAAGCTTGGACTG Repressed ELL TCAAAGAGACTGGCCTGGAT CAAAAGCCGTTTCTGAGGAG Induced MAF 4094 CAGAACTGGCAATGAGCAAC CCGGTTCCTTTTTCACTTCA Unresponsive gene CXCR CCAGTCTGGGTGGTCAGTCT CTCATGCACGTGAGGAAGAA Induced FKBP AGGCTGCAAGACTGCAGATC CTTGCCCATTGCTTTATTGG Induced DKK CCTTGGATGGGTATTCCAGA CAGTCTGATGACCGGAGACA Repressed FST TGTGCCCTGACAGTAAGTCG CCGAAATGGAGTTGCAAGAT Repressed PHLDA TCACTGTGGTGATGGCAGAG GATGGCCTGACGATTCTTGT Repressed FGFBP AAGGACACTCTGGGCAACAC GAGAGAGATGCCCTCCTCCT Repressed NUP TCGTGCTAGAAGGGGAAGAA CAGATGCCAGTTTCAAAGCA Unresponsive gene MYC 4609 CCGAGGAGAATGTCAAGAGG TAACTACCTTGGGGGCCTTT Repressed TP73L 8626 GAAACGTACAGGCAACAGCA CACAGAAGGCAGCTTGTTCA Repressed AREG 374 GTGTGGGGAAAAGTCCATGA AACAGCAACAGCTGTGAGGA Repressed RIPK GCTCATGATCTGGACGTGAA TCGGAGAGGCTGTAGTCGTT Induced ADARB1 104 TCGTGGAGCCCATTTACTTC CAAAGGCTTGTTGAGGGTGT Induced AADAT TTAATGCTCCCTGGAAATGC GAAGGCCACATCCATCTGTT Unresponsive gene ARL4C CCCTGCATATCGTCATGTTG CCGTTGCTCAGCTTGATCTT Repressed ANTXR CATTAACTGGAGACAGAGGC TTCAAGCCTCCTGCTTTCTG Repressed DUSP AAGGTCCTGGTCCACTGTGA GAGACCATGCTCCTCCTCTG Repressed EDG ACAACGAGTCCATTGCCTTC AGATTGCCACCATGACCAAT Repressed ELK CTTCTTCACCGCACAGACAC AGTGTGGGGAACTGGAACAG Repressed EPB41L4B ATGGCTCCAGGTTACGAAGA TGCTATCACTGGGTTGCTTG Induced CYLD 1540 TCCCTTGCCAGAATATGGAG AAGCCATTCTGACCACCATC Induced CORO2B AGGATGAGATTCGACGGTTG AACAGTTCTTGGGGCTGTTG Induced CYP24A GAAGAATGCAGGGGATGAAG TTGGTGTTGAGGCTCTTGTG Repressed BAIAP TCAAGCAGAGGCCCTACAGT ATCACACCTGTCGTTGGTCA Induced CXCL CACCCCAAGAACATCCAAAG TAACTATGGGGGATGCAGGA Repressed CA2 760 TCTGGAATGTGTGACCTGGA CACCCTCCCCATTGAAGTTA Induced FN GATGCTCCCACTAACCTCCA CGGTCAGTCGGTATCCTGTT Induced FOXO1A 2308 CTTGGATGGCATGTTCATTG CTGTGTGGGAAGCTTTGGTT Induced GATA TGACGGAGAGCATGAAGATG GGCACATAGGAGGGGTAGGT Induced IRF2BP GACACCCATTTTGTGCAGTG TCCACTAGCTCCCTGCTGTT Repressed KIAA TCATACCTCCTCACCCCAAG TGGCGTCAGTGGTAGAAACA Unresponsive gene KLF GTCTCTTCGTGCACCCACTT TGCTCAGCACTTCCTCAAGA Induced KCNN CATGCAGAGATGCTGTGGTT GAACAGCTGGACCTCTTTGG Repressed KIAA TGAGTTGTTTTCGGCAGATG CTCTTTCTGTCCCTCCCACA Induced TBC1D9B CCAACCTGAAAGACCGTGAT GAGCTGGGGAAGACTCTGTG Unresponsive gene with ARBR DNMBP TAAAAAGCACCACACGTCCA GTCAGCTTCGAGTCCAGGTC Repressed KIAA CCTGGAGGAAGATGACGATG TGCAAGCCATAACCACACTC Repressed PLEKHA CTCCATCTCTGTGCCTCCAT TCACTGTGACTCGCTCTGCT Induced C7orf TGCTGATGCTCTGGCTATTG AGGAACTGGCAGCTAACACG Unresponsive gene MAPKAPK TCTGTACGCAGGGAGGAAGT CTGTGAATGCCTGGTCTCCT Induced MGC GAGCAAGGGATCCTGACTTG GGACGAGCTCAATTTCGATG Induced KLHDC8B GGAGAGCTTTAGCCTTGCAC CACCCCCAATAACAAACAGC Induced MPST 4357 ACATCCCAGGTACCGTGAAC CCACCAGTGGCTTAGACAGG Induced MME 4311 CCGAACCTACAAGGAGTCCA GCAAATGCTGCTTCCACATA Induced MTX ACTGGTGCTCCACTGAAGGT CTCTTTTCGAAGGTGGGTGA Unresponsive gene NFKBIA 4792 CTACACCTTGCCTGTGAGCA AGACACGTGTGGCCATTGTA Induced OSBPL ACGGAGCTTCTCACTTGCAT CCACAAGATGGTCTGGAGGT Induced FAM84B GAGTACCTACACGCCCGAGA CCACCTGGAAGTTACCCACA Repressed PAG AAAACATGGTGGAGGACTGC AGATTTTGCCTTGCCACTGT Induced CD GCTATGGTGGTGCCGACTAC TGCTTGTCCAGATGACTTCG Repressed PLAU 5328 AGCCCCACTACTACGGCTCT CCAGCTCACAATTCCAGTCA Repressed PLEC CCAGCTTCGAGGAGATTGAG GATTGGTAGATGCCCTTGGA Repressed PLK AAAGTTGGGGACTTCGGTCT CACAGCCATGTCCTTGTTTG Repressed PTHLH 5744 CAAGATTTACGGCGACGATT GGAGAGGGCTTGGAGTTAGG Repressed PRNP 5621 ACAACTTTGTGCACGACTGC ACGCGCTCCATCATCTTAAC Repressed PMVK TTGGAGCTGATGTCTGTGCT CTTGTAGGTGCTGGTGTCCA Induced TMEM TTGTGCGAGGACAAACACTC ACATTAGGGTGAAGCCGATG Induced ZMIZ AGAGAGGGACCTGGAGGTGT AAACTCGGAGTGTTGGATGG Repressed GPRC5A 9052 GCCCACATCTACCTCACGAT GGAGCTGAGGATGGTGTCAT Repressed RNF ACCAGGAGCACAGAAAATGG AGATGCCGAAAGCAAACTGT Unresponsive gene SLCO2A CCAACTTCCTCATTGGTGCT CGGGGAATGGTTTGTAGAGA Induced PRSS ACTTACGAAGAGGCCAAGCA CCTGAAGACCCTGAGTCTCG Induced SSX2IP AAGCAGAGAGAGTATGTGGG GGTGTACCTTTGAAACTTGG Induced 19

20 HUGO Gene Symbol ENTREZ GeneID Forward Primer Reverse Primer Response to R1881 in HPr-1AR ST TTACTGCCAAGTGGGAAAGG CGCTCCACCTCATCTAGGAC Induced S100P 6286 GATGCCGTGGATAAATTGCT TTGTGACAGGCAGACGTGAT Induced SLC12A GAAAACAGCCCATTCCTCAA TGGGGTTACTGTCCATCTCC Induced SLC16A GTCTATGCGGGATTCTTTGG AGGACAGGACAGCATTCCAC Unresponsive gene SLC22A TCATAGCCCCATTTCTGCTC TCAGGCAAAAGCATCACAAG Induced GRHL CCTCTCACAAGCGAATGGAT GCAATGGGAAGGACATGAGT Induced TIPARP CCATCAGAAACCCTCAGTGG TCTTGGAAAATGCCTGGAAC Induced TM4SF TGCAGGATCTGGCTACTGTG GGTGCTGGCAAAGGTGTAGT Repressed UAP AACCAGTTGGAGTGGTTTGC GAAATGGTTGGCAATGTTCC Repressed AKAP CAAGTCTGGCCACAGAGTCA TTAAGAGCATCGGACCCATC Induced ACSS TGATGAGATCCTGGTGGTGA GGGTCCTCCAAGGTGGTAGT Induced ACSL TTGGGAAGGATTCTGGTCTG GTCAGAAGGCCATTGTCGAT Induced AQP3 360 TTGGCTTTGCTGTCACTCTG GTAGATGGGCAGCTTGATCC Induced DDIT CCTGGACAGCAGCAACAGT TCACTGAGCAGCTCGAAGTC Repressed BMP2 650 ATGGAAAAGGGCATCCTCTC AGCCACAATCCAGTCATTCC Repressed FOXC AGCAGCAGAACTTCCACTCG GGAAGGCCATTTGACAGCTA Induced NSDHL CTATCCCTGCTGGTGATGGT GGCTCTCTCGCAGCTGTAGT Induced ETNK TCACGCTTGTGAAGAACGAG TCTTGCAGAGCAGGTCATTG Induced IL6R 3570 CTCCTGCCAGTTAGCAGTCC AGGCTGCAAGATTCCACAAC Repressed KIAA GTAGCAGCGACCCTGAGAAC GTTCCCTCTGAGCAGTCCAG Induced PCYOX1L CTACTCACTGGGGGAGTCCA GGACAGCAGAAACGACATCA Induced PGM2L CAGAGATGTGTGCGTCACCT AACGCCAGATCAGTCCATTC Induced PTP4A ACACACAATCCAACCAATGC AGGCCAATCAAGAACATGGA Unresponsive gene SGK 6446 TGCTGCTGAAATAGCCAGTG CTCCTTGCAGAGTCCGAAGT Induced TFPI ACTGTGCTCTGCCAATGTGA ATTGTCATTCCCTCCACAGC Induced TLE AATGGTGAGCTTTGGAGCTG AGTACGCTGGTTTTCCTCCA Induced ITGB TGGCAGCTGTGTCTGTATCC CCCCGGTCAAACTTCTTACA Unresponsive gene NT5E 4907 CTGGGAGCTTACGATTTTGC GCTGAACCTTGGTGAAGAGC Repressed ADAMTS CGTCTCACCAAAGGACAGGT GAGGTGGAATCTGGGCTACA Repressed UCK ACAGCTAGCGGCAAGTCTTC CTGCTCCGAGGTAAGGACAC Unresponsive gene with ARBR SEMA3G GGTGCTGGTGAACAAATGG CACAGCAGGAACACATCCTC Induced HMGB ACAACCGAGACAAACCCTTG GTCAACAGGTCCAGCCTCAC Induced SPTLC GGCTCGGTTTTGTGTTTCAG GACGTGCTGACTTCTTGTGC Induced C17orf GGAGCAAAGGAAATGCAGAG CTGTGACTGAATCCCTGGTG Induced TGFB CCACGGAGAAGAACTGCTG CCAGGCTCCAAATGTAGGG Unresponsive gene NEDD ATCTCGGGTGCCTATGAATG GTCCAGGCGATTAAAACAGG Induced BIN1 274 CCTGCTGTGGATGGATTACC TTGAAGGGACTCGTAGTGGTG Induced IGFBP GTTCCTCAATGTGCTGAGTCC GCTGCCCATACTTATCCACAC Induced LASS AAGACGCAATCAGGAGAAGC ATGCCTCGTATTCCACAACC Induced MPHOSPH ATGCCCTCTCAAACTTCCAC CACTCACTGGGGCTACTTCC Induced INHBA 3624 AAAGGAGCAGTCGCACAGAC TTGACCTTGCCATCACACTC Repressed CMTM ATGATCCTCGCCTTTTACCTG CGCTCTGGTTGTAACTCTTGG Unresponsive gene SEMA4B TGCAGACGCAGAGAAGAAAC AGGTACACATGGGGCTGAAG Repressed DCBLD CCTTCTCCATGCCTCTGTTC TGTAAGGGTTCCACTCTCAGG Repressed ST6GALNAC GTGGCCTGTTCAATCTCTCC CATACGGGGCTTTGTGTTG Unresponsive gene PNKD TCTTCCTCTCTGGCTGTGG AAGCCCAGGTTCTCCTCTG Repressed ARID5B ACCATGCCCACAAAGAAGAG GACCTGGCCTCACATTTCAC Repressed ACOT GACCCTGTTGTGGACAGCTC TTCTTCTCGTCCTCGGTCTC Unresponsive gene with ARBR PYGO GGCTCTTGGTCCTTGTTTCC GTGAGCAATCCAGGCTCTTC Unresponsive gene ADAM CTCAGAAGCCACCAGAGCAC TCAGCCACAATCACCAACTC Unresponsive gene EFNA CTACATCTCGGTGCCCACTC GGATCTTGGGAGGACTCTGG Induced EFNA GCCTTGTGGCTCTGGTAATG ACGCTTCGATTTGCTCTCTC Induced SLC30A TGGAAGAAGATAGGGCTGGAC CAGGGAAACATGGATTCACAC Induced PXDN 7837 ATGTCAGCCTTGCCTTGTTC AAAGGTTCTGTTCCGTTTGC Induced GLI GTAAGCAGGAGGCTGAGGTG GGATGTGCTCGTTGTTGATG Unresponsive gene INPP CAGAGGAAGAAACAGCAGAGC TGGGTCAGTAAAGGCAACATC Induced ARL8B GTTCCGTTCGCTCTTCTGG GAATTGACCTGACGCGATG Unresponsive gene BAP ATAGGAAGCTGGCACCACTC AACGGGTTGTCTTGATCCTG Unresponsive gene TMCC CTAGCCTGGGTGAGTGAAGC CCAAGGAATGGAAATTGGAG Unresponsive gene with ARBR SSR CTCATCGCCCTCCTGTCTAC CTTGTGTCTCCCACCCTGAC Induced IL GACAAGAGCCAGGAAGAAACC ACTGCACCTTCACACAGAGC Repressed TRIO 7204 ATATCCGCGAAGTCATCCAG TGTCCAGATCCTCTCCATCC Induced FAM105A CCCGTAACAAGCTGATGAGG ATGCCCTGGCTGAATATCTG Induced ANKH TCTGCATGGCTCTGTCACTC GTTCTGCAAAGGCAAAGTCC Unresponsive gene with ARBR LHFPL GGCTGTTTCTGCTCCAACAC GTCTTGCTGTGCCCTTTAGC Unresponsive gene PHF TTTGGACTGGACTGTTGCTG AGCTGCCTGAAGAAGATTGC Repressed FLJ GGAAGTAGCCCTCGTTCTCC ATCCCTCCATCCTCATTTCC Unresponsive gene with ARBR GRPEL GCTTTGGCTCACTCTCACTTG AATACCCAACTGCCTGATTCC Unresponsive gene with ARBR GMDS 2762 CCTACCTGGCTGAGTTCCTG TGAGATCGCCATAGTGCAAC Unresponsive gene with ARBR 20

21 HUGO Gene Symbol ENTREZ GeneID Forward Primer Reverse Primer Response to R1881 in HPr-1AR DSP 1832 TCAGGAGGGTGGAAGAAGAC TTTGGCAGCATCATCAAGAG Unresponsive gene with ARBR ITGB AGAATCAAAGGCTGCAAACC CTTTCTGGACCCATCTGGAC Induced NOL CTCCATGCTGGTTGTCTTCC GCAAAGAGGGATCTGAGCTG Unresponsive gene with ARBR ZFAND CCCGTGAAACCAGTATTTGG AAGACCCTGTTGTTGGCTTG Induced C10orf CAGAGAACCAGGAGGTTTGG ACAGGACAGACGCCATTCAG Unresponsive gene CAMK2G 818 CTGCAACACCACCACAGAAG CAGGCTCAAAGGAAGTGAGG Unresponsive gene FER1L TAATGGCTGGGAAAGTGGAG GAGGTTTCTGGTCGATTTGG Unresponsive gene with ARBR KIAA AGTTCATGCCCTTCAACCTG CGCTCATAGACAGCAAAGGAC Unresponsive gene with ARBR BAG ATGACCCATCGAGAAACTGC ACCTCTTTGCGGATCACTTG Unresponsive gene with ARBR EPS8L CGGATGTCCACTTCTTCCAC CCGAATCTTCTCCTGGTGTC Repressed CCND1 595 GGAAGTGTTGAAGGGAGGTG AACGGTAGCAGCGCAATAAG Repressed BIRC2 329 CTGGGAACCAAAGGATGATG ATTCGAGCTGCATGTGTCTG Unresponsive gene FGD TGGTGTGCAGCTTTCTTCAG ATAGCCCAGAAGTGGAATGG Induced KRT TTCTTTGATGCGGAGCTGTC TCATACTGGGCCTTGACCTC Induced SPRYD GTGATCCTGTCTCCGACTGC GCTCCGGCTCTATCTCTTCC Unresponsive gene with ARBR IGFBP ACAGGATGTGAACCGCAGAG GGCCCATCTCAGTGTCTTG Unresponsive gene FBXO ATAACGTGCTGGTGGAGGAC TGCGTTTGGGATGTAGTGAG Induced PLEKHG GCATGAGCCACTCTTCATCC CCTTCACCAGCTCTTTCAGC Repressed GRAMD GCTCCTTGAGTTGGGATGAC CAGGGATCATTGGTGGAGAC Induced BNC1 646 ACAGTGGAGGGCTGTAATGC AGGTAAGCTGCACGGAAATG Unresponsive gene with ARBR CDH AAACCAGCACACCCTGTACG GCCTCATTCACATCCTCCAC Repressed OSGIN ATGCAGAAGAAGCGAAGAGG CTACAGCACCGGACACAAAG Induced BCAS AAGCAAGGAGGTCGCTGTAG CATCAAGCTGGGAAGAGGAG Unresponsive gene FAM104A CCCCTCTGTTCCCTCTCTTC GTCTTCTTCGTTGCCATTCC Induced CYGB CGTGGAGAACCTGCATGAC TCCAGAATGACCCCAGAGAG Unresponsive gene TBC1D AGGTGGAGGAGGAGTACAAGC GTAATAGCGCAGCAGGAAGG Unresponsive gene with ARBR RAB11B 9230 GGTGTACGACATCGCCAAG CGCAGGTCACTCTTGTTGC Unresponsive gene with ARBR FLJ ATGTGTTGGTGGTTGGTGTC CCCTCGTTCCTTCATTTCAG Unresponsive gene with ARBR TMEPAI CAGCCATCTGGAGCAAAGAG CTAAGAAGCGCGGAGTGTTC Repressed COMT 1312 ACGCTTCTCTTGGAGGAATG CCAGGAACGATTGGTAGTGTG Unresponsive gene SFI CGTCGCTGGAAAGAGAACAC TCTGCACTGACACAGCTTCC Induced PISD CAACCTCAGCGAGTTCTTCC CCTGCTCCACCTCACAGTTC Unresponsive gene TSC22D CCCAGCTAGAGCGTGAGAAC AGCCACTTACACCGCAGAAC Induced PLAC TCCATGAGAGTAGCCAGCAAG AAGACACAAGGTGGACAATCG Induced CETN GTTGCCACAAGCAGTCATTC ACACATCCAAATGCCAGTGC Unresponsive gene ZBTB GCTGTTTGTGTGTGAGGAGTG CAGGTGCATGTTGAGATTGG Unresponsive gene KLHL GAACGAATGGGACAAGATCC CACGTCCGAGAGTTCAAATG Unresponsive gene with ARBR SOX TCCTGGGACTGTAGCTCTCC GTCCAGCTTCTCCTGGCTTC Unresponsive gene with ARBR MCEE TCAAGCTCCCATTCCAACAG CTGCTATGGCTACATGGTTGAG Induced DPP CCAAAGACTGTACGGGTTCC TTGCATTGGTGACTGAGCTG Induced LNX TAAAGGTCAACGGGATGGAC ATCACAGTCAGCCACAGCAC Induced FGF TGTGCTTCCAGGTACAGGTG GCTGCTTACGGCTCACATC Induced C6orf TGGAGTGCAAACAGGATGAG GTAGCCCAGCAACACGATG Induced ZNF TGACCCAGGAACAGAGAATG GTTGAATGAAATGGGCCTTG Induced TUBAL GCTGACAAAGCCTACCATGAG AGCCGAGGATCACACTTGAC Unresponsive gene with ARBR SFMBT AATCAACGCAGCCTACAAGC TATTTGGCCTTCAGCGTCTC Induced ANK3 288 TCTGCTGGAATATGGTGCTG TTCGCATTTCTACCGAGGAG Unresponsive gene with ARBR MMP AACGCTGGACGGATGGTAG GATCTCCATTTCCATAGGTTGG Induced EPSTI AAACCGCTGAGTTCTTGAGC TTCCATGTTGAGGATTGTGG Induced DNAJC CTACGCATTTCGGATCTGG GACCAGCTTCTCGCCTACTC Induced SERPINB AGTGAAGCCAACACCAAGTTC TGCTGATGTGATGCTGATAGG Induced ANGPTL TGGCTCAGTGGACTTCAACC GTGATGCTATGCACCTTCTCC Repressed THBD 7056 GCTTGCTCATAGGCATCTCC CGCGCACTTGTACTCCATC Induced IL TACATCCTCGACGGCATCTC CCATCTTTGGAAGGTTCAGG Repressed SOX ACAGCGACAAGATCCCTTTC CGGACTTCACCTTCTTCCTG Unresponsive gene DST 667 TTCTGCTGCGTCACCTACC CTCCTGTGGCTGGAATAACC Unresponsive gene with ARBR KRT ACAACAAGTTCGCCTCCTTC TGGGCTCCAGGTTATTCTTG Induced LAMB GGCAATCCATCAGAAGTTGG CACCTCCCAGTCTCCTTGTC Unresponsive gene with ARBR TNFRSF1A 7132 GCCAGGAGAAACAGAACACC TCCTCAGTGCCCTTAACATTC Unresponsive gene with ARBR ALOX5AP 241 ACTTTCCTCGCTGTGCTCTG GTGCTCTGCGTTCTCTCTCC Induced PGS TCACCAGGAGCAAGAGCAG TCTTCACCCACAGCTTCACC Unresponsive gene with ARBR TMEM CCACACGGTCATGCTAACAC TGAGAAGCCAGCAAATCCAG Induced B3GNT GGTCGTGTTCATCGTGGTG CGGCTGTGTAGTCAGGGTAAG Unresponsive gene with ARBR SEC14L GGGAGATGACAGAGGTGCTG AAACCGCAGGACATAGATGC Induced SCAP AGGAGCTTGGAGGAGTTGTG AAGCGTTCCCAGTCATTCTG Unresponsive gene with ARBR NPAS AAGGAGGTTTGCTTCATTGC TTGGAGGTGCTCTGTGATCC Unresponsive gene with ARBR PDE4C 5143 ATCATGGCCGAGTTCTTCC CACTGAGGCCGTATGCTTG Induced PITPNC GTTGAGGGAAGGCTGGAGAG AGCAGAATGTCTCGGACCAC Unresponsive gene with ARBR AP3S GATCAAGGCGATCCTAATCTTC CTCCCTGATGATTTGCTGTTG Induced 21

22 HUGO Gene Symbol ENTREZ GeneID Forward Primer Reverse Primer Response to R1881 in HPr-1AR PIK3R ACTCACCTTCTGCTCCGTTG TTGACAATCTGGTCCTGCTG Unresponsive gene with ARBR DLGAP ACTCGGATACCCAGGATGC TCGATGCTGATGGAGTTGG Unresponsive gene with ARBR ERN TCCCTCAACATCGTTCACAG TTCTTGCAGAGGCCAAAGTC Induced TNS GAGAGAACTGGGAGGTGCAG AGCTCAGGTACAGGGTGTGG Repressed TULP ATTCAGTACGAGGGCAGGTG TGACAGACCCAACCAGGAC Unresponsive gene with ARBR LTBR 4055 AGAACCAAGGTCTGGTGGAG TGGCAAGGAGCAGAAAGAAG Unresponsive gene with ARBR TNFAIP GCACGCTCAAGGAAACAGAC TGTCCCATTCATCATTCCAG Unresponsive gene with ARBR UFD1L 7353 GAACTGCGTGTGATGGAGAC CATGCTGGACTTGTCTTTCG Induced DNER CAGCTTGTTGCAGATCCTTG AAATGCAGAGGTAGCCATCG Induced NDRG ATTATTGGCATGGGAACAGG CAAGGGTTCACGTTGATAAGG Induced DYRK1A 1859 TGCAAACCTTCATCTGTTCG AAGGCAGAAACCTGTTGGTC Induced C1QBP 708 TTGGGAGCTGGAACTGAATG ATCAAATGTTGGTGGGATGC Unresponsive gene with ARBR TNFAIP CGTGGTCAGTTTCCATCAGG TGTGACTTGGCAGTGAGGTG Unresponsive gene with ARBR DPF AAAGGGTCCTGATGGATTGG AATTGGAGGCAAGATGGATG Unresponsive gene with ARBR RHOU CCGGACAGGATGAATTTGAC GACGTTCTGGAAGGATGAGG Induced SERTAD TCGAGGACGACGATGACAC CCAAGGCAGAGGAGAAACTG Unresponsive gene with ARBR JMJD1A CAGGGTTGCTCTCAAAGTCC TTAGGAAGGCCAGTCAATGC Unresponsive gene HYAL GCACAGGGAAGTCACAGATG GCAGAAAGTGGTTTGTCGTG Unresponsive gene NAT CTACACACCGGATGGAGCTG TCATCTCTGGTTGGCATGTG Repressed HYAL AACCAACTCGGCCTCTATCC GCTGTGGTGGATCTGGTAGG Unresponsive gene PHF GGTGGTGCCTCATTCTGTG CACTCCTCACACTCCCATTTC Induced WWTR CAGCAATGTGGATGAGATGG TCAAGGAAATCAGGGAAACG Unresponsive gene LPP 4026 TGGACGCTGAGATTGACTCC AACTGGAGGAGAGGCTGTTG Induced CD ACTGCCAAAGTGTATGGGATG GCAAGGTACGGTCTGAGTTCC Unresponsive gene MTHFD2L CACTACCAGACCACGTTGATG GGCAGGTATGAGAGAATGCTG Unresponsive gene with ARBR EPGN TGACAGCACTGACCGAAGAG CCAGGCAAAGGTGTGAGAAC Repressed TLR TGGAAGCTGGTGGCAATAAC GGGATGGAGAGTCACACAGG Induced SLC26A GCAATTATGGTTGGCAGCAC AATCCACTCAGCAAGGCATC Induced LEMD TGGTTGAAAGGAGAAGACCAG GATGAGCACGTTGGTGACAG Unresponsive gene with ARBR DEF CTGGTGCCTCTTCAACTTCC CACCTCCAAGCTCATGCTG Repressed PPARD 5467 TGATGCCCAGTACCTCTTCC TCTCGGTTTCGGTCTTCTTG Unresponsive gene TBC1D22B TCCCTGCCAACTACAAGGTC GGAGTGATTGCTGTTTGTGG Induced RNF AGCATCTTCAGGGTTTGGAG CCTTCTTGCTGCGATTTAGC Unresponsive gene with ARBR TJAP TGCCCGAGTGTTAGAGAAGC CCACATGCACAAAGACATCC Induced VEGFA 7422 CCTTGCTGCTCTACCTCCAC GAAGATGTCCACCAGGGTCTC Induced UST ACTATCCCGAGTGCTCCAAC TCATTCACGTTCAGCTTTGC Induced PBXIP CTCTTCTCAGGTGGCCTCTC AGCTGTTCCCTTAGCCCATC Induced FLAD CTTCACCCATGTCCTCACAG ATAGCTTCTCCCAGCCTTCC Unresponsive gene LENEP CCAAACCCTACCCTTCTTCC CCAGCAGTTCCTTGATACACC Unresponsive gene ZBTB7B ACCTCAAAGGCCAGAACTGC AGAGAGAGGCGGAAGGTGTC Induced DCST TTATCCGACACCTCCTCCTC CCAGCGTAGCCAGTACCTTC Induced RAG1AP GCAGTGTCTTCACCATCAGC GAAACCCATAGAGGCACCAG Induced DPM ACTGTGGGCTATCGTGTGG AATGGGAGGAAGGGCTGTC Unresponsive gene C1ORF TGCAGCTCAGAAACAGATGC CTCCTCGTAGTCCCATGACTG Unresponsive gene SCAMP CCAGGATGTGCTCTTTGTCC CACGGATACTGCTGTGTTGC Unresponsive gene CKS1B 1163 CGGACAAATACGACGACGAG GCCAAGATTCCTCCATTCAG Unresponsive gene SHC AGGAGGAAGAGCCACCTGAC TCCCGAAGCCTCATGTCTAC Induced CXCL ACCGAAGTCATAGCCACACTC CCCCTTGTTCAGTATCTTTTCG Repressed EREG 2069 CATCTTCTACAGGCAGTCCTC CCATTCATGTCAGAGCTACAC Repressed SERPINB CGCGGTCTCGTGCTATCTG GTAGGTGATGATCCGAATCC Induced GBA 2629 CAGCCTCACAGGATTGCTTC TACCAAGGGCAGGAAAGGTC Induced PDE6A 5145 TGACTTTGTTTGCACCTTCG TCTTCTCCTCCTGCACCTTC Induced WDR AGTTCCTGCCCTTCCACTTC CATTCAGAGCTGCCACAATC Unresponsive gene with ARBR PKHD GGCCTGAGAGGACAGAACAC TATTTCCAGGGCAACAGAGC Unresponsive gene with ARBR AKR1D AGAGATGAGAATGGCAAATGG CCTGGCTTGTTCAGGATGAG Induced MGAM 8972 TACCAATCCCAACTGTGCTG GAACATCCTGAGACCGAACC Unresponsive gene with ARBR LRRC8A TCTTCTACGGCCTCATCTGC AAGGCGAAGTCGTTCTTGAC Induced HKDC CCTTTGGGCTTCACATTCTC TTCTCCTCTTGATGGCTTCC Unresponsive gene with ARBR DCLRE1A 9937 GTTCTCTTCTTGCGGACCAG AGGCAGTGTTGATGGCAAAC Induced SCNN1A 6337 TCTGCACCCTCAATCCCTAC ACAGGTCAAAGAGCGTCTGC Unresponsive gene with ARBR SOAT GGACCATGCTGTTTCTAGGC GTATGGCAGGACCAAGATCG Induced NAP1L GTGAAGAAGCGGATGAGGAAG CACTCTGCTGGGTTTTGATCC Unresponsive gene NTN ATTTGGCCTGGAAGATGATG TCGTATGGTGGTGACAAAGC Unresponsive gene with ARBR NFYB 4801 AGGTGCCATCAAGAGAAACG GTGACTGCTCCACCAATTCC Unresponsive gene DAAM CTTACCCGCATTTCATGTCC GGGTCCTGTCCTTTGTCATTC Unresponsive gene with ARBR CRISPLD GGGAACTGGATTGGAGAAGC CCGTTTCCACCTCATTCATC Induced DCST GCAACTTTGGGCAGACCTAC CTCCCAGCTTGTGTTGAGC Induced KRTCAP CTTTGCTGGGATGCAGATG AGCGAGAACACGAAGAGACC Unresponsive gene TRIM ACCTGTCCTGCACTTCTTCC CCTGGAACACTCCGTACTGC Unresponsive gene 22

23 HUGO Gene Symbol ENTREZ GeneID Forward Primer Reverse Primer Response to R1881 in HPr-1AR MUC CTGGTCTGTGTTCTGGTTGC GGGTACTCGCTCATAGGATGG Induced THBS AGGACACCAAGGACAACTGC GGACCAGGAGGCACATAATC Induced CREB3L GTGTCAATGGAGGTGGAACC GGTGGTAATGTGGGTGAAGG Induced THOC GCAACAAGGATGATGTGGTG CAACCATTGCCATTTGTCAG Unresponsive gene ANXA8 244 GCTCATTGTGGCCCTTATGT AGGCCAGGATCTCAATGATG Repressed ASB CACCTGGGCACTCCACTCTA ACCAGCTCCACAGGACGTTT Induced ARHGAP GTGGTGTTTGGACCCACTCT TATCGGGCACGGTGTTAAAT Induced F CCGACGAGATTGTGAAGGAT CCGAGGTTTGTCTCCAGGTA Induced GPR CCTTGGAGTTCCTGCTGTGT AGGTGGACACGAAGACCTTG Induced IGFBP TCACAGCAGACAGTGTGAGAC AGACCCAGGGATCCTCTTC Induced SLC9A TGATGATAAATATCTGCGGAAGC TTCACGACAATCATTTAGAGATGC Induced IRS GGCTTCGTGAAGCTCAACTG AGAAGAAGCTGTCCGAGTGG Induced CPEB GTCAGCAACAGGAACAGCAA GACGATGCGCTAGATGTCAA Induced ATP1A1 476 AGACCGAATTCCTGCTGACC AGAAGGCAATGTTCCTCGTCTC Induced RAB11FIP CAAGGAGCGAGGAGAAATTG GGTGTCTGACCCACTGTCCT Induced SDPR 8436 CAGCGTGTTTGTGAAACAGC GGGCAAATCATCATCTGAGG Induced SLC19A TTTGGGTGTGCTATGCATCC GCCACTGGCATCTACCACAA Induced PYGB 5834 CTGCTTCAAGGACATCGTGA TGACCTTCTTGGTCCACTCC Induced BRF CAGAAGTGGAGACCCGAGAG GACAGTGGAGACAGGGGGTA Induced STOM 2040 CAGACACTGACCACCATTGC TTCATGCTTGGAAGGCTAGC Induced MT1X 4501 AAGAGTGCAAATGCACCTCC ACAGCAGCTGCACTTGTCTG Induced AMIGO CATGCCCCTGCAAGTGTAAA TCAGGGGTTCCAAAAACACC Repressed VIPR ATTCAAGGCCGCAATGTAAG AAACTCGCTGCCTTGTCATC Repressed CXCL GCAGGGAATTCACCTCAAGA GACAAGCTTTCTGCCCATTC Repressed SEC22C 9117 TGCTCCTAATTTCCGAATGG AAATGCAGGCTACGAAAGGA Repressed ANKRD TTAGCGCCCGAGATAAGTTG CACAGGCGATAAGATGCTCC Repressed GPR AAGCTTGGTTCTTTGCCTGA AAACCACACATTGGCAGTCA Repressed AIM1 202 TCCTTTCATCCTGGAACCTG GGCAAATGATCGTCTCCAGT Repressed CYR GTGGAGTTGACGAGAAACAATG CTGGGACCATGAAGTTGTTTG Repressed CYP4B GGCTTTCTCAAGCTCATCCAC GCCCAGGACACCACTTTG Unresponsive gene with ARBR DHCR TGTTGCCTGAGCTTGATGAC GACCAGGGTACGGCATAGAA Unresponsive gene with ARBR PDE4DIP 9659 TCCGGGATGTTGGTATGAAT TTATTGGCAAAGGAGCCATC Unresponsive gene with ARBR EFNA TACCTGGTGGAGCATGAGGA AACCTCAAGCAGCGGTCTTC Unresponsive gene with ARBR CDC42EP TCTGTGTTCACAGAAACGCC CATGAGAGCTTGGGATCCTC Unresponsive gene with ARBR NET GACCTGCAGGATGGAGATGT CTTGTGGAACACGTCATTGG Unresponsive gene with ARBR ASB CATGAGACTGCCCTTCACCA CCAGTGGCCTTCCTCAAGTT Unresponsive gene with ARBR DEAF TGTTTGAGCAAGCCAAGCAT GCTGGTGATCCTTCCAGTCC Unresponsive gene with ARBR BIRC3 330 GACAGGAGTTCATCCGTCAAG TTCCACGGCAGCATTAATC Unresponsive gene with ARBR VGCNL ATGTGAAAGATCGCTGGTGTG ATAATCAGTGGCCGTGGAATC Unresponsive gene with ARBR ABHD ATGAATGCCATGCTGGAGAC TCAGGTTCAAACACCGGAC Unresponsive gene with ARBR CDH1 999 TGGAGAGACACTGCCAACTG TTAGGGCTGTGTACGTGCTG Unresponsive gene with ARBR ENTPD6 955 AACAGGACATTCCGTTCGAC TTCCGTTCATGATGGAAACA Unresponsive gene with ARBR ID AATCCTACAGCGCGTCATC GGCTGTCTGGATGGGAAG Unresponsive gene JUN 3725 GCTCGGGCTGGATAAGGGCTCA GATTTGCAGTTCGGACTATACTG Unresponsive gene LAD AGATACCACACGGCCATACG TGAGCCTTGATGTCACAACC Unresponsive gene BHLHB TGAAGGCCATTTCACTAGCAG GTAGAAGGGCAGGCAGAAAG Unresponsive gene CNO AAGAAGCTCCTGCACACGAT CAGAGCTGAGGCCTTTCACT Unresponsive gene KRT6A 3853 TGCTGCCTACATGAACAAGG TGTCTGAGATGTGGGTCTGC Unresponsive gene MT2A 4502 GCAAATGCAAAGAGTGCAAA ATCCAGGTTTGTGGAAGTCG Unresponsive gene MLYCD AAACTGGATGGACATGAAGC ACGATTGCCTGGATGTTG Unresponsive gene MTP TCTAGCCTCTGTGGCCATTC TTCCCCACTGTTGGGTAGAG Unresponsive gene 23

24 Supplemental Table 4. Real-time quantitative PCR (qpcr) primers for AR binding region (ARBR) validation DESCRIPTION/ARBR FORWARD PRIMER REVERSE PRIMER Control, NO_ARBR_HSPA1A promoter TCTGGAGAGTTCTGAGCAGG CCCTTCTGAGCCAATCACCG ARBR2.16 ACAGGTAGGGCTGGAGAAGG AAAAAGGAGGAGCACCACAG ARBR2.17 GACAGCTCCCGAATCATCAG GTTGCCAGGGTTGTACCAAG NO_ARBR_SGK (+471 from +1) TGCCAGCGGTAAACATTCTC CCAGTTCAAGCCAAATCAGC NO_ARBR_SGK (-282 from +1) GCGGCGGAATAAATGAGAG AGGGCCGTTATCAGTCTCC ARBR6.35 AGGGTTTTCAGGTGGGACAG TGAGGAGGTAACAAGCGAAGG NO_ARBR_SGK (-1924 from +1) TTCCCTTCTCTCGGTTTGG GCCTTTTCCTTGGTCTTGG NO_ARBR_SGK (-2576 from +1) GTGCAGGTTGATGTTTCTGG GTGACTTTGTTATTGCCAGTGC NO_ARBR_SGK (-3049 from +1) GCAATGGGGAGAATAAATGG AGACGGCTATCCCTGTTCTG NO_ARBR_SGK (-3667 from +1) ACCAGGGAGTATGAGGAAAGG TTAACACTGCACAGGCATTAGG NO_ARBR_SGK (-899 from +1) CCTTCCTCCTCTTCCAAGTTC AAGATTTCCTGCCCCGAGTC NO_ARBR_SGK (-1482 from +1) CCTCTCTTCCCTGCCTTCTG ACCTCGCTCGCTCTTTATGTG NO_ARBR_TIPARP (-3152 from +1) TTAGACAGGGTCCTGTTCTG ACGGTGGCATGCATCTCTAG NO_ARBR_TIPARP (-2993 from +1) CGTAGAGATAGGGGTCTCAC GTCTCATTTTGGCCACGCAG NO_ARBR_TIPARP (-2581 from +1) AAGTCTAGAGGCAGCCAGTC GATGGAAGCAACAAGCTAAGG NO_ARBR_TIPARP (-2065 from +1) GAGGCTGAGAAACTAGCCAG GGTATAGCCCAGTTCCAAC NO_ARBR_TIPARP (-1734 from +1) CTTGCACATTGAGGCTTGTC CCACGAGCTAAGGATGCTG NO_ARBR_TIPARP (-1128 from +1) CACTGACCAAGATCTTGTCTC CAGGGCCATTAAACTAAGGTG ARBR3.30 ACAAGGCCCACGAAATAGTC CACCCTGTGAGGAAGCAAAC NO_ARBR_TIPARP (+503 from +1) GGCAGGTGTATACATCAGACC TCCTAGGCGCCTCAGAGCAC NO_ARBR_SGK (+1346 from +1) TTCTAGCCAAGTCCTTCTCAGC AAACTGCCCTTTCCGATCAC ARBR6.34 TTGGCGGTAGACACTCCTTG GGGTGCTTCACATTCTTCAAC NO_ARBR_SGK (+2201 from +1) GGCTCGTTTCTATGCTGCTG ACAGCCAGTGCTACGTCTCC NO_ARBR_SGK (+2659 from +1) TCGGACTCTGCAAGGAGAAC GGGTAAACCAGGCACCAAAC NO_ARBR_SGK (+3223 from +1) AGGACTGTGGACTGGTGGTG GTAGGGAGCAGGCAGTGTTC NO_ARBR_SGK (-8870 from +1) CCATGCCAGGAGAGTAGTTCTG AAGTGAGGAATGTGCTGGAAAG ARBR2.15 GTCATCAGCCCAGTCATGC GGATCAAAGTGCCCAAGAAG ARBR3.31 CGGTTTGGTATGGTGGAGAG CACGAAGAAGGAGGGATCTG NO_ARBR_TIPARP (+2024 from +1) GCTGTGTGCATTGTGGTAGC ATTGTGCCAGGTGAGAAAGG ARBR3.22 TCCAGCCCCATAACTCCTAC ACTGTTCTGTCCCCTCATCAC ARBR3.23 CCCTCTTCTGTGCTACTCTGG ACGGTGTGTTTCTGAGAGAGTG ARBR3.24 AGCGCATCTGAAATGTTACACC GCTGACCCTAGAAGGGATTATG ARBR3.25 ACACACTCGTGCTGGACAAC TCCTGCCAACTGAACTACCAG ARBR3.26 AAACAGTCCAACTGGCAAGG CGGAACAAGATGAGAATGGTC ARBR3.27 GGCCTGCTCTAGGGTCTTATG CAAACCACAAGGGAATGAAAG ARBR3.28 TGAGCAAAGGCAACTTGAGTG GGAATCCTGCAAAGAGACAGG ARBR3.29 AGGCTTTCCCAAGTGATTCTG AGCGGTAGTTATGCAATGGTG ARBR1.15 TTATCTTCAAGCTGCCTCACC GACTGGCTGAACCTCTTAGGC NO_ARBR_SSX2IP TGCACATTTGGATCTCATGTC CAAACTGCCACATGCTTAGTTG ARBR1.23 TGCCCTCCATTAGGTATTGC AATCTCCTCTTCCTGCTGTCC ARBR1.24 GCCAGCCAAAGCTAGTCATC AGGCTACGGCAAGTCAAGG ARBR1.52 TCCCGTGGTTAAACTTGGTC TGTGGCTTCTGTAGGTTCTGC NO_ARBR_SLC30A1 TGAGGCTCTATCTGGGAAGC TAAAGCAGGACCAGCCAAAC NO_ARBR_SLC30A1 CCAGTGCTAGGAGAAGGAGAAC CTTTATCTGGTGGAGCGACATC NO_ARBR_MPHOSPH10 GAGAAGAGATGGGACAAGATGC TGTCGAGGTTGAGTTGGTTTC ARBR2.10 TTCCCAACAGAAAGCTGGTC TACCGCGCACCAATAAAGAG ARBR2.31 CCAAAGGCTGGAGAGAGATAAG GATGTTCCTCAAGACACAGCAG FALSE_NEGATIVE_ARBR_INPP1 AATCTTCCCAGAGCAGGAGAG TGTTGCTGTCATTACCTCATGG ARBR2.39 AACGGATGGAAGCGATACAC TTTGCAGACACCAGCAGAAG ARBR2.40 TCATTCCCTGATGTCTGTGC GAGAAAGTGAGTGGTGGATGG ARBR2.41 TGCTCTCACAGAACAAACAGG TTGAGTTACCCAGGCTTTCAC ARBR3.09 CCTGTGGGAATAGCAAGGTC AACCCAGGAGTTGCAGAAGC ARBR3.10 AGGACAGAGGGTGACAGTGC CCTGATTCGGTGACCTGTG FALSE_NEGATIVE_ARBR_OSBPL11 TTCCATTCAGCATCCAAAGAG ACTGGTAGGGAAGGCACAGAG ARBR3.12 GCAGCCTACGAAAGTCCATC ACCCAGAGTTGGAACACAAAG ARBR5.01 TGTGGTGGGAGAGACAACAG ATTTCAGCAGGCGAAGTAGC 24

25 DESCRIPTION/ARBR FORWARD PRIMER REVERSE PRIMER ARBR5.14 CCCTTGGAGTTAGCCACAAC AATGGAGCAGTGTGCATGAG ARBR5.15 GGGAAGAACAGCTTGCTCAC TGCCTCACCCTAGTCAAAGC ARBR5.33 CCGAAGCTGAAGGGAACAC GCCAGGTCGTCCAGTTTATC ARBR6.29 CAAGCATAAGACAGGCAGAAAC CCAGAGAGAACAGTGCAGAATG ARBR6.30 ACCCTCCCAGGAAGAATTATG CCTTAGATGAGCGGTAGAGAGG ARBR6.31 AACTGCCCAGGACACCAAG CCAACTCCAGCATTGTAACG ARBR1.12 TCCACTTCCAGAATGTCCATC AGGAGCAGGGTAGCAAGACC ARBR1.49 CCGTGTTTGTGTCCTTCAGC GACCCATTGCTTCACTTTCC ARBR2.04 ACTGTCACTCAACGCCCATC CAGCTTGGACCTCAGGAAAC ARBR2.07 ATCTGTCCCACAGCAAACACTC GGGACCAAGTGTCTCTTCACTC ARBR2.23 GCACAGTCAAGAGAGGCATTAC TGGTCTCTCAACCTATTGATGG ARBR2.24 CTAGTGGTCCTTGGCTGGTG ATCCTCAGATGCTGGCTTTG ARBR2.25 TCCTTGAAGTTCCCTTGATCTC ATACATGCCAACACCACTGAAG ARBR4.08 GGTGACAAATGGTCCAATCC GTGTCTCATGCCTGTTCAGC ARBR4.09 ATAAGACTGCGATGCCTTGG ATCTGTCCCTGCCAACAGAG ARBR4.10 GCCACACACAGTCAAAGTCG GGAGTTGCTGGTGATCTAGGC ARBR4.01 TTGGGTATTCAGAGGCATCC CCAGGACTGTGGGAATGAAG ARBR4.05 CCATACCCTTTCCATTCTCG CTTCTGAGTGCTTGTTGTGG ARBR3.20 ATTTCTGAGCCGCCACTTC CAGCCAGTCTCCAAGGATTAG FALSE_NEGATIVE_ARBR_MME TTAAACTGCTCGGCCAATATG CCCAGAAAGGACAAAGTGAGG FALSE_NEGATIVE_ARBR_MME TGGGTGTTGCTAGTGTTTGTG TGATGCAGAACAGCAAGAGAG FALSE_NEGATIVE_ARBR_SLCO2A1 ACTTCCACCACACCAAGCTG AGACAGCCAGGAAGGCATAG FALSE_NEGATIVE_ARBR_SLCO2A1 GCAGCCAGTGAGGTGAAGAG CAAGTTCCCAGGTCAGGAAG ARBR3.16 CTCCTGAAACTCTGGCAACAC CTGGAAACCCAATCATTTCTG FALSE_NEGATIVE_ARBR_AREG CTGTATTGTATGCCGTGGTG GCAGCTCCTTTAGGCCAAG ARBR4.14 GCAACACAAGGAACAAGCAG ACAAGGTGCTGGGAGATAGG ARBR4.15 CCCAGCGCAATAACAGATTAC TTGTCCAGCCTCAGAAAGAAC ARBR4.16 GCATTTCCAAGCCTGTTTAGC AGTCTGCATCCACCAGGAAG ARBR5.16 CAAGTGACGCCATCCAGTC CAGGAACGCAAGATTCACAG ARBR6.33 TCCCACGAAGTGCTTGTCTAC GGCAGGGTTCTGTGTTTCTTC ARBR8.04 ATCTTCTGGCTCTTGGATCG GCAGCACTAAAGGGCATCTC ARBR9.04 CCTGCCAAACGAACTGAAC CTGGTAGCCTCTCTGCTTCC ARBR10.04 TCCACCTACCTCGGGTTTC GCCTGCACTTTCCTGATCC ARBR10.05 ACTGTGTGAGGTCAGCAACG CCACTCTGCACGGAAACAC ARBR10.06 GCTCAGGCTCACTCTGTTCC CCCCATCTCTACCCATTCTG ARBR12.09 CCTCTTTCACAACCATGTGC GACAGAAACGCAGGAACCAG ARBR4.17 GCAGCAAGCCAGAGGATTAG CTGTTCCTGGTCCTGACACC ARBR4.18 TCCTAACCAAGTGCGTGAGC CTAGACTGACGGGTGGAAGG ARBR4.19 TTCTGAGCACGGGTACATTTC TTGGAGCAACTTCATTTCAGC ARBR1.25 GAGCCTGCAACTCATTACTGC TGCATCTGACCACTCTCCTG ARBR1.26 GGGAACAAGAGGACATGCTG GCCTCATTCTCACCCTTGAC ARBR1.27 CTGGCAAAGGTCCAGACAC GCAACAGCTCCAAGAAGTGC ARBR1.28 GAGTTTCCTGTCACTGCTCTCC CTAAGGGCACAAGGTCAAGC ARBR1.29 CTGCGAGTGCTGTAGGTGTC GGAAACAGGTGCTCATGCTC ARBR1.30 GGGCTTAGAAAGGATGAGGTC CCCTTCCCATTAGCAGTTCC ARBR1.31 GATGTGTGTGCTTGGGTGTC CCAGGGATTCTCACTCCTACTC ARBR1.32 GAGCGGTCAGATGGAAAGC AGTGCGGGCAGTAAATATCC ARBR1.33 GGTGGAGCATGAGGAGTACC CAGGGTGAAAGGTGTGAAGC ARBR1.34 TTAGGTGGTGGGAAGAATGC TGCAGTGAGCTATGATTGTGC ARBR1.35 AATGTTTCGTTCTGGCCTTG GCTTGTAAGAGCAACGTGGTG ARBR1.36 GACCTTGCACACAGGCTTC CCGAGTGACAGAGTGACATCC ARBR1.37 AGGCTGGGTTGTTATGATGC CCAAGGAAGACAGGGAGGTG ARBR4.13 TGTGATGACTCAGGTTTGC TGTGCCTTATGGAGTGCTCC ARBR1.41 CCGGAATGTCCATTCAGTTT TCCTGGGCTTCTGATGTCTT ARBR1.48 AGTGAATGGGATTTGGCAGT AAGGGGTAGAGCAGAGCACA ARBR2.32 ACCCAGTGGGAGGAGAAGTT GGGGAACAGGAAAGAAAAGC ARBR2.33 GCCACATATTTGTGCGAGAA AGTTCAAAAGCAGGGCAAGA ARBR4.29 AGCTGATGTTCAGCACAGGA CCTGGTGTGGTCTCTTGCTT 25

26 SUPPLEMENTAL REFERENCES Baugh, L.R., Hill, A.A., Brown, E.L., and Hunter, C.P Quantitative analysis of mrna amplification by in vitro transcription. Nucleic Acids Res 29(5): E29. Ben-Dor, A., Shamir, R., and Yakhini, Z Clustering gene expression patterns. J Comput Biol 6(3-4): Bohlander, S.K., Espinosa, R., 3rd, Le Beau, M.M., Rowley, J.D., and Diaz, M.O A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics 13(4): Cleutjens, K.B., van der Korput, H.A., van Eekelen, C.C., van Rooij, H.C., Faber, P.W., and Trapman, J An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter. Mol Endocrinol 11(2): DePrimo, S.E., Diehn, M., Nelson, J.B., Reiter, R.E., Matese, J., Fero, M., Tibshirani, R., Brown, P.O., and Brooks, J.D Transcriptional programs activated by exposure of human prostate cancer cells to androgen. Genome Biol 3(7): RESEARCH0032. Fox, M.S., Ares, V.X., Turek, P.J., Haqq, C., and Reijo Pera, R.A Feasibility of global gene expression analysis in testicular biopsies from infertile men. Mol Reprod Dev 66(4): Hughes, T.R., Mao, M., Jones, A.R., Burchard, J., Marton, M.J., Shannon, K.W., Lefkowitz, S.M., Ziman, M., Schelter, J.M., Meyer, M.R., Kobayashi, S., Davis, C., Dai, H., He, Y.D., Stephaniants, S.B., Cavet, G., Walker, W.L., West, A., Coffey, E., Shoemaker, D.D., Stoughton, R., Blanchard, A.P., Friend, S.H., and Linsley, P.S Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19(4): Li, H., Rhodius, V., Gross, C., and Siggia, E.D Identification of the binding sites of regulatory proteins in bacterial genomes. Proc Natl Acad Sci U S A 99(18): Nelson, P.S., Clegg, N., Arnold, H., Ferguson, C., Bonham, M., White, J., Hood, L., and Lin, B The program of androgen-responsive genes in neoplastic prostate epithelium. Proc Natl Acad Sci U S A 99(18): Patil, C.K., Li, H., and Walter, P Gcn4p and novel upstream activating sequences regulate targets of the unfolded protein response. PLoS Biol 2(8): E246. Tusher, V.G., Tibshirani, R., and Chu, G Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98(9): Zaret, K.S. and Yamamoto, K.R Reversible and persistent changes in chromatin structure accompany activation of a glucocorticoid-dependent enhancer element. Cell 38(1):

27 SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. The 157 putative androgen-responsive genes (ARGs) identified by expression microarray analysis. The colorimetric representation shows GenBank accession numbers corresponding to genes, indicated by HUGO Gene Nomenclature Committee gene symbols, whose transcripts were repressed (A, green) and induced (B, red) by androgen. The color intensity reflects the relative fold change in transcript level for androgen- versus vehicletreated cells. The time course spanned 4, 8, 15, and 24 hours. Supplemental Figure 2. DNase I hypersensitive site (DHS) mapping revealed an androgendependent hypersensitive site at the ARE upstream of SGK. Androgen increased DNAse I accessibility at two DNase I hypersensitive sites (black triangles). Cleavage of the 5.5 kb parental Xba I fragment (-4,579 to +946) was androgen-stimulated at the ARBS (-1174 to -1160) of the ARE resulting in a 2.1 kb fragment (DHS1); an additional androgen-independent 1.0 kb fragment (DHS2) was also generated by DNase I cleavage at the transcription start site (+1) of SGK. Supplemental Figure 3. Distribution analysis of androgen response elements (AREs) located near ARGs. (A) The distribution of AREs identified by ChIP-chip was plotted relative to the predicted transcription start site (+1) of the shortest transcriptional variant of the nearest ARG. AREs reside great distances upstream and downstream as well as near the promoter. Notably, fewer AREs were found upstream than downstream of ARG transcription start sites of the shortest transcriptional variants. Relative distances upstream (negative) and downstream (positive) of the transcription start sites (vertical black line) were assigned to 10 kb bins. AREs 27

28 were generally found beyond 10 kb of the transcription start site (B) The location of AREs is shown relative to the predicted gene substructure of the shortest transcriptional variant of the nearby ARGs. When the distribution of all AREs near all ARGs was assessed, most AREs were situated downstream of the transcription start site. These distributions were subdivided based on whether the ARG was repressed or induced, suggesting biases in terms of ARE location and the mean AREs/ARGs ratios. Supplemental Figure 4. The number of AREs containing putative ARBSs. All 524 ARE sequences were scored for putative ARBSs using a predicted position weight matrix (PWM) representative of the ARBS (Figure 7A). The percent of ARBRs predicted to contain ARBSs (red squares) with varying score cutoffs was plotted. The false positive rate (blue triangles) was calculated by randomly sampling unbound sequences at varying score cutoffs. 28

29 Fold Repression > >8 Fold Induction A B SEMA3G AA TMEM37 N93191 S100P R32952 AQP3 R91904 CXCR7 N53172 KRT73 W74418 SLCO2A1 AA MGC16121 R32951 TIPARP AA SGK AA RIPK4 W72972 ACSL1 T73556 KLF4 H45711 LNX1 T84156 EPB41L4B H94262 SERPINB3 AA ACSS1 N67766 PAG1 N50114 PRSS23 R76394 C17orf80 AA SPTLC3 AA HMGB3 AA FOXO1A AA FKBP5 W86653 SLC22A3 AA ST5 AA NEDD4 AA PXDN W72043 FBXO21 R39464 CREB3L2 R34297 SLC30A1 R31168 NFKBIA W55872 SLC12A7 AA KIAA1727 H56655 ITGB8 R74357 INPP1 H52141 TFPI2 AA FOXC1 AA KLHDC8B T70612 KIAA0232 AA BAIAP2 H46962 MME R98851 GRHL1 R63971 PCYOX1L AA GATA2 R32406 ADARB1 AA MAPKAPK2 AA SSX2IP H20847 PMVK H09914 ETNK2 H77535 FN1 R62612 NSDHL AA FAM104A T58729 PGM2L1 AA TLE3 W32778 OSBPL11 R22420 LASS6 W85843 MPHOSPH10 AA BIN1 AA CORO2B N92783 CA2 H23187 AKAP13 T50096 MPST AA PLEKHA7 R44496 CYLD AA SFMBT2 R52082 PTP4A1 AA MTX1 N69540 THOC3 AA TBC1D9B AA AADAT N75669 TGFB1 R36467 RABGAP1L H10709 H92588 POMT1 R37635 ARVCF H17975 AP1G2 T49401 MBL2 T69359 DUS1L AA PTPN13 R85643 TIGD3 H10344 C4orf29 W91885 C4orf30 R00166 AA hr 8hr 15hr 24hr FST AA DKK1 AA ARL4C N35301 DDIT4 AA CYP24A1 N21576 ADAMTS1 R76553 FGFBP1 AA KCNN4 AA PLK2 AA CXCL1 W46900 PHLDA1 H26271 GPRC5A AA AREG AA MYC W87741 EDG2 AA CD274 R78533 BMP2 AA PLAU AA NT5E R60343 SNAI2 H57309 IL6R T52330 ANTXR2 T59658 PTHLH AA FAM84B N66178 INHBA R66924 ZMIZ1 AA PHF15 AA TM4SF1 AA IRF2BP2 AA PLEC1 AA DCBLD2 AA TP73L AA DUSP5 W65461 ANXA8 W81668 ARID5B W93382 KIAA1434 H91615 ELK3 H97146 DNMBP AA PNKD R01094 UAP1 N68465 SEMA4B AA PRNP AA ST6GALNAC2 AA LOC AA CMTM7 AA SOX4 N23606 RNF126 AA ITGB3 AA SLC16A1 AA UCK2 AA NUP88 AA DST H44784 KIAA1522 AA PSEN2 AA ALPI AA R73672 CPA4 AA AA ZIC2 H40921 DTX4 AA SFRP1 T68892 TMEM158 AA RCCD1 T96829 LIMA1 AA LARP2 T82817 RAB3A H14231 AA PLAC8 AA hr 8hr 15hr 24hr Bolton_et_al._Supplemental_Figure1

30 al_figure2 134,532, ,535, ,538, ,541,000 SGK Probe: -375 to +544 DHS2 DHS Xba I ARE -4,579 Xba I DNAse I: 0 Vehicle Androgen minutes 0 minutes 5.5 kb fragment (-4,579 to +946) 2.1 kb DHS1 (-1,174 to +946) 1.0 kb DHS2 (+1 to +946)

31 A Bolton_et_al._Supplemental_Figure3 B 15% 44% Repressed Induced % 6% Total AREs Total ARGs AREs/ARG

32 al_figure4

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