Coregulator Recruitment and Histone Modifications in Transcriptional Regulation by the Androgen Receptor

0888-8809/04/$15.00/0 Printed in U.S.A. Molecular Endocrinology 18(11):2633–2648 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0245 Coregulator Recruitment and Histone Modifications in Transcriptional Regulation by the Androgen Receptor ZHIGANG KANG, OLLI A. JÄNNE, AND JORMA J. PALVIMO We have used chromatin immunoprecipitation (ChIP) assay to follow transcription factor loading and monitor changes in covalent histone modifications associated with the prostate-specific antigen and kallikrein (KLK2) genes in response to androgen and antiandrogen in LNCaP cells. The dynamics of testosterone (T)-induced loading of androgen receptor (AR) onto the proximal promoters of the genes differed significantly from that onto the distal enhancers. Significantly more holo-AR was loaded onto the enhancers than the promoters, but the receptor’s residence time was more transient on the enhancers. Even though holo-AR recruited some RNA polymerase II (Pol II) onto the enhancers, the principal Pol II transcription complex was assembled on the promoters. The pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the prostate-specific antigen promoter, but not that of the enhancer, whereas the partial antagonists cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading also onto the enhancer. In contrast to the CDX-occupied receptor, both CPA- and RU486-bound AR recruited Pol II and coactivators p300 and glucocorticoid receptor-interacting protein 1 (GRIP1) onto the promoter and enhancer. However, CPA and RU486 also brought about a simultaneous recruitment of the nuclear receptor corepressor (NCOR) onto the promoter as efficiently as CDX. There were dynamic changes in covalent modifications of histone H3: acetylation of lysine 9 and 14, methylation of arginine 17, phosphorylation of serine 10 as well as di- and tri-methylation at lysine 4 of the H3 N-terminal tail were enhanced in response to T, but not after CDX treatment. Collectively, these results indicate that transcriptional activation by AR is accompanied by a cascade of distinct covalent histone modifications and that the pure antiandrogen CDX and the partial antagonists CPA and RU486 exhibit clear differences in their ability to promote recruitment of histone-acetylating and histone-deacetylating complexes in human prostate cancer cells. (Molecular Endocrinology 18: 2633–2648, 2004) T cancer (3, 4). Upon hormone binding, cytoplasmic AR dissociates from chaperones and translocates to the nucleus where it binds to androgen response elements (AREs) of target genes and modulates the rate of transcription initiation typically through bridged interactions with the transcription machinery and the chromatin remodeling complexes (5–9). There are two distinct classes of activities that regulate the accessibility of promoters to transcription and DNA replication machinery, both of which contribute to the generation of a dynamic chromatin structure (10). The first class includes ATP-dependent chromatin remodeling complexes, such as SWI/SNF, ISWI, NURD, WINAC, that are thought to reorganize chromatin structure through DNA sliding or conformational changes in the nucleosomes to expose DNA (11–13). The second class includes enzymes that catalyze posttranslational modifications in histones (14). Dynamic changes in multiple posttranslational modifications of the N-terminal tails of core histones, i.e. the histone code, can control chromatin packaging and create binding sites for chromatin-associated proteins HE ANDROGEN RECEPTOR (AR), a member of the nuclear receptor superfamily that functions as a ligand-regulated transcription factor, mediates a variety of developmental processes that create the male phenotype (1, 2). AR is a central player both in the development and maintenance of normal prostate as well as in the initiation and progression of prostate Abbreviations: AR, Androgen receptor; ARE, androgen response element; CARM1, coactivator-associated arginine methyltransferase 1; CBP, cAMP response element binding protein-binding protein; CDX, bicalutamide; ChIP, chromatin immunoprecipitation; CPA, cyproterone acetate; DHT, dihydrotestoserone; FCS, fetal calf serum; GRIP1, glucocorticoid receptor-interacting protein 1; HAT, histone acetyltransferase; HDAC, histone deacetylase; KLK2, kallikrein 2; NCoR, nuclear receptor corepressor; nt, nucleotide; PCAF, p300/ CBP-associated factor; Pol II, RNA polymerase II; PSA, prostate-specific antigen (KLK3); SET, Su(var)3–9, enhancer-ofZeste, Trihorax; SMRT, silencing mediator for retinoic and thyroid hormone receptors; T, testosterone. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 2633 Biomedicum Helsinki, Institute of Biomedicine (Z.K., O.A.J., J.J.P.), and Department of Clinical Chemistry (O.A.J.), University of Helsinki and University of Helsinki Central Hospital, FI-00014 Helsinki; and Department of Medical Biochemistry (J.J.P.), University of Kuopio, FI-70211 Kuopio, Finland 2634 Mol Endocrinol, November 2004, 18(11):2633–2648 within gene regulatory regions in vivo (31). In this work, we have examined the kinetics of various histone H3 tail modifications associated with the promoter and enhancer of prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes during androgen induction in human prostate cancer cells. We have also compared AR transcription complex assembly onto these regulatory regions in cells exposed to pure or partial antiandrogens by utilizing quantitative ChIP assays. RESULTS Comparison of AR Loading and RNA Polymerase (Pol) II Recruitment onto the Promoters and Enhancers of the PSA and KLK2 Genes Quantitative ChIP assays were used to monitor coregulator recruitment and histone modifications by the androgen receptor in LNCaP cells. PSA and KLK2 were chosen as model genes because they are wellrecognized targets of androgen action in vivo (32, 33). Reporter gene assays and in vitro binding studies have indicated that the PSA promoter containing two AREs [ARE I at nucleotide (nt) ⫺170 and ARE II at nt ⫺394] cooperates with the upstream enhancer region harboring several low affinity AREs (collectively termed ARE III, at nt ⫺4200) in androgen regulation (34). A related member of the human kallikrein gene family, the KLK2 gene, located 12 kb downstream of the PSA gene on 19q13.2-q13.4, has an analogous organization of AR responsive regulatory sequences (35). We first compared loading of AR and recruitment of Pol II between the promoter and the enhancer regions of the two genes. As shown in Figs. 1 and 2, the dynamics of holo-AR loading and that of Pol II recruitment onto the PSA promoter and enhancer were essentially indistinguishable from those occurring on the analogous regions of the KLK2 gene. In agreement with our previous results (36), initial loading of holo-AR onto the promo (...truncated)