Novel ATPase of SNF2-like Protein Family Interacts with Androgen Receptor and Modulates Androgen-dependent Transcription

Abstract

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein.ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido- [␥-32 P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoterdependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays. INTRODUCTIONThe androgen receptor (AR) belongs to the superfamily of nuclear receptors that are ligand-activated transcription factors capable of regulating transcription of genes containing appropriate response elements, usually within or around the proximal promoter regions (Beato et al., 1995;Quigley et al., 1995;Perlmann and Evans, 1997). After hormone binding, the receptors associate with their cognate DNA motifs and modulate transcription initiation. Nuclear receptors may interact directly with the basal transcription factors associated with RNA polymerase II, such as TFIIB (Ing et al., 1992;Blanco et al., 1995;Hadzic et al., 1995), TFIIF (McEwan and Gustafsson, 1997), and TFIIH (Lee et al., 2000), or elicit their actions indirectly via auxiliary regulatory proteins, called coactivators and corepressors (Torchia et al., 1998;Freedman, 1999;McKenna et al., 1999;Glass and Rosenfeld, 2000).DNA is folded in the nucleus into a tight chromatin structure that often renders important regulatory sequences inaccessible for sequence-specific transcription factors, including steroid receptors (Kingston et al., 1996;Näär et al., 2001). As a consequence, different chromatin-modifying complexes are required to counteract this repressive effect (Bjö rklund et al., 1999;Kingston and Narlikar, 1999;Lemon and Freedman, 1999;Vignali et al., 2000). The protein complexes can be classified into two main categories: 1) ATP-dependent chromatin-remodeling complexes, which use the energy of ATP hydrolysis to alter the association of histones with DNA; and 2) complexes that alter chromatin by covalent modification of its components. These modifications include histone acetylation, methylation, phosphorylation, and ADP-ribosylation. Yeast SWI/SNF was the first ATP-dependent complex shown to facilitate the function of gene regulatory proteins in a...

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

“…The Epstein-Barr virus nuclear protein EBNA3C 507DDDVIEV513 is also critical for SUMO interaction (25). ARIP4 and a closely related SNF-2-domain-containing protein, SRCAP, have similar sequences (15,26), and ARIP4 has now been identified to bind to SUMO-2, with dependence on K33, K35, and K42. These acidic SUMO-interacting amino acids may bind directly to the SUMO lysine-rich repressor patch.…”

“…Protein names, gene identifiers, calculated molecular mass, sizes at point(s) in gel where identified, and estimated peptide abundances are indicated in Table 2. The identified proteins include SAE2, PIASx␤, and arkadialike protein 1, which are involved in SUMO͞ubiquitin conjugation, SETDB1 and LSD1, which are a histone lysine methyltransferase and a histone lysine demethylase, respectively, PELP1, ARIP4, and MTA-2, which are coactivators or repressors, p53BP1, zinc-finger protein 198 (ZNF198), the nuclear matrix protein NXP-2, and HSP70 (13)(14)(15)(16). Because WT SUMO-2 was used in these experiments, proteins could have been retained by GST-SUMO-2 affinity resins because of either binding or covalent linkage to GST-SUMO-2.…”

Chromatin remodeling by ATP‐dependent molecular machines

“…(34,35,59) ARIP4 is an androgen receptor-interacting protein in the Rad54/ATRX subfamily that possesses chromatin-remodeling activity. (60) CSB (Cockayne Syndrome protein B) is a chromatin-remodeling protein that is involved in nucleotide excision repair. (61) Also, the plant DDM1 protein is a chromatinremodeling factor that is important for the maintenance of DNA methylation and genome stability.…”

“…(69,82,85,88,90) (6) A variety of remodeling factors have been observed to generate superhelical torsion in DNA or chromatin. (35,60,91) (7) RSC complex, ISWI and Rad54 have been found to exhibit triplex DNA displacement activity and thus appear to translocate along the DNA. (65,92) [In the triplex DNA displacement assay, a short oligonucleotide that binds in the major groove of a pyrimidine-rich target sequence is displaced by motor proteins that translocate through the sequence.]…”

Androgen receptor co-regulatory networks in castration-resistant prostate cancer

“…Upon androgen stimulation, AR translocates from the cytoplasm into the nucleus, dimerizes, and binds to specific DNA recognition sequences termed androgen response elements (AREs), where it subsequently recruits a series of co-regulatory proteins that either enhance (coactivators such as p160/SRC and CBP/p300) or repress (corepressors such as NCoR and SMRT) the transcription of target genes (Bennett et al 2010). Co-regulator proteins can influence AR-mediated transcription in a number of ways, including coordinating changes in the structure of the chromatin or facilitating interactions with the RNA polymerase transcriptional machinery (Rouleau et al 2002, Shang et al 2002, Louie et al 2003.…”