ATP-dependent Chromatin Remodeling Protein Research Articles

Abstract 2959: The tumor suppressor CHD5 is an epigenetic regulator of neuronal cell fate

Abstract Genomic lesions affecting the 1p36 chromosomal region are prevalent in variety of human malignancies, yet the tumor suppressor in this region had not been identified. We used chromosome engineering to create a series of mouse models with lesions spanning the 1p36-analagous region. These gain and loss-of-function models allowed us to pinpoint a potent tumor suppressive interval that when deleted predisposes to cancer and when duplicated causes excessive tumor suppression. We identified Chromodomain Helicase DNA-binding domain protein 5 (CHD5) as the causative gene in the region, defined CHD5 as an inducer of the tumor suppressive network encoded at the Cdkn2 locus, and demonstrated that CHD5 is frequently deleted in human glioma. It is now appreciated that CHD5 is mutated in cancers of the breast, ovary, colon, and prostate, as well as in melanoma, glioma, and neuroblastoma, and furthermore, that robust CHD5 expression is a favorable indicator of patient survival following anti-cancer therapy. Although ample evidence from human studies indicates that CHD5 plays a pivotal role in cancer, the mechanism responsible for tumor suppression is not well understood. CHD5 belongs to the CHD family of ATP-dependent chromatin remodeling proteins, yet CHD5's role in modulating chromatin and the consequence of CHD5 deficiency in vivo was unknown. We discovered that CHD5's tumor suppressive function is dependent on its dual plant homeodomains (PHDs)_motifs that bind specifically marked histones. The PHDs of CHD5 preferentially bind unmodified histone 3 (H3), an interaction essential for CHD5's ability to inhibit proliferation, to modulate transcription, and to suppress tumorigenesis in vivo. We recently defined CHD5 as a regulator of chromatin-mediated dynamics essential for neuronal differentiation. Whereas neural stem cells (NSCs) normally have the capacity to differentiate into neurons, NSCs from Chd5-deficient mice retain stem-like characteristics and differentiate into astrocytes at the expense of neurons. We discovered that H3K27me3_a repressive chromatin mark implicated in cancer_is depleted by CHD5 deficiency. We show that CHD5 rescues the de-differentiated phenotype of CHD5 deficient NSCs, while re-establishing global levels of H3K27me3. Our findings define CHD5 as a dosage-sensitive tumor suppressor, identify CHD5 as a member of a newly appreciated class of H3-binding PHD-containing proteins, and reveal a critical role for CHD5 in dictating stem cell fate. This provides new insight into the epigenetic mechanisms responsible for CHD5's tumor suppressive role that when perturbed, set the stage for cancer. Citation Format: Alea A. Mills, Dong-Woo Hwang. The tumor suppressor CHD5 is an epigenetic regulator of neuronal cell fate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2014-2959

Structure, function and regulation of CSB: A multi-talented gymnast

The Cockayne syndrome complementation group B protein, CSB, plays pivotal roles in transcription regulation and DNA repair. CSB belongs to the SNF2/SWI2 ATP-dependent chromatin remodeling protein family, and studies from many laboratories have revealed that CSB has multiple activities and modes of regulation. To understand the underlying mechanisms of Cockayne syndrome, it is necessary to understand how the biochemical activities of CSB are used to carry out its biological functions. In this review, we summarize our current knowledge of the structure, function and regulation of CSB, and discuss how these properties can impact the biological functions of this chromatin remodeler.

Abstract SY20-01: CHD5: Chromosome engineering, chromatin dynamics and cancer

Abstract Although 1p36 loss has been documented for decades and a variety of malignancies have this particular lesion, the tumor suppressor mapping to this genomic interval had not been identified. Using chromosome engineering to create models with gain and loss of the region of the mouse genome corresponding to human 1p36, we pinpointed a potent tumor suppressive interval, identified Chromodomain Helicase DNA-binding domain protein 5 (CHD5) as the causative gene in the region, elucidated molecular pathways induced by CHD5, and demonstrated that CHD5 is frequently deleted in human cancer. The discovery of CHD5 as a tumor suppressor has had a major impact in the cancer field. Indeed, it is now appreciated that CHD5 is mutated in cancers of the breast, ovary, colon, and prostate, as well as in melanoma, glioma, and neuroblastoma. Furthermore, CHD5 status is a prognostic indicator of patient survival following anti-cancer therapy. Still, the mechanism by which CHD5 exerts its tumor suppressive role is barely understood. CHD5 belongs to the CHD family of SWI-SNF-like ATP dependent-chromatin remodeling proteins; however, the role of CHD5 in modulating chromatin dynamics has not been reported. Here, we show that CHD5's tumor suppressive function is dependent on its tandem plant homeodomains (PHDs)—modules that in several other chromatin-remodeling proteins regulate transcription by binding to specific covalent modifications on histone tails. Each of the two PHDs of CHD5 preferentially bind unmethylated H3K4 (H3K4me0), an interaction abrogated by methylation of H3K4 but unaffected by modification of H3K9. Consistent with its binding specificity in vitro, genome-wide ChIP-sequencing indicates that CHD5 binds loci lacking highly methylated H3K4 marks in vivo. Both loss- and gain-of-function studies reveal that CHD5 modulates transcription, and furthermore, that CHD5 is a potent inhibitor of cellular proliferation. Mutation of residues within the PHDs of CHD5 that abolish H3K4me0 binding effectively abrogate the ability of CHD5 to modulate expression of target genes as well as to inhibit proliferation, leading to tumorigenesis in vivo. These findings identify CHD5 as a member of a newly appreciated class of H3K4me0-binding PHD containing proteins and reveal a critical role for this interaction in facilitating CHD5's cellular function, providing new insight into the molecular mechanism responsible for the tumor suppressive role of CHD5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY20-01. doi:1538-7445.AM2012-SY20-01

Regulación de la expresión génica: cómo operan los mecanismos epigenéticos

Cell phenotype does not only depend on the nucleotide sequence, but is determined by those genes that are expressed and those that are not. One way to regulate this gene expression pattern is by modifying the chromatin structure via diverse epigenetic mechanisms. In the present article we present the most important epigenetic mechanisms: deoxyribonucleic acid methylation, histone post-translational modifications, non-coding ribonucleic acid mediated gene-silencing, ATP-dependent chromatin remodeling, and Polycomb and Trithorax group proteins.

Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis

Abstract 1300Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

ATRX in chromatin assembly and genome architecture during development and disease

The regulation of genome architecture is essential for a variety of fundamental cellular phenomena that underlie the complex orchestration of mammalian development. The ATP-dependent chromatin remodeling protein ATRX is emerging as a key regulatory component of nucleosomal dynamics and higher order chromatin conformation. Here we provide an overview of the role of ATRX at chromatin and during development, and discuss recent studies exposing a repertoire of ATRX functions at heterochromatin, in gene regulation, and during mitosis and meiosis. Exciting new progress on several fronts suggest that ATRX operates in histone variant deposition and in the modulation of higher order chromatin structure. Not surprisingly, dysfunction or absence of ATRX protein has devastating consequences on embryonic development and leads to human disease.

Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression

p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow, including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover, the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases, and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes, including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes.

A Chromatin-remodeling Protein Is a Component of Fission Yeast Mediator

The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15(+) and hrp1(+) causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters.

Epigenetic Mechanisms Modulate Thyroid Transcription Factor 1-mediated Transcription of the Surfactant Protein B Gene

Epigenetic regulation of transcription plays an important role in cell-specific gene expression by altering chromatin structure and access of transcriptional regulators to DNA binding sites. Surfactant protein B (Sftpb) is a developmentally regulated lung epithelial gene critical for lung function. Thyroid transcription factor 1 (Nkx2-1) regulates Sftpb gene expression in various species. We show that Nkx2-1 binds to the mouse Sftpb (mSftpb) promoter in the lung. In a mouse lung epithelial cell line (MLE-15), Nkx2-1 knockdown reduces Sftpb expression, and mutation of Nkx2-1 cis-elements significantly reduces mSftpb promoter activity. Whether chromatin structure modulates Nkx2-1 regulation of Sftpb transcription is unknown. We found that DNA methylation of the mSftpb promoter inversely correlates with known patterns of Sftpb expression in vivo. The mSftpb promoter activity can be manipulated by altering its cytosine methylation status in vitro. Nkx2-1 activation of the mSftpb promoter is impaired by DNA methylation. The unmethylated Sftpb promoter shows an active chromatin structure enriched in the histone modification H3K4me3 (histone 3-lysine 4 trimethylated). The ATP-dependent chromatin remodeling protein Brg1 is recruited to the Sftpb promoter in Sftpb-expressing, but not in non-expressing tissues and cell lines. Brg1 knockdown in MLE-15 cells greatly decreases H3K4me3 levels at the Sftpb promoter region and expression of the Sftpb gene. Brg1 can be co-immunoprecipitated with Nkx2-1 protein. Last, Nkx2-1 and Brg1 with intact ATPase activity are required for mSftpb promoter activation in vitro. Our findings suggest that DNA methylation and chromatin modifications cooperate with Nkx2-1 to regulate Sftpb gene cell specific expression.

New functions for an ancient domain

Macrodomains function as binding modules for metabolites of NAD+, including poly(ADP-ribose). Three new studies explore how binding of poly(ADP-ribose) by the macrodomains of histone variant macroH2A1.1 and the ATP-dependent chromatin-remodeling protein ALC1 (also called CHD1L) leads to the modulation of chromatin structure, regulating nuclear functions such as DNA-damage detection and repair.

A Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) Complex Represses Expression of the Gene Encoding Tumor Necrosis Factor α-induced Protein 8 (TNFAIP8)

The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) plays key roles in development and homeostasis. A tandem affinity purification procedure revealed that COUP-TFI associated with a number of transcriptional regulatory proteins in HeLa S3 cells, including the nuclear receptor corepressor (NCoR), TIF1beta/KAP-1, HDAC1, and the SWI/SNF family member Brahma. The proapoptotic protein DBC1 was also identified in COUP-TFI complexes. In vitro experiments revealed that COUP-TFI interacted directly with NCoR but in a manner different from that of other nuclear receptors. DBC1 stabilized the interaction between COUP-TFI and NCoR by interacting directly with both proteins. The gene encoding the anti-apoptotic protein TNFAIP8 (tumor necrosis factor alpha (TNFalpha)-induced protein 8) was identified as being repressed by COUP-TFI in a manner that required several of the component proteins of the COUP-TFI complex. Finally, our studies highlight a central role for COUP-TFI in the induction of the TNFAIP8 promoter by TNFalpha. Together, these studies identify a novel COUP-TFI complex that functions as a repressor of transcription and may play a role in the TNFalpha signaling pathways.

Characterization of novel isoforms and evaluation of SNF2L/SMARCA1 as a candidate gene for X-linked mental retardation in 12 families linked to Xq25-26

BackgroundMutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.MethodsWe used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.ResultsThe SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.ConclusionOur results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.

Rtf1 Is a Multifunctional Component of the Paf1 Complex That Regulates Gene Expression by Directing Cotranscriptional Histone Modification

Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.

Isw1 Acts Independently of the Isw1a and Isw1b Complexes in Regulating Transcriptional Silencing at the Ribosomal DNA Locus in Saccharomyces cerevisiae

Transcriptional silencing of Pol II-transcribed genes in Saccharomyces cerevisiae occurs at the HM loci, telomeres and ribosomal DNA (rDNA) locus. Gene silencing at these loci requires histone-modifying enzymes as well as factors that regulate local chromatin structure. Previous work has shown that the ATP-dependent chromatin remodeling protein Isw1 is required for silencing of a marker gene inserted at the HMR locus, but not at telomeres. Here we show that Isw1 is required for transcriptional silencing of Pol II-transcribed genes in the ribosomal DNA locus. Our results indicate that Isw1 associates with the rDNA and that this interaction is not altered in cells lacking other members of the Isw1a and Isw1b chromatin remodeling complexes. Further, the association of Isw1 with the rDNA is not altered in cells lacking the histone deacetylase Sir2 or the histone methyltransferase Set1, two factors that are required for gene silencing at the rDNA. Notably, the loss of transcriptional silencing at the rDNA in cells lacking Isw1 is correlated with a change in rDNA chromatin structure. Together, our data support a model in which Isw1 acts independently of the previously characterized Isw1a and Isw1b complexes to maintain a heterochromatin-like structure at the rDNA that is required for gene silencing.

Schimke immuno‐osseous dysplasia: A cell autonomous disorder?

SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like protein 1) encodes a SWI/SNF ATP-dependent chromatin remodeling protein. Mutations in SMARCAL1 cause the autosomal-recessive multisystem disorder Schimke immuno-osseous dysplasia (SIOD); this suggests that the SMARCAL1 protein is involved in the development or maintenance of multiple organs. Disease within these many tissues could arise by a cell autonomous or a cell non-autonomous mechanism. Consistent with a cell autonomous mechanism, we did not find any disease recurrence in transplanted organs or protection of other tissues by the organ grafts. In order to better understand the role of SMARCAL1 during normal development and in the pathogenesis of SIOD, we characterized the spatial and temporal expression of the murine homolog (Smarcal1). The Smarcal1 mRNA and protein were expressed throughout development and in all tissues affected in patients with SIOD including the bone, kidney, thymus, thyroid, tooth, bone marrow, hair, eye, and blood vessels. Significantly, the expression profile of Smarcal1 in the mouse has led us to reexamine and identify novel pathology in our patient population resulting in changes in the clinical management of SIOD. The expression of Smarcal1 in affected tissues and the non-recurrence of disease in grafted organs lead us to hypothesize a cell autonomous function for SMARCAL1 and to propose tissue-specific mechanisms for the pathophysiology of SIOD.

Human but Not Yeast CHD1 Binds Directly and Selectively to Histone H3 Methylated at Lysine 4 via Its Tandem Chromodomains

Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin "marks" on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein.

A Novel Transcription Regulatory Complex Containing Death Domain-associated Protein and the ATR-X Syndrome Protein

Death domain-associated protein (Daxx) is a multi-functional protein that modulates both apoptosis and transcription. Within the nucleus, Daxx is a component of the promyelocytic leukemia protein (PML) nuclear bodies (NBs) and interacts with a number of transcription factors, yet its precise role in transcription remains elusive. To further define the function of Daxx, we have isolated its interacting proteins in the nucleus using epitope-tagged affinity purification and identified X-linked mental retardation and alpha-thalassaemia syndrome protein (ATRX), a putative member of the SNF2 family of ATP-dependent chromatin remodeling proteins that is mutated in several X-linked mental retardation disorders. We show that substantial amounts of endogenous Daxx and ATRX exist in a nuclear complex. Daxx binds to ATRX through its paired amphipathic alpha helices domains. ATRX has ATPase activity that is stimulated by mononucleosomes, and patient mutations in the ATPase domain attenuate this activity. ATRX strongly represses transcription when tethered to a promoter. Daxx does not affect the ATPase activity of ATRX, however, it alleviates its transcription repression activity. In addition, ATRX is found in the PML-NBs, and this localization is mediated by Daxx. These results show that the ATRX.Daxx complex is a novel ATP-dependent chromatin-remodeling complex, with ATRX being the core ATPase subunit and Daxx being the targeting subunit. Moreover, the localization of ATRX to the PML-NBs supports the notion that these structures may play an important role in transcription regulation.

Novel ATPase of SNF2-like protein family interacts with androgen receptor and modulates androgen-dependent transcription.

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-[gamma-32P]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 promoter-dependent 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.