NFI Transcription Factors Interact with FOXA1 to Regulate Prostate-Specific Gene Expression

ORIGINAL RESEARCH NFI Transcription Factors Interact with FOXA1 to Regulate Prostate-Specific Gene Expression Magdalena M. Grabowska, Amicia D. Elliott, David J. DeGraff, Philip D. Anderson, Govindaraj Anumanthan, Hironobu Yamashita, Qian Sun, David B. Friedman, David L. Hachey, Xiuping Yu, Jonathan H. Sheehan, Jung-Mo Ahn, Ganesh V. Raj, David W. Piston, Richard M. Gronostajski, and Robert J. Matusik Department of Urologic Surgery (M.M.G., G.A. H.Y., Q.S., X.Y., R.J.M.), Department of Molecular Physiology and Biophysics (A.D.E., D.W.P.), and Vanderbilt-Ingram Cancer Center (R.J.M.), Vanderbilt University Medical Center, Nashville, Tennessee 37232; Department of Pathology (D.J.D.), Penn State University College of Medicine, Hershey, Pennsylvania 17033; Department of Biological Sciences (P.D.A.), Salisbury University, Salisbury, Maryland 21801; Mass Spectrometry Research Center (D.B.F., D.L.H.), Department of Biochemistry, Department of Biochemistry and Center for Structural Biology (J.H.S.), and Department of Cell and Developmental Biology (R.J.M.), Vanderbilt University, Nashville, Tennessee 37232; Department of Chemistry (J.-M.A.), University of Texas Dallas, Dallas, Texas 75080; Department of Urology (G.V.R.), University of Texas Southwestern, Dallas, Texas 75390; and Department of Biochemistry (R.M.G.), Developmental Genomics Group, NY State Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, Buffalo, New York 14203 Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression. (Molecular Endocrinology 28: 949 –964, 2014) ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received July 19, 2013. Accepted April 18, 2014. First Published Online May 6, 2014 doi: 10.1210/me.2013-1213 Abbreviations: AR, androgen receptor; ARE, androgen response element; ChIP, chromatin immunoprecipitation; ChIP-Seq, chromatin immunoprecipitation-sequencing; DHT, dihydrotestosterone; DBD, DNA-binding domain; ER, estrogen receptor; EtOH, ethanol; FBS, fetal bovine serum; FOX, forkhead box; FRET, Förster resonance energy transfer; GR, glucocorticoid receptor; GST, glutathione S-transferase; HA, hemagglutinin; LC-MS/MS, liquid chromatography-tandem mass spectrometry; mCer3, mCerulean3; NFI, nuclear factor I; PDB, Protein Data Bank; PSA, prostate-specific antigen; Q-RT-PCR, quantitative real-time PCR; siRNA, small interfering RNA; TF, transcription factor. Mol Endocrinol, June 2014, 28(6):949 –964 mend.endojournals.org 949 950 Grabowska et al NFI Family Members Regulate AR/FOXA1 Gene Expression t is well recognized that signaling by the androgen receptor (AR) has important roles in normal prostate development, growth, and differentiation (1–3), as well as in benign and neoplastic conditions of the prostate (4). However, AR alone is not sufficient to mediate tissuespecific gene expression. Rather, it is the combinatorial control (5, 6) and activity of multiple factors that determine tissue-specific gene expression. Specifically, the ability of AR to engage other transcription factors (TFs) in a physical complex dictates tissue-specific gene expression in the prostate (7). In addition to the prostate, the AR is expressed in various tissues where it exhibits a distinct role for normal gene expression and physiology. For example, the AR in the skeletal muscle dictates anabolism of that tissue (8). Therefore, in addition to epigenetic mechanisms, it is the ability of AR to interact with other TFs that determines AR function in a given tissue. Our interest in identifying factors that mediate tissue specificity of AR target gene expression led to identification of forkhead box (FOX) A1 (FOXA1) as an AR interacting protein (9, 10) and showed that this interaction is essential for the expression of AR-regulated, prostatespecific genes (for review, see Ref. 11). The FOXA family of proteins (FOXA1, FOXA2, and FOXA3) bind with differing affinity to the consensus DNA sequence [(A/C)AA(C/T)] and have been implicated in various developmental, homeostatic, and disease processes (12–14). Our focus has been on FOXA1 because FOXA2 is expressed only in neuroendocrine cells of the adult prostate and FOXA3 is not expressed in adult prostate (15). FOXA1 works as a “pioneer factor” and acts to increase TF accessibility to the DNA by displacing linker histones from nucleosomes, allowing for chromatin unfolding (16). Further studies by us and others have validated the importance of this AR/FOXA1 interaction in prostate cancer (14, 17–20) and demonstrated the interaction between FOXA1 and other steroid receptors (21–24). The loss of FOXA1 in prostate cancer cell lines that express AR results in dramatic reprogramming of AR to different binding sites (20, 25). The ability of FOXA1 to interact with AR and specify binding to specific androgen response elements (AREs) suggests that other TFs involved with the AR/FOXA1 complex may further regulate tissuespecific gene expression. To identify novel TFs involved in the AR/FOXA1 transcription complex, we expressed a dualtagged FOXA1 construct in an androgen-regulated prostatic cell line, LNCaP, and performed tandem affinity purification and mass spectrometry to identify a novel set of FOXA1 interacting proteins. Sixteen (...truncated)