Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer

BackgroundGene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach.ResultsEvolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP.ConclusionOverall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells.

[1]  Paolo Magni,et al.  Activation of the Y1 receptor by neuropeptide Y regulates the growth of prostate cancer cells. , 2006, Endocrinology.

[2]  R. Viger,et al.  Role of the GATA family of transcription factors in endocrine development, function, and disease. , 2008, Molecular endocrinology.

[3]  Tao Zhang,et al.  SOX9 is expressed in normal prostate basal cells and regulates androgen receptor expression in prostate cancer cells. , 2007, Cancer research.

[4]  Arul M Chinnaiyan,et al.  Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. , 2003, Journal of the National Cancer Institute.

[5]  S. Hahn,et al.  Implications for Radiotherapy VEGF Expression and Increases Tumor Oxygenation : and α Nelfinavir Down-regulates Hypoxia-Inducible Factor 1 , 2006 .

[6]  G. Saunders,et al.  The Wilms' tumor gene WT1 can regulate genes involved in sex determination and differentiation: SRY, Müllerian-inhibiting substance, and the androgen receptor. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

[7]  M. Sporn,et al.  Repression of the transforming growth factor-beta 1 gene by the Wilms' tumor suppressor WT1 gene product. , 1994, Molecular endocrinology.

[8]  Rosalie Nolley,et al.  The prostate specific antigen era in the United States is over for prostate cancer: what happened in the last 20 years? , 2004, The Journal of urology.

[9]  Xin Chen,et al.  TRANSFAC: an integrated system for gene expression regulation , 2000, Nucleic Acids Res..

[10]  S. Prabhakar,et al.  Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Rajiv Dhir,et al.  Differences in gene expression in prostate cancer, normal appearing prostate tissue adjacent to cancer and prostate tissue from cancer free organ donors , 2005, BMC Cancer.

[12]  Sudhir Kumar,et al.  Genomic clocks and evolutionary timescales. , 2003, Trends in genetics : TIG.

[13]  C. Y. Wang,et al.  WT1 modulates apoptosis by transcriptionally upregulating the bcl‐2 proto‐oncogene , 1999, The EMBO journal.

[14]  Thomas Werner,et al.  MatInspector and beyond: promoter analysis based on transcription factor binding sites , 2005, Bioinform..

[15]  W. Miller,et al.  Comparison of five methods for finding conserved sequences in multiple alignments of gene regulatory regions. , 1999, Nucleic acids research.

[16]  I-Min A. Dubchak,et al.  Active conservation of noncoding sequences revealed by three-way species comparisons. , 2000, Genome research.

[17]  G I Bell,et al.  Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. , 1992, Science.

[18]  V. Sukhatme,et al.  Transcriptional repression of the insulin-like growth factor I receptor (IGF-I-R) gene by the tumor suppressor WT1 involves binding to sequences both upstream and downstream of the IGF-I-R gene transcription start site. , 1994, The Journal of biological chemistry.

[19]  Ping Zhang,et al.  NDRG1, a growth and cancer related gene: regulation of gene expression and function in normal and disease states. , 2007, Carcinogenesis.

[20]  S. Hewitt,et al.  Differential Function of Wilms Tumor Gene WT1 Splice Isoforms in Transcriptional Regulation (*) , 1996, The Journal of Biological Chemistry.

[21]  P. Nelson,et al.  Suppression of growth and tumorigenicity in the prostate tumor cell line M12 by overexpression of the transcription factor SOX9 , 2004, Oncogene.

[22]  J. Licht,et al.  E-cadherin Is a WT1 Target Gene* , 2000, The Journal of Biological Chemistry.

[23]  S. Orkin,et al.  DNA-binding specificity of GATA family transcription factors , 1993, Molecular and cellular biology.

[24]  X. Xia,et al.  Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus. , 1993, The Journal of biological chemistry.

[25]  荻島 達也.  Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer , 2005 .

[26]  W. Miller,et al.  Long human-mouse sequence alignments reveal novel regulatory elements: a reason to sequence the mouse genome. , 1997, Genome research.

[27]  E. Devilard,et al.  FGFR1 and WT1 are markers of human prostate cancer progression , 2006, BMC Cancer.

[28]  K. Tchou-Wong,et al.  Egr-1 mediates hypoxia-inducible transcription of the NDRG1 gene through an overlapping Egr-1/Sp1 binding site in the promoter. , 2007, Cancer research.

[29]  Rajiv Dhir,et al.  Gene expression analysis of prostate cancers , 2002, Molecular carcinogenesis.

[30]  Adrian V. Lee,et al.  The WT1 Wilms' tumor suppressor gene product interacts with estrogen receptor-alpha and regulates IGF-I receptor gene transcription in breast cancer cells. , 2005, Journal of molecular endocrinology.

[31]  E. Denamur,et al.  Cross-species characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene reveals multiple levels of regulation. , 1997, The Biochemical journal.

[32]  T. Curran,et al.  Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence. , 1990, Science.

[33]  S. Ho,et al.  Estrogens and Antiestrogens as Etiological Factors and Therapeutics for Prostate Cancer , 2006, Annals of the New York Academy of Sciences.

[34]  W Miller,et al.  Conserved E Boxes Function as Part of the Enhancer in Hypersensitive Site 2 of the β-Globin Locus Control Region , 1997, The Journal of Biological Chemistry.

[35]  E. Lander,et al.  Gene expression correlates of clinical prostate cancer behavior. , 2002, Cancer cell.

[36]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[37]  D. Cox,et al.  Noncoding sequences conserved in a limited number of mammals in the SIM2 interval are frequently functional. , 2004, Genome research.

[38]  P. de Coppet,et al.  The Human Copper-Zinc Superoxide Dismutase Gene (SOD1) Proximal Promoter Is Regulated by Sp1, Egr-1, and WT1 via Non-canonical Binding Sites* , 1999, The Journal of Biological Chemistry.

[39]  G. Fraizer,et al.  Regulation of vascular endothelial growth factor, VEGF, gene promoter by the tumor suppressor, WT1. , 2007, Frontiers in bioscience : a journal and virtual library.

[40]  S M Hewitt,et al.  Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1. , 1995, Cancer research.

[41]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[42]  D. Mercola,et al.  EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity. , 1996, Critical reviews in oncogenesis.

[43]  L. Ellis,et al.  Activator protein 2alpha inhibits tumorigenicity and represses vascular endothelial growth factor transcription in prostate cancer cells. , 2004, Cancer research.

[44]  P. Humphrey,et al.  Impaired prostate tumorigenesis in Egr1-deficient mice , 2001, Nature Medicine.

[45]  Christopher D. Brown,et al.  Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance. , 2004, Genome research.

[46]  S. Hewitt,et al.  Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter. , 1994, The Journal of biological chemistry.

[47]  Pengbo Liu,et al.  Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells. , 2006, Cancer research.

[48]  M. Goodman,et al.  Embryonic ε and γ globin genes of a prosimian primate (Galago crassicaudatus): Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints , 1988 .

[49]  G. Fraizer,et al.  Suppression of prostate tumor cell growth in vivo by WT1, the Wilms' tumor suppressor gene. , 2004, International journal of oncology.

[50]  P. Farnham,et al.  Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. , 2002, Methods.

[51]  Y. Wu,et al.  GATA-1 Transactivates the WT1 Hematopoietic Specific Enhancer (*) , 1995, The Journal of Biological Chemistry.

[52]  M. Rivera,et al.  Wilms' tumour: connecting tumorigenesis and organ development in the kidney , 2005, Nature Reviews Cancer.

[53]  S. Horvath,et al.  Insulin growth factor-binding protein 2 is a candidate biomarker for PTEN status and PI3K/Akt pathway activation in glioblastoma and prostate cancer , 2007, Proceedings of the National Academy of Sciences.

[54]  Ivan Ovcharenko,et al.  rVISTA 2.0: evolutionary analysis of transcription factor binding sites , 2004, Nucleic Acids Res..

[55]  G. Saunders,et al.  Transactivation of an Intronic Hematopoietic-specific Enhancer of the Human Wilms’ Tumor 1 Gene by GATA-1 and c-Myb* , 1997, The Journal of Biological Chemistry.

[56]  L. Ellis,et al.  Activator Protein 2α Inhibits Tumorigenicity and Represses Vascular Endothelial Growth Factor Transcription in Prostate Cancer Cells , 2004, Cancer Research.

[57]  T. Werner.  Computer-Assisted Analysis of Transcription Control Regions , 2000 .

[58]  M. Nagpal,et al.  Overexpression of CXCL10 in human prostate LNCaP cells activates its receptor (CXCR3) expression and inhibits cell proliferation. , 2006, Biochimica et biophysica acta.

[59]  D. Feldman,et al.  Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action , 2006, Molecular Cancer Therapeutics.

[60]  Francesca Chiaromonte,et al.  Experimental validation of predicted mammalian erythroid cis-regulatory modules. , 2006, Genome research.

[61]  S. Madden,et al.  Transcriptional repression mediated by the WT1 Wilms tumor gene product. , 1991, Science.

[62]  W. Miller,et al.  Mulan: multiple-sequence local alignment and visualization for studying function and evolution. , 2005, Genome research.

[63]  V. Sukhatme,et al.  Increased expression of the insulin-like growth factor I receptor gene, IGF1R, in Wilms tumor is correlated with modulation of IGF1R promoter activity by the WT1 Wilms tumor gene product. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[64]  D. Tenen,et al.  Down regulation of PSA by C/EBPα is associated with loss of AR expression and inhibition of PSA promoter activity in the LNCaP cell Line , 2006, BMC Cancer.

[65]  Erwin G. Van Meir,et al.  Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms. , 2006, Cancer research.

[66]  Mei Li,et al.  MultiPipMaker and supporting tools: alignments and analysis of multiple genomic DNA sequences , 2003, Nucleic Acids Res..

[67]  E. Diamandis,et al.  The emerging roles of human tissue kallikreins in cancer , 2004, Nature Reviews Cancer.

[68]  J. Kench,et al.  Aberrant Neuropeptide Y and Macrophage Inhibitory Cytokine-1 Expression Are Early Events in Prostate Cancer Development and Are Associated with Poor Prognosis , 2006, Cancer Epidemiology Biomarkers & Prevention.

[69]  R. Nagle,et al.  Membrane-type 1 matrix metalloproteinase is regulated by sp1 through the differential activation of AKT, JNK, and ERK pathways in human prostate tumor cells. , 2007, Neoplasia.

[70]  K. Lindblad-Toh,et al.  Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals , 2005, Nature.

[71]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[72]  B. Köhler,et al.  Coexpression of Wilms' tumor suppressor 1 (WT1) and androgen receptor (AR) in the genital tract of human male embryos and regulation of AR promoter activity by WT1. , 2007, Journal of molecular endocrinology.

[73]  J. Tremblay,et al.  Novel roles for GATA transcription factors in the regulation of steroidogenesis , 2003, The Journal of Steroid Biochemistry and Molecular Biology.

[74]  M. Soloff,et al.  Proliferation of DU145 prostate cancer cells is inhibited by suppressing insulin‐like growth factor binding protein‐2 , 2004, International journal of urology : official journal of the Japanese Urological Association.

[75]  Z. Wang,et al.  The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene. , 1992, The Journal of biological chemistry.

[76]  N. Thompson,et al.  Identification of Sp2 as a transcriptional repressor of carcinoembryonic antigen-related cell adhesion molecule 1 in tumorigenesis. , 2004, Cancer research.

[77]  Michael M Lieber,et al.  PDLIM4 repression by hypermethylation as a potential biomarker for prostate cancer. , 2006, Clinical cancer research : an official journal of the American Association for Cancer Research.

[78]  X. Zhang,et al.  AP-2 regulates the transcription of estrogen receptor (ER)-β by acting through a methylation hotspot of the 0N promoter in prostate cancer cells , 2007, Oncogene.

[79]  B. Birren,et al.  Sequencing and comparison of yeast species to identify genes and regulatory elements , 2003, Nature.

[80]  L. Fulton,et al.  Finding Functional Features in Saccharomyces Genomes by Phylogenetic Footprinting , 2003, Science.

[81]  K. Miyagawa,et al.  Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing , 1995, Cell.

[82]  Brad Saville,et al.  Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor α and SP proteins , 2004, Oncogene.

[83]  K. McFann,et al.  DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells. , 2006, American journal of physiology. Endocrinology and metabolism.

[84]  Francesca Chiaromonte,et al.  Evaluation of regulatory potential and conservation scores for detecting cis-regulatory modules in aligned mammalian genome sequences. , 2005, Genome research.

[85]  D. Tindall,et al.  Expression of early growth response genes in human prostate cancer. , 1998, Cancer research.

[86]  Martin Vingron,et al.  Annotating regulatory DNA based on man-mouse genomic comparison , 2002, ECCB.

[87]  Jun Kawai,et al.  Evolutionary turnover of mammalian transcription start sites. , 2006, Genome research.

[88]  Andreas Prlic,et al.  Ensembl 2007 , 2006, Nucleic Acids Res..

[89]  A. Jemal,et al.  Cancer Statistics, 2007 , 2007, CA: a cancer journal for clinicians.

[90]  Bernard A. Roos,et al.  A role for GATA transcription factors in the androgen regulation of the prostate-specific antigen gene enhancer , 2000, Molecular and Cellular Endocrinology.

[91]  V. Srikantan,et al.  Characterization of the androgen receptor in a benign prostate tissue-derived human prostate epithelial cell line: RC-165N/human telomerase reverse transcriptase , 2007, Prostate Cancer and Prostatic Diseases.

[92]  D. Haber,et al.  WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. , 1995, The EMBO journal.

[93]  J. Touchman,et al.  Vertebrate genome sequencing: building a backbone for comparative genomics. , 2002, Trends in genetics : TIG.

[94]  W. Miller,et al.  Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. , 2000, Science.

[95]  D. Polsky,et al.  Altered N-myc Downstream-Regulated Gene 1 Protein Expression in African-American Compared with Caucasian Prostate Cancer Patients , 2004, Clinical Cancer Research.

[96]  A. Ward,et al.  Different isoforms of the Wilms’ tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus , 2006, Cell proliferation.

[97]  N. Hastie,et al.  Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one , 2003, Journal of Cell Science.

[98]  Hong Wu,et al.  Expression signature of the mouse prostate. , 2006, The Journal of biological chemistry.

[99]  Ewan Birney,et al.  Ensembl Genome Browser , 2010 .

[100]  A. Clark,et al.  Evolution of transcription factor binding sites in Mammalian gene regulatory regions: conservation and turnover. , 2002, Molecular biology and evolution.