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• W2014209222 abstract "It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation. It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation. Amplification at 20q13 occurs in a variety of tumor types, such as breast (1Collins C. Rommens J.M. Kowbel D. Godfrey T. Tanner M. Hwang S.I. Polikoff D. Nonet G. Cochran J. Myambo K. Jay K.E. Froula J. Cloutier T. Kuo W.L. Yaswen P. Dairkee S. Giovanola J. Hutchinson G.B. Isola J. Kallioniemi O.P. Palazzolo M. Martin C. Ericsson C. Pinkel D. Albertson D. Li W.B. Gray J.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8703-8708Crossref PubMed Scopus (273) Google Scholar), gastric (2Yang S.H. Seo M.Y. Jeong H.J. Jeung H.C. Shin J. Kim S.C. Noh S.H. Chung H.C. Rha S.Y. Clin. Cancer Res. 2005; 11: 612-620PubMed Google Scholar), ovarian (3Iwabuchi H. Sakamoto M. Sakunaga H. Ma Y.Y. Carcangiu M.L. Pinkel D. Yang-Feng T.L. Gray J.W. Cancer Res. 1995; 55: 6172-6180PubMed Google Scholar), lung (4Zhu H. Lam D.C. Han K.C. Tin V.P. Suen W.S. Wang E. Lam W.K. Cai W.W. Chung L.P. Wong M.P. Cancer Lett. 2007; 245: 303-314Crossref PubMed Scopus (68) Google Scholar), prostate (5Bar-Shira A. Pinthus J.H. Rozovsky U. Goldstein M. Sellers W.R. Yaron Y. Eshhar Z. Orr-Urtreger A. Cancer Res. 2002; 62: 6803-6807PubMed Google Scholar), and colon (6Lassmann S. Weis R. Makowiec F. Roth J. Danciu M. Hopt U. Werner M. J. Mol. Med. 2007; (in press)PubMed Google Scholar), and is associated with aggressive tumor behavior (7Tanner M.M. Tirkkonen M. Kallioniemi A. Holli K. Collins C. Kowbel D. Gray J.W. Kallioniemi O.P. Isola J. Clin. Cancer Res. 1995; 1: 1455-1461PubMed Google Scholar). The mapping of the amplified region at 20q13.2 led to the positional cloning and characterization of ZNF217 (1Collins C. Rommens J.M. Kowbel D. Godfrey T. Tanner M. Hwang S.I. Polikoff D. Nonet G. Cochran J. Myambo K. Jay K.E. Froula J. Cloutier T. Kuo W.L. Yaswen P. Dairkee S. Giovanola J. Hutchinson G.B. Isola J. Kallioniemi O.P. Palazzolo M. Martin C. Ericsson C. Pinkel D. Albertson D. Li W.B. Gray J.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8703-8708Crossref PubMed Scopus (273) Google Scholar), which is considered to be one of the driver genes at 20q13.2, promoting selection during the early stages of tumor development. Initial comparative genomic hybridization studies showed that ZNF217 is amplified and overexpressed in ∼40% of breast cancer cell lines and 18% of primary breast tumors (8Kallioniemi A. Kallioniemi O.P. Piper J. Tanner M. Stokke T. Chen L. Smith H.S. Pinkel D. Gray J.W. Waldman F.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2156-2160Crossref PubMed Scopus (698) Google Scholar). Further comparative genomic hybridization studies of various tumor specimens report that ZNF217 amplification and overexpression at the 20q13 locus can display tumor type-specific profiles. For example, an analysis of 22 sporadic colorectal carcinomas detected DNA copy number changes for ZNF217 in 45% of the CIN (chromosomal instability)-type but not the MIN (microsatellite instability)-type sporadic colorectal carcinoma colon tumors (6Lassmann S. Weis R. Makowiec F. Roth J. Danciu M. Hopt U. Werner M. J. Mol. Med. 2007; (in press)PubMed Google Scholar). Evidence in support of a causal role for ZNF217 in tumor formation comes from studies using normal human mammary epithelial cells. Nonet et al. (9Nonet G.H. Stampfer M.R. Chin K. Gray J.W. Collins C.C. Yaswen P. Cancer Res. 2001; 61: 1250-1254PubMed Google Scholar) showed that introduction of ZNF217 into early passage human mammary epithelial cells can lead to a rare event of immortalization. It has been proposed that overexpression of ZNF217 may give a selective advantage to tumor cells by interfering with pathways associated with normal regulation of cell growth, cell death, differentiation, or DNA repair. DNA sequence analysis suggests that ZNF217 encodes a transcription factor having eight C2H2 Kruppel-like zinc finger DNA binding motifs and a proline-rich transactivation domain at the C terminus (1Collins C. Rommens J.M. Kowbel D. Godfrey T. Tanner M. Hwang S.I. Polikoff D. Nonet G. Cochran J. Myambo K. Jay K.E. Froula J. Cloutier T. Kuo W.L. Yaswen P. Dairkee S. Giovanola J. Hutchinson G.B. Isola J. Kallioniemi O.P. Palazzolo M. Martin C. Ericsson C. Pinkel D. Albertson D. Li W.B. Gray J.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8703-8708Crossref PubMed Scopus (273) Google Scholar). Biochemical studies support a role for ZNF217 in transcriptional regulation. For example, ZNF217 has been identified in complexes that contain repressor proteins such as CtBP 4The abbreviations used are: CtBP, C-terminal binding protein; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; DAVID, Database for Annotation, Visualization and Integrated Discovery; nt, nucleotides; PWM, positional weight matrix. 4The abbreviations used are: CtBP, C-terminal binding protein; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; DAVID, Database for Annotation, Visualization and Integrated Discovery; nt, nucleotides; PWM, positional weight matrix. and coREST (10You A. Tong J.K. Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1454-1458Crossref PubMed Scopus (382) Google Scholar, 11Lee M.G. Wynder C. Cooch N. Shiekhattar R. Nature. 2005; 437: 432-435Crossref PubMed Scopus (591) Google Scholar), histone deacetylases, the histone methyltransferase G9a, and the histone demethylase LSD1 (11Lee M.G. Wynder C. Cooch N. Shiekhattar R. Nature. 2005; 437: 432-435Crossref PubMed Scopus (591) Google Scholar, 12Hakimi M.A. Dong Y. Lane W.S. Speicher D.W. Shiekhattar R. J. Biol. Chem. 2003; 278: 7234-7239Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 13Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 14Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (626) Google Scholar). The direct interaction of ZNF217 with CtBP (15Quinlan K.G. Nardini M. Verger A. Francescato P. Yaswen P. Corda D. Bolognesi M. Crossley M. Mol. Cell. Biol. 2006; 26: 8159-8172Crossref PubMed Scopus (63) Google Scholar) suggests that ZNF217 could be recruited to a variety of transcription complexes through the interaction of CtBP with numerous site-specific DNA-binding proteins (16Chinnadurai G. Mol. Cell. 2002; 9: 213-224Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Although both biochemical and structural studies have linked ZNF217 to transcriptional regulation, a detailed analysis of its role in transcription has been limited due to a lack of known ZNF217 target genes. Therefore, we used a ChIP-chip method to identify thousands of ZNF217 target genes in three cancer cell lines: the breast cancer line MCF7, the colon cancer line SW480, and Ntera2, a teratocarcinoma line that can differentiate into neurons. To investigate the role of ZNF217 in transcriptional regulation, we examined the expression level of ZNF217 target genes in Ntera2 cells before and after reduction of the levels of ZNF217 using siRNAs and examined colocalization of ZNF217 with CtBP family members using ChIP-chip assays. Gene ontology analysis indicated that some ZNF217 target genes in Ntera2 cells are transcription factors that are involved in cell differentiation and organ development. We show that ZNF217 was down-regulated upon treatment of Ntera2 cells with retinoic acid, suggesting that the inappropriate expression of ZNF217 in differentiated adult cells may suppress differentiation, leading to tumorigenesis. Cell Culture—SW480 cells were grown in McCoy's 5A modified medium (Invitrogen) supplemented with 10% fetal bovine serum (NovaTech) and 1% penicillin/streptomycin (Invitrogen). MCF7 and Ntera2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 1% penicillin/streptomycin, and 10% fetal bovine serum. All cells were incubated at 37 °C in a humidified 5% CO2 incubator. For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMART-pool; Dharmacon, catalog number M-004987-00) or si-GLO RISC-Free (Dharmacon, catalog number D-001600-01) as a nonspecific control was transiently transfected into Ntera2 cells (100 nm) plated on 100-mm dishes. Transfections were carried out using Invitrogen Lipofectamine2000 according to the manufacturer's recommendations. After 72 h, cells were replated at 30–50% density for retransfection and harvested after another 72 h. For ZNF217 knockdown RNA analysis, Ntera2 cells were transfected with the siRNAs (100 nm) in 6-well dishes, and RNA was harvested at 72 h (experiments A and B) or retransfected and harvested 48 h later (experiment C); RNA was prepared using a Qiagen RNAeasy kit (catalog number 74104). Ntera2 cells were differentiated using 10–5 m retinoic acid (Sigma). Antibodies used on Western blots were anti-ZNF217 polyclonal (17Huang G. Krig S. Kowbel D. Xu H. Hyun B. Volik S. Feuerstein B. Mills G.B. Stokoe D. Yaswen P. Collins C. Hum. Mol. Genet. 2005; 14: 3219-3225Crossref PubMed Scopus (53) Google Scholar) and anti-OCT4 goat polyclonal (Santa Cruz Biotechnology, catalog number 8628 N-19). ChIP Assays and Amplicon Preparation—ChIP assays were performed as described previously (18Weinmann A.S. Bartley S.M. Zhang M.Q. Zhang T. Farnham P.J. Mol. Cell. Biol. 2001; 21: 6820-6832Crossref PubMed Scopus (331) Google Scholar) with minor modifications. A complete protocol can be found in Oberley et al. (19Oberley M.J. Tsao J. Yau P. Farnham P.J. Methods Enzymol. 2004; 376: 316-335Google Scholar). Antibodies used in this study include a ZNF217 rabbit polyclonal antibody that was generated using a glutathione S-transferase fusion peptide sequence (17Huang G. Krig S. Kowbel D. Xu H. Hyun B. Volik S. Feuerstein B. Mills G.B. Stokoe D. Yaswen P. Collins C. Hum. Mol. Genet. 2005; 14: 3219-3225Crossref PubMed Scopus (53) Google Scholar). Immunoserum was purified on a Pierce Aminolink peptide column constructed with the fusion peptide sequence. CtBP1 (catalog number 612043) and CtBP2 (catalog number 612044) antibodies were purchased from BD Transductions. The secondary rabbit anti-mouse IgG (catalog number 55436) was purchased from MP Biomedicals. Standard PCRs using 2 μl of the immunoprecipitated DNA were performed. PCR products were separated by electrophoresis through 1.5% agarose gels and visualized by ethidium bromide intercalation. Amplicons, prepared using 50–80% of a ChIP sample, were generated using a Sigma Whole Genome Amplification kit; see our published ChIP protocol (20O'Geen H. Nicolet C.M. Blahnik K. Green R. Farnham P.J. BioTechniques. 2006; 41: 577-580Crossref PubMed Scopus (124) Google Scholar) for details. ChIP-Chip Assays—ENCODE and promoter arrays were produced by NimbleGen Systems, Inc. (Madison, WI). The 5-kb human promoter array design is a two-array set, containing 5.0 kb of each promoter region (from build HG17) that extends 4.2 kb upstream and 800 bp downstream of the transcription start site. Where individual 5.0-kb regions overlap, they are merged into a single larger region, preventing redundancy of coverage. The promoter regions thus range in size from 5.0 to 50 kb. These regions are tiled at a 110-bp interval using variable length probes with a target Tm of 76 °C. NimbleGen ENCODE oligonucleotide arrays contained ∼380,000 50-mer probes per array, tiled every 38 bp. The regions included on the arrays encompassed the 30 Mb of the repeat masked ENCODE sequences, representing ∼1% of the human genome. All NimbleGen arrays were hybridized, and the data were extracted according to standard operating procedures by NimbleGen Systems Inc. Signal Map software provide by NimbleGen Systems, Inc. was used to visualize the array peaks. PCR Analysis of Target Genes—Primers were designed to flank ZNF217 binding sites identified on the arrays. PCR was performed using low cycle numbers to ensure linear amplification of the input. Bio-Rad Quantity One software was used for quantification of gene enrichment over input DNA. RNA Illumina Expression Arrays—RNA samples were harvested using a Qiagen RNAeasy kit (Qiagen) and then assayed using the Agilent Systems Bioanalyzer to ensure that high quality RNA was used for the array experiments. The Illumina TotalPrep RNA Amplification kit from Ambion (AMIL1791) was used to generate biotinylated, amplified RNA for hybridization with the Illumina Sentrix Expression BeadChips, Human 6-v1. The Sentrix gene expression beadchips used for this study consisted of a six-array, two-stripe format comprising ∼48,000 probes/array. In this collection 24,000 probes were from RefSeq sequences, and 24,000 were from other GenBank™ sequences (Illumina). Arrays were processed according to the manufacturer's instructions, scanned at medium photomultiplier tube (PMT) settings as recommended by the manufacturer, and analyzed using Bead Studio Software version 2.3.41. Data were normalized using the “average” method that simply adjusts the intensities of two populations of gene expression values such that the means of the populations become equal. Differential expression was calculated for the control versus siZNF217 data sets using an algorithm provided by Bead Studio. -Fold enrichment values were used to obtain the list of candidates with greater than 1.5-fold change. DAVID Analysis—Functional annotations were performed using the program DAVID 2.1 (Ref. 21Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar; see also david.abcc. ncifcrf.gov/). The same parameters were used for all analyses presented in this study. These parameters were gene ontology molecular function term, level 2; Interpro name in the Protein Domains section; and SP_PIR_Keywords in the Functional Categories section. Before performing the analyses, hypothetical genes and genes with no known function were removed from the list. After performing the analyses, all categories that represented less than 4% of the total number of genes were eliminated. In addition, redundant terms (e.g. transcriptional regulation and transcription factor activity) and non-informative terms (e.g. multigene family) were also eliminated. Peak Finding—For identification of the ZNF217 binding sites on the ENCODE arrays, we used the Tamalpais program described in Bieda et al. (22Bieda M. Xu X. Singer M. Green R. Farnham P.J. Genome Res. 2006; 16: 595-605Crossref PubMed Scopus (263) Google Scholar) and chose the L1 set of high confidence peaks for further analysis. Briefly these binding sites are identified as peaks that have a minimum of six consecutive probes in the top 2% of all probes on the array. Only those peaks that were identified in at least two of three biological replicates were considered to be binding sites. The human 5-kb promoter array consists of a set of two arrays encompassing a total of ∼24,000 human promoters. We refer to the two arrays in the set as “promoter 1” and “promoter 2” arrays. Most promoters encompass ∼5 kb of genomic sequence tiled with a 50-mer probe every ∼100 nt. Given that amplicons were ∼300–500 nt in length, a binding site should be the center of a hill waveform of ∼600–1000 nt in total length for an average of 6–10 probes per binding sites. Given that the probes on the “edges” of the hill will have little amplification, we reasoned that the center four probes (∼400 nt) should be well enriched above background. Hence we assigned a value to each promoter based on the highest mean of four consecutive probes, a procedure we termed “Maxfour.” To calculate these “Maxfour values,” custom software was written in Perl and bash shell. 5M. C. Bieda, L. G. Acevedo, and P. J. Farnham, manuscript in preparation. A small portion of promoters were represented by <4 probes, and hence no Maxfour value was possible; we considered analysis of these promoters to be unreliable, and these promoters were assigned “–100” as a value. Further statistical exploration of this procedure will be presented elsewhere. 5M. C. Bieda, L. G. Acevedo, and P. J. Farnham, manuscript in preparation. Location Analysis of ZNF217 Binding in the Human Genome— Although ZNF217 is predicted to contain eight zinc fingers and thus is thought to be a DNA-binding protein, its role in transcriptional regulation has not been well characterized due to a lack of known target genes. Using the ChIP-chip approach, previous studies have identified target genes of transcription factors in a global and unbiased manner (23Ren B. Robert F. Wyrick J.J. Aparicio O. Jennings E.G. Simon I. Zeitlinger J. Schreiber J. Hannett N. Kanin E. Volkert T.L. Wilson C.J. Bell S.P. Young R.A. Science. 2000; 290: 2306-2309Crossref PubMed Scopus (1566) Google Scholar, 24Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H.-M. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (388) Google Scholar). Commonly used platforms for ChIP-chip are arrays that contain CpG islands (25Heisler L.E. Torti D. Boutros P.C. Watson J. Chan C. Winegarden N. Takahashi M. Yau P. Huang T.H. Farnham P.J. Jurisica I. Woodgett J.R. Bremner R. Penn L.Z. Der S.D. Nucleic Acids Res. 2005; 33: 2952-2961Crossref PubMed Scopus (78) Google Scholar), core promoters, or 5–10 kb of upstream promoter sequences (26Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3430) Google Scholar). However, we did not know whether ZNF217 binds to CpG islands or close to transcription start sites. Therefore, to initiate our studies we first needed to perform an unbiased location analysis of ZNF217 binding. For these ChIP-chip analyses, we used ENCODE oligonucleotide arrays that represent 1% of the human genome and include ∼400 genes and intergenic regions (see “Experimental Procedures” for details). Using a rabbit polyclonal antibody to ZNF217, we performed ChIP assays in three different human cancer cell lines: the MCF7 breast cancer line, the SW480 colon cancer line, and the teratocarcinoma cell line Ntera2. Our classification of a peak as a binding site on ENCODE arrays required that the region be bound in at least two of three independent experiments (see Ref. 22Bieda M. Xu X. Singer M. Green R. Farnham P.J. Genome Res. 2006; 16: 595-605Crossref PubMed Scopus (263) Google Scholar). Therefore, three biological replicate ChIP samples from each of the three cell lines were hybridized to ENCODE arrays (see supplemental Table S1). After array normalization, the ZNF217 hybridization signals were divided by the total input signals to provide a -fold enrichment value for each 50-bp oligomer on the array. Sites that were bound by ZNF217 were identified with the Tamalpais peaks program (22Bieda M. Xu X. Singer M. Green R. Farnham P.J. Genome Res. 2006; 16: 595-605Crossref PubMed Scopus (263) Google Scholar) at the L1 level (p < 0.0001); this requires that at least six oligos in a row be in the top 2% of all probes on the array. We identified a total of 61 ZNF217 binding sites in SW480 cells, 175 binding sites in MCF7 cells, and 178 binding sites in Ntera2 cells. To determine the location of the binding sites relative to the nearest gene, we used the Gencode Database (27Harrow J. Denoeud F. Frankish A. Reymond A. Chen C.K. Chrast J. Lagarde J. Gilbert J.G. Storey R. Swarbreck D. Rossier C. Ucla C. Hubbard T. Antonarakis S.E. Guigo R. Genome Biol. 2006; 7: S4.1-S4.9Crossref Google Scholar). This analysis identified 44 genes in SW480 cells, 103 genes in MCF7 cells, and 101 genes in Ntera2 cells. We found that a significant percentage of the binding sites (39% in SW480, 41% in MCF7, and 49% in Ntera2) fell within 2 kb upstream or downstream of the transcription start site of the nearest gene, although binding was also observed in the regions greater than 2 kb upstream from the start site as well as within genes and in intergenic regions (Fig. 1A). Although most of the binding sites covered about 500 bp to 1 kb, there was one region on the ENCODE array of more than 150 kb that was bound by ZNF217 in Ntera2 cells (Fig. 1B). Because these experiments are the first to identify genomic binding sites of ZNF217 using ChIP-chip assays, we thought it was important to demonstrate the specificity of the ZNF217 antibody. Therefore, we treated Ntera2 cells with siRNAs to ZNF217 (see Fig. 7 for a Western blot indicating the degree to which ZNF217 protein can be reduced by the siRNA treatment) and then performed ChIP experiments using the ZNF217 antibody in the control versus the knockdown cells. Amplicons prepared from these ChIP samples were hybridized to ENCODE arrays. As shown in Fig. 1B, ZNF217 binding throughout the HOXA gene cluster on chromosome 7 was greatly reduced in the cells treated with siRNAs to ZNF217. Thus, because reduction of ZNF217 RNA reduced the signals obtained in the ChIP assay, we are confident that we are identifying bona fide ZNF217 target genes.FIGURE 7ZNF217 is down-regulated by retinoic acid in Ntera2 cells. Western blot of retinoic acid time course in Ntera2 shows down-regulation of ZNF217 by Day 4 of treatment to the same levels as siZNF217 treatment. OCT4 down-regulation indicates Ntera2 cells are differentiating. A section of the ponceau stain indicates levels of protein loaded in each lane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) De Novo Identification of a Putative ZNF217 Binding Site Motif—The peaks that we identified above represent the first collection of in vivo ZNF217 binding sites. It is not yet known whether ZNF217 is directly bound to the DNA at each of these sites or whether it is recruited to the sites indirectly via interaction with partners such as CtBP. Because CtBP has been shown to bind to numerous DNA-binding proteins, an indirect recruitment mechanism might result in the identification of multiple motifs in the collection of binding sites. To search for common motifs, we used a de novo motifs discovery approach termed ChIPMotifs (28Jin V.X. O'Geen H. Iyengar S. Green R. Farnham P.J. Genome Res. 2007; (in press)Google Scholar). Briefly the ChIPMotifs approach incorporates a statistical bootstrap resampling method to identify the top motifs detected from a set of ChIP-chip training data using ab initio motif-finding programs such as Weeder (29Pavesi G. Mereghetti P. Mauri G. Pesole G. Nucleic Acids Res. 2004; 32: W199-W203Crossref PubMed Scopus (390) Google Scholar) and MEME (multiple EM for motif elicitation) (30Bailey T.L. Gribskov M. J. Comput. Biol. 1997; 4: 45-59Crossref PubMed Scopus (58) Google Scholar). To obtain a training data set, we first selected the common ZNF217 binding sites identified using the ENCODE arrays from all three cell lines. This provided a set of 53 very high confidence ZNF217 binding regions (each region was identified as a binding site in at least two of three biologically independent experiments in all three of the different cell types). A set of 506 sequences from the ENCODE regions of 500 bp in length that did not bind to ZNF217 was selected as a negative control data set. After applying the ChIPMotifs approach to these training sets, we identified the motif shown in Fig. 2. An 8-base consensus is defined as ATTCCNAC (reverse complement counterpart is GTNGGAAT, Fig. 2A) with a 5-base core consensus as ATTCC (reverse complement counterpart is GGAAT). The score cutoff for this motif positional weight matrix (ZNF217_PWM, Fig. 2B) built from the ChIPMotifs was determined as 1.00 for core score and 0.86 for PWM score with a significant Fisher p value of 0.001 determined by our ChIPMotifs approach. Using these cutoff scores, 47% (25 of 53) of the ZNF217 binding regions included this motif, whereas 81% of the regions in the negative control data set lacked the motif. After finding a motif common to the ZNF217 binding sites of all three cell lines, we wanted to know whether we would identify the same or different motifs if we analyzed the three cell lines individually. An AP1-like motif, ANGAGTCA, was identified in MCF7 cells with a significant p value of 0.009 for the cutoff of 1.0 for core score and 0.86 for PWM score, and a core binding motif, CATTCC, was identified in SW480 cells with a p value of 1.1 × 10–5 for the cutoff of 1.0 for core score and 0.85 for PWM score. The SW480 core sequence is much like the motif identified using the combined data sets. This may be due to the fact that the majority of the 61 binding sites identified in SW480 were included in the 53 common binding sites in the training data set from all three cell lines. Unfortunately we were unable to identify any significant motif using only the Ntera2 binding sites; an E2F core consensus was initially identified by the ab initio programs but failed to pass the significance test using a bootstrap resampling statistical approach. It is likely that the “common motif” was not identified using the Ntera2 set because the common 53 targets were a small portion of the total number of ZNF217 binding sites in Ntera2 cells. A recent study used in vitro casting experiments to identify a 15-base consensus sequence bound by zinc fingers 6 and 7 of ZNF217 (31Cowger J.J. Zhao Q. Isovic M. Torchia J. Oncogene. 2007; (in press)PubMed Google Scholar). Although we could not identify the full consensus site in any of the in vivo ZNF217 binding sites, the core sequence of CAGAAY (where Y is a pyrimidine) was found in 64% of the MCF7, 50% of the Ntera2, and 54% of the SW480 sites identified by ChIP-chip. Identification of ZNF217 Target Genes Using Promoter Arrays—Having shown that ∼40–50% of the ZNF217 binding sites identified using ENCODE arrays localized within 2 kb upstream or downstream of the transcription start site, we felt that the 5-kb NimbleGen promoter arrays would be appropriate for the identification of a large set of ZNF217 target genes. Using this two-array set, the region spanning 4.2 kb upstream to 0.8 kb downstream of the transcription start sites of 24,000 human promoters can be analyzed with each promoter being represented by 50 probes spaced ∼100 bp apart. We next performed ChIP-chip assays using the ZNF217 antibody and two independent cultures of Ntera2 cells (see supplemental Table S1). After array normalization, the ZNF217 hybridization signals were divided by the total input signals to provide a -fold enrichment value for each 50-bp oligomer on the array. To identify binding sites on these promoter arrays, we developed a analysis program (termed “Maxfour”) that ranks each of the promoters based on the highest average intensity value for four consecutive probes (which corresponds to ∼ 400 nt). This ranking system was used for the analysis of all promoter arrays in this study. The Maxfour values from the two independent ZNF217 ChIP-chip experiments for the 14,000 promoters on promoter 1 array (the promoters are present on two different arrays termed promoter 1 and promoter 2 arrays) were aligned to examine the reproducibility of the ChIP assays. The correlat" @default.
• W2014209222 created "2016-06-24" @default.
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• W2014209222 creator A5025287489 @default.
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• W2014209222 date "2007-03-01" @default.
• W2014209222 modified "2023-10-16" @default.
• W2014209222 title "Identification of Genes Directly Regulated by the Oncogene ZNF217 Using Chromatin Immunoprecipitation (ChIP)-Chip Assays" @default.
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