ZIP: a novel transcription repressor, represses EGFR oncogene and suppresses breast carcinogenesis
2009; Springer Nature; Volume: 28; Issue: 18 Linguagem: Inglês
10.1038/emboj.2009.211
ISSN1460-2075
AutoresRuifang Li, Hua Zhang, Wenhua Yu, Yupeng Chen, Bin Gui, Jing Liang, Yan Wang, Luyang Sun, Xiaohan Yang, Yu Zhang, Lei Shi, Yanyan Li, Yongfeng Shang,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoArticle30 July 2009free access ZIP: a novel transcription repressor, represses EGFR oncogene and suppresses breast carcinogenesis Ruifang Li Ruifang Li Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Hua Zhang Hua Zhang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Wenhua Yu Wenhua Yu Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yupeng Chen Yupeng Chen Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Bin Gui Bin Gui Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Jing Liang Jing Liang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yan Wang Yan Wang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Luyang Sun Luyang Sun Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Xiaohan Yang Xiaohan Yang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yu Zhang Yu Zhang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Lei Shi Lei Shi Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yanyan Li Yanyan Li Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yongfeng Shang Corresponding Author Yongfeng Shang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Ruifang Li Ruifang Li Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Hua Zhang Hua Zhang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Wenhua Yu Wenhua Yu Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yupeng Chen Yupeng Chen Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Bin Gui Bin Gui Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Jing Liang Jing Liang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yan Wang Yan Wang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Luyang Sun Luyang Sun Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Xiaohan Yang Xiaohan Yang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yu Zhang Yu Zhang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Lei Shi Lei Shi Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yanyan Li Yanyan Li Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Yongfeng Shang Corresponding Author Yongfeng Shang Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China Search for more papers by this author Author Information Ruifang Li1, Hua Zhang1, Wenhua Yu1, Yupeng Chen1, Bin Gui1, Jing Liang1, Yan Wang1, Luyang Sun1, Xiaohan Yang1, Yu Zhang1, Lei Shi1, Yanyan Li1 and Yongfeng Shang 1 1Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, China *Corresponding author. Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, China. Tel.: +86 10 8280 5118; Fax: +86 10 8280 1355; E-mail: [email protected]n The EMBO Journal (2009)28:2763-2776https://doi.org/10.1038/emboj.2009.211 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite the importance of epidermal growth factor receptor (EGFR) in animal development and malignant transformation, surprisingly little is known about the regulation of its expression. Here, we report a novel zinc finger and G-patch domain-containing protein, ZIP. We demonstrated that ZIP acts as a transcription repressor through the recruitment of the nucleosome remodelling and deacetylase complex. Transcriptional target analysis revealed that ZIP regulates several cellular signalling pathways including EGFR pathways that are critically involved in cell proliferation, survival, and migration. We showed that ZIP inhibits cell proliferation and suppresses breast carcinogenesis, and that ZIP depletion leads to a drastic tumour growth in vivo. We found that ZIP is downregulated in breast carcinomas and that its level of expression is negatively correlated with that of EGFR. Our data indicate that ZIP is a novel transcription repressor and a potential tumour suppressor. These findings may shed new light on the EGFR-related breast carcinogenesis and might offer a potential new target for breast cancer therapy. Introduction Growth factors and their transmembrane receptor kinases have important functions in an array of cellular behaviours including cell proliferation, survival, adhesion, migration, and differentiation (Yarden and Sliwkowski, 2001). The epidermal growth factor receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3), and HER4 (erbB-4) (Yarden and Sliwkowski, 2001). These proteins are composed of an extracellular ligand-binding domain and an intracellular tyrosine kinase domain, joined by a transmembrane segment. On ligand binding, EGFR family proteins undergo conformational changes in the ectodomain, which facilitate the formation of homo/heterodimers or oligomers triggering tyrosine kinase phosphorylation (Zandi et al, 2007). As a consequence, second-messenger pathway cascades, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are activated, ultimately leading to the alteration of cellular behaviours (Zandi et al, 2007). Since the identification of a link between EGFR and a transforming viral oncogene v-erb-B (Downward et al, 1984), it has been well established that EGFR is involved in malignant transformation and progression of a broad variety of cancers (Holbro et al, 2003; Chan et al, 2006). Indeed, EGFR overexpression have been reported in cancers originating from bladder, brain, breast, cervical, uterine, colon, esophageal, glioma, lung, ovarian, pancreatic, and renal cell (Chan et al, 2006). This deregulation is often associated with a more aggressive phenotype and accordingly worse survival of the cancer patients (Nicholson et al, 2001). This scenario makes the EGFR family an ideal target to be exploited for cancer therapeutics. Current anti-EGFR therapies include monoclonal antibodies, such as cetuximab, panitumumab, and matuzumab, which target the extracellular domain of EGFR, and small-molecule tyrosine kinase inhibitors, such as gefitinib (Iressa) and erlotinib, which target the receptor catalytic domain (Mendelsohn and Baselga, 2006). Despite the extensive molecular and functional characterization of EGFR and a continuing effort in pursuing anti-EGFR cancer therapies, little is known about the mechanism underlying the regulation/deregulation of EGFR expression. This issue is of particular importance as it is noted that amplifications in the EGFR gene were restricted to region of the regulatory sequence in the 5′-end of intron 1 and associated with EGFR expression in epithelial breast tumours (Brandt et al, 2006), implying the importance of transcriptional regulation of EGFR in breast carcinogenesis. Transcriptional repression can be mediated by several mechanisms. One repression mechanism involves the recruitment of corepressor complexes (Hu and Lazar, 2000; Rosenfeld et al, 2006), many of which contain subunits that possess histone deacetylase (HDAC) activity. HDACs act to deacetylate histones and hence convert chromatin into a repressive state (Rosenfeld et al, 2006). The Mi-2/nucleosome remodelling and deacetylase (NuRD) complex has important functions in animal development and physiology (Ahringer, 2000). This complex is a multi-subunit protein assembly with both histone deacetylation and chromatin remodelling ATPase activities and functions primarily in gene transcriptional repression (Zhang et al, 1998; Denslow and Wade, 2007). To date, the NuRD complex has been documented to mediate the transcription repression by distinct sequence-specific transcription factors, including p53, Ikaros, Hunchback, Tramtrack69, KAP-1, BCL-6, and FOG-1 (Bowen et al, 2004; Denslow and Wade, 2007). It is believed that every subunit of this complex exhibits heterogeneity at the protein and/or gene level and that the functional specialization of the NuRD complex is largely determined by incorporation of unique gene products into the complex (Bowen et al, 2004; Denslow and Wade, 2007). In this work, we describe the identification and functional characterization of ZIP, a novel zinc finger and G-patch domain-containing protein. We demonstrated that ZIP recruits the NuRD complex to EGFR promoter and represses EGFR transcription. We show that ZIP inhibits cell proliferation and suppresses breast carcinogenesis. These data support a role for ZIP as a novel transcription repressor and a potential tumour suppressor. Results Cloning and characterization of ZIP We cloned a gene, ZIP (for ZInc finger and G-Patch domain-containing of its protein product), from a mammary cDNA library. The cDNA of ZIP is 1882 bp in length (GeneBank ID BC032612) and contains an open reading frame encoding for a protein of 511 amino acids. The predicted molecular mass of this protein is ∼55.6 kDa, with a theoretical isoelectric point of 5.49. The corresponding gene is mapped to chromosome 20q13.3 and consists of seven exons and six introns. Bioinformatics analysis indicates that ZIP harbours a CCCH or C3H1 type of zinc finger, a TUDOR domain, a G-patch domain, a coiled-coil domain, and a nuclear localization signal (Figure 1A). Amino-acid sequence alignment reveals that human ZIP shares 77.9% identity with its mouse homologue and the similarity of the amino-acid sequence of ZIP with homologues in other organisms is 76.7% in Rattus norvegicus, 49.2% in Danio rerio, 19.4% in Caenorhabditis elegans, and 24.1% in Drosophila melanogaster (Figure 1B). Phylogenetic analysis also indicates that ZIP is an evolutionarily well-conserved gene (Figure 1C). Figure 1.Cloning and characterization of ZIP. (A) A schematic representation of the structure of ZIP. The following conserved domains are shown: ZnF (zinc finger), TUDOR, G-patch, and coiled coil. (B) Amino-acid sequence alignment of ZIP from different species. Shaded residues represent conserved regions (upper panel), and conserved domains of ZIP homologues from different species are indicated (lower panel). (C) Phylogenetic analysis of evolutionary relationships among homologues of ZIP proteins from different species. Download figure Download PowerPoint To confirm the existence of ZIP transcript(s) and to examine the expression profile of ZIP, we analysed the expression of ZIP mRNA by Northern blotting with Clontech's human multiple tissue blots. The results indicate that ZIP gene transcribes an ∼1.8 kb message in various tissues (Figure 2A). In the liver and kidneys, additional transcripts were detected (Figure 2A). We focused our research on the ∼1.8 kb transcript because it is the transcript that we initially cloned and it is the transcript that exhibits a broader tissue distribution. Figure 2.Expression and subcellular localization of ZIP. (A) Northern blotting analysis of ZIP mRNA expression in different tissues. (B) Western blotting analysis of ZIP protein expression. MCF-7 cells were transfected with empty vector or FLAG-ZIP or EGFP-ZIP. Cellular proteins were prepared and western blotting was performed with anti-FLAG (upper panel) or anti-ZIP (lower panel). MCF-7 (ZIP): MCF-7 cells overexpressing ZIP. (C) RT–PCR (left panel) and western blotting (right panel) analysis of ZIP expression in different cancer cell lines. GAPDH and β-actin were used as internal controls. (D) Subcellular localization of ZIP protein. MCF-7 cells were transfected with EGFP-ZIP (upper panel) or FLAG-ZIP (lower panel). Twenty-four hours after transfection, EGFP fluorescence and rhodamine staining of FLAG were visualized by fluorescence microscopy. DAPI staining was included to visualize the cell nucleus. Download figure Download PowerPoint To examine the expression of ZIP protein, a FLAG-tagged ZIP expression construct (FLAG-ZIP) was transfected into mammary carcinoma MCF-7 cells. Twenty-four hours after transfection, cellular proteins were extracted and analysed by western blotting with a monoclonal antibody against FLAG. The results indicate that ZIP is expressed as a protein of ∼56 kDa (Figure 2B, upper panel). Western blotting analysis of endogenous ZIP along with overexpressed FLAG-ZIP or enhanced green fluorescent protein (EGFP)-tagged ZIP (EGFP-ZIP) proteins with polyclonal antibodies against ZIP, which we generated with recombinant ZIP (364–511 aa), indicate that ZIP has an apparent Mr of ∼56 kDa (Figure 2B, lower panel), confirming its predicted molecular weight. In addition, both reverse transcriptase (RT)–PCR (left panel) and western blotting (right panel) analyses detected ZIP expression in various cell lines (Figure 2C). To gain insight into the biological function of the ZIP protein, we first examined its subcellular localization. Both fluorescent imaging of EGFP-ZIP and immunostaining of FLAG-ZIP in MCF-7 cells indicate that ZIP is primarily a nuclear protein (Figure 2D), suggesting that ZIP may function primarily in the nucleus. ZIP binds DNA and recognizes specific DNA sequences Transcriptional regulation is a primary research focus in our laboratory (Shang and Brown, 2002; Zhang et al, 2004, 2006, 2007; Wu et al, 2005, 2006; Shang, 2006; Shi et al, 2007; Liang et al, 2009). The presence of a zinc finger domain in ZIP prompted us to investigate the hypothesis that ZIP might recognize and bind to specific genomic sequences. We, therefore, performed cyclic amplification and selection of target (CASTing) assays to search for putative DNA-binding sequences for ZIP by screening double-stranded random oligonucleotides using a glutathione S-transferase fusion protein (GST–ZIP) immobilized on glutathione Sepharose 4B beads. As shown in Figure 3A, GST–ZIP fusion protein was found to bind DNA sequences specifically after the second round of binding and amplification reaction; DNA products were only detected with GST–ZIP, but not with GST, after this round. We performed a total of nine rounds of binding and amplification reactions. After that, the final PCR products were cloned and sequenced. Of 93 sequences that were cloned and sequenced, 80 contained a GA-rich DNA element GGAGG/AAG/AA (Figure 3A). Figure 3.ZIP is a DNA-binding protein and possesses intrinsic transcription repression activity. (A) CASTing assay. Binding and amplification reactions were done with GST or GST–ZIP fusion proteins. Coomassie blue staining of the purified GST and GST–ZIP fusion proteins and the results from PCR amplification of bound DNA up to four rounds are shown on the left. The computational result using Weblogo (http://weblogo.berkeley.edu) for conserved nucleotides within ZIP-binding sequences is shown on the right. (B) Transcription repression by ZIP. The schematic diagram shows the GAL4-luciferase reporters. For reporter assays, MCF-7 cells were transfected with different amounts of GAL4–ZIP expression construct together with the indicated GAL4-luciferase reporter. Twenty-four hours after transfection, cells were harvested and luciferase activity was measured and normalized to that of renila. Each bar represents the mean±s.d. for triplicate experiments. (C) Reporter assays with FLAG-ZIP transfection (left panel) or with TSA treatment (right panel). MCF-7 cells were transfected with indicated plasmids and were treated with TSA or left untreated. Cells were then harvested and luciferase activity was measured and normalized to that of renila. Each bar represents the mean±s.d. for triplicate experiments. (D) In vitro HDAC activity assay for the ZIP complex. Nuclear extracts from HeLa cells stably transfected with FLAG-ZIP were immunoprecipitated with anti-FLAG antibody. Increased amounts of immunoprecipitates (IPs) were incubated either with [3H]acetate-labelled HeLa histones for deacetylase activity determination by liquid scintillation counting of released [3H]acetate (left panel) or with calf thymus histones followed by immunoblotting analyses with antibodies against acetylated H3 and total H3 (right panel). Download figure Download PowerPoint ZIP possesses intrinsic transcription repression activity accompanied by histone deacetylation The fact that ZIP harbours a zinc finger and the result of CASTing assays suggest that ZIP may indeed be a DNA-binding protein and may thus be involved in transcriptional regulation. To determine whether ZIP does in fact possess a trans-acting activity, we fused ZIP to the C-terminus of GAL4 DNA-binding domain and tested the transcription activity of the fused construct in MCF-7 cells. We used three different GAL4-driven luciferase reporter systems, which differ in basal promoter elements (Figure 3B). The results show that ZIP drastically repressed the reporter activity in a dose-dependent manner in all of the three reporter systems. In the meanwhile, overexpression of FLAG-ZIP did not affect the activity of GAL4-driven reporter (Figure 3C, left panel), suggesting that ZIP must be physically associated with DNA to exert its transcription repression activity. Similar results were also obtained in the endometrial carcinoma cell line ECC-1 and the lung carcinoma cell line A549 (data not shown). As stated above, one common mechanism of gene transcription repression is through the recruitment of corepressor complexes that contain subunits with HDAC activities (Hu and Lazar, 2000; Rosenfeld et al, 2006). To determine whether HDAC activity is required for ZIP-mediated gene repression, we measured the reporter activity in cells treated with trichostatin A (TSA), a specific HDAC inhibitor. The results indicate that TSA treatment was able to almost completely alleviate the repression of the reporter activity by ZIP construct (Figure 3C, right panel), suggesting that ZIP-mediated repression was associated with a HDAC activity. To further support this, nuclear extracts from HeLa cells stably expressing FLAG-ZIP were immunoprecipitated with the anti-FLAG antibody. The ZIP-containing complex was then tested for HDAC activity by incubating the immunoprecipitates with [3H]acetate-labelled HeLa histones. In vitro HDAC activity was measured by quantifying the release of radiolabelled acetyl groups from purified hyperacetylated HeLa histones. We found that FLAG-ZIP immunoprecipitates from HeLa cell extracts had HDAC activity and that treatment of the immunoprecipitates with TSA reduced HDAC activity to background levels (Figure 3D, left panel). In addition, incubating the immunoprecipitates with calf thymus bulk histones followed by immunoblotting also indicates that the acetylation level of H3 was greatly reduced (Figure 3D, right panel). All these experiments support the hypothesis that the ZIP complex is associated with a histone deacetylation activity. ZIP is physically associated with the NuRD complex To further elucidate the molecular mechanism underlying ZIP-mediated transcription repression, ZIP-containing protein complexes were affinity purified from nuclear extracts of HeLa cells stably expressing FLAG-ZIP with the anti-FLAG antibody that was immobilized on agarose beads. The purified protein complex was resolved on SDS–PAGE and silver stained (Figure 4A). Mass spectrometry analysis identified, in addition to ZIP, DHX15 [DEAH (Asp-Glu-Ala-His) box polypeptide 15], and CBP80 (nuclear cap-binding complex subunit 1), protein components of the NuRD complex, including Mi-2α, Mi-2β, RbAp46/48, MTA2, HDAC1, HDAC2, and MBD3 (Figure 4A), suggesting that ZIP is physically associated with the NuRD complex in vivo. Figure 4.Physical association of ZIP with the NuRD complex. (A) Mass spectrometry analysis of ZIP-associated proteins. Nuclear extracts from HeLa cells stably expressing FLAG-ZIP were prepared and subjected to affinity-purification with anti-FLAG antibody that was immobilized on agarose beads. The purified protein complex was resolved on SDS–PAGE and silver stained, and the bands were retrieved and analysed by mass spectrometry. DHX15: DEAH (Asp-Glu-Ala-His) box polypeptide 15; CBP80: nuclear cap-binding complex subunit 1. Complete amino-acid sequences from mass spectrometry analysis are included in Supplementary data 3. (B) Co-immunoprecipitation of ZIP and the components of the NuRD complex. Whole-cell lysates from HeLa cells were prepared and immunoprecipitation was performed with anti-ZIP followed by immunoblotting with antibodies against indicated proteins (upper panel), or immunoprecipitated with antibodies against Mi-2, MTA2, RbAp46/48, HDAC1, HDAC2, or IgG followed by immunoblotting with anti-ZIP (lower panel). (C) ZIP interacts directly with Mi-2 in vitro. GST pull-down assays were performed with GST–ZIP and in vitro transcribed/translated components of the NuRD complex (left panel) or with GST–ZIP (1-511) or GST-fused ZIP deletion mutants (number represents the amino-acid position; ΔZnF: ZIP without zinc finger; ΔCC: ZIP without coiled-coil domain) and in vitro transcribed/translated Mi-2 (right panel). (D) Co-fractionation of ZIP and the NuRD complex by FPLC. Cellular extracts from HeLa cells were fractionated on Superose 6 size exclusion column. The chromatographic profile with the elution positions of calibrating proteins of known molecular mass is shown. The chromatographic fractions were analysed by western blotting with antibodies against indicated proteins or were first incubated with bulk histones and then analysed by western blotting with anti-acetylated H3 (AcH3) or anti-H3. Equal volumes from each fraction were analysed. Download figure Download PowerPoint To confirm an in vivo interaction between ZIP and the NuRD complex, total proteins from HeLa cells were extracted and immunoprecipitated with the antibodies against ZIP. The immunoprecipitates were then immunoblotted with antibodies against the components of the NuRD complex and also against mSin3A. The results show that the components of the NuRD complex, but not mSin3A, could be efficiently co-immunoprecipitated with ZIP (Figure 4B, upper panels). Reciprocal immunoprecipitations with antibodies against the components of the NuRD complex, including Mi-2, RbAp46/48, MTA2, HDAC1, and HDAC2, and immunoblotting with anti-ZIP also revealed that ZIP is co-immunoprecipitated with the components of the NuRD complex (Figure 4B, lower panel). GST pull-down assays were then performed to investigate the molecular details of the interaction between ZIP and the NuRD complex. Bacterially expressed GST–ZIP proteins were purified and incubated with in vitro transcribed/translated components of the NuRD complex. The results of these experiments indicate that ZIP only interacts directly with Mi-2, suggesting that the recruitment of the NuRD complex by ZIP in its transcription repression is through an interaction of ZIP with Mi-2 (Figure 4C, left panel). Further analyses by GST pull-down assays with deletion mutants of ZIP revealed that the coiled-coil domain of ZIP is responsible for the interaction of ZIP with Mi-2 (Figure 4C, right panel). To further consolidate the in vivo association of ZIP and the NuRD complex, protein fractionation experiments were carried out through a high salt extraction and size exclusion approach by fast protein liquid chromatography (FPLC) using Superose 6 size columns. The result of the experiment revealed that native ZIP in HeLa cells could be eluted in chromatographic fractions with apparent molecular masses much greater than that of the monomeric protein; ZIP immunoreactivity could be detected in elutes with high molecular masses and with a relatively symmetrical peak centred around ∼669 kDa, and the elution pattern of ZIP in chromatographic fractions with high molecular masses was largely overlapped with that of the NuRD complex proteins, including MTA2, HDAC1, HDAC2, RbAp46/48, and Mi-2 and was accompanied by HDAC activities, as assayed by incubating these fractions with the bulk histones and then immunoblotted with anti-acetylated H3 (Figure 4D), supporting the hypothesis that ZIP is associated with the NuRD complex in vivo. Identification of the transcriptional targets for ZIP On the basis of the DNA-binding element that we identified in CASTing assays, we searched the Eukaryotic Promoter Database (EPD) (http://cmgm.stanford.edu/help/manual/databases/epd.html) for genes that might be potentially targeted by ZIP. The search yielded 383 genes containing the putative ZIP-binding sites in their 5′-upstream regulatory regions, including EGFR (Supplementary data 1). Next, we decided to identify potential downstream targets of ZIP in the human genome using Chromatin ImmunoPrecipitation-DNA selection and ligation (ChIP-DSL). ChIP experiments were first conducted in MCF-7 cells with ZIP antibodies. After ChIP, ZIP-associated DNAs were amplified using nonbiased conditions, labelled, and hybridized to AVIVA's Hu20K arrays. Relative confidence prediction scores were generated by quantile normalization across each probe followed by an analysis using a two-state Hidden Markov model (Mukherjee and Mitra, 2005). These scores included both probe intensity and width of probe cluster. Triplicate experiments eliminated stochastic false positives, after which peaks that reproducibly appeared at least twice in the three replicates were included. The detailed results of the ChIP-DSL experiments are deposited in GEO Datasets (accession
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