Identification of P100 Target Promoters by Chromatin Immunoprecipitation-Guided Ligation and Selection (Chip-GLAS)

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Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)

Xin Liu1,2,3,6, Lijie Dong1,2,3,6, Xuejun Zhang1,2,3, Baoya Wang1,2,3, Xinting Wang1,2,3,4,HuLi1,2,3,4, Jinyan He1,2,3, Lin Ge1,2,3, Xiang Jing5, Zhi Yao1,2,3 and Jie Yang1,2,3,4

The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery. To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100, we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS). From this assay, we determined that a set of promoter fragments, including several factors in the transforming growth factor beta (TGF-b) signaling pathway, exhibited interaction with p100. The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines. Cellular & Molecular Immunology (2011) 8, 88–91; doi:10.1038/cmi.2010.47; published online 4 October 2010

Keywords: chromatin immunoprecipitation; microarray; p100 protein

INTRODUCTION These studies suggest that p100 may play several important roles in Transcriptional regulation involves protein complexes specifically cellular events and that it is likely to be a potential coactivator in assembled at a given promoter to activate or suppress gene transcrip- distinct signal transduction pathways. Because promoter microarrays tion. Transcriptional activation is regulated by coactivators that con- are an essential tool for global transcription factor binding studies, we nect sequence-specific DNA-binding transcription factors to the adopted the ChIP-on-chip microarray technique to discover potential general transcriptional machinery to initiate gene transcription. transcription complexes that interact with p100. Coactivators are broadly divided into two classes according to their function: (1) chromatin remodeling enzymes that promote the DNA MATERIALS AND METHODS binding ability of transcription factors; and (2) adaptors that recruit Cell culture and transfection transcription factors to the transcriptional apparatus. HeLa cells were cultured as previously described.6 The HeLa-p100- p100 is a highly homologous protein expressed in Histoplasma cap- Flag stable cell line was generated by co-transfecting pSG5-p100-Flag sulatum, Saccharomyces pombe, Mus musculus, Bos taurus and Homo plasmid with hygromycin B resistance gene using the FUGENE6 sapiens. Human p100 was first identified as a coactivator of Epstein– Transfection Reagent (Roche Diagnostics). The breast cancer cell line Barr virus nuclear protein 21 and was subsequently discovered as a MDA-MB-231 was transiently transfected with pSG5-p100-Flag plas- coregulator of c-Myb and pim-1.2 In mammary epithelial cells, p100 mid by using Lipofectamine 2000 (Invitrogen). levels increased in response to Prolectin (PRL) during lactation and correlated with the induction of b-casein gene expression.3 In our Chromatin preparation and chromatin immunoprecipitation previous study, we found that p100 functions as a transcriptional HeLa-p100-Flag stable cells were fixed with crosslinking solution con- coactivator for STAT5-dependent gene regulation and discovered taining 1% formaldehyde (Sigma). The crosslinking reaction was the existence of a positive regulatory loop in PRL-induced transcrip- then quenched by adding glycine solution. After washing with cold tion in which PRL stabilizes p100.4 Other groups have found that p100 phosphate buffered saline, cells were resuspended in lysis buffer5 serves as a core protein in the STAT6 enhanceosome,5,6 which is com- at 4 uC for 5 min. After sonication, the lysate was centrifuged at posed of STAT6, p100, CBP, RNA pol II and SRC-1. 14 000 r.p.m. for 10 min. The chromatin were immunoprecipitated

1Department of Immunology, Basic Medical College, Tianjin Medical University, Tianjin, China; 2Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin, China; 3Key Laboratory of Educational Ministry of China, Tianjin, China; 4Laboratory of Molecular Immunology, Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, China and 5Department of Ultrasonography, Tianjin Third Central Hospital, Tianjin, China 6 These authors contributed equally to the work. Correspondence: Dr J Yang and Dr Z Yao, Department of Immunology, Tianjin Medical University, Heping District Qixiangtai Road No. 22, Tianjin 300070, China. E-mail: [email protected], [email protected] Received 10 November 2009; revised 7 July 2010; accepted 21 July 2010 p100 protein is involved in regulating TGF-b pathway X Liu et al 89 with anti-Flag M2 agarose (Sigma) or polyclonal rabbit anti-IgG (as Quantification of gene expression by real-time PCR negative control) overnight at 4 uC. After washing, the agarose was Total RNA was isolated from non-transfected and transfected MDA- incubated with elution buffer at 65 uC for 15 min. The eluted samples MB-231 cells using Trizol (Invitrogen) according to the manufac- were incubated at 65 uC overnight to reverse the crosslinking. The turer’s instructions. The RNA was used for cDNA synthesis using samples were then amplified, labeled and hybridized to a chromatin oligo(dT) primers and One Step RNA PCR Kit (M-MLV; Takara immunoprecipitation-guided ligation and selection (ChIP-GLAS) Biotechnology). microarray containing 20 000 unique human promoter regions The PCR reactions were performed as previously described.6 The (Aviva Systems Biology, San Diego, CA, USA). cycling conditions (30 cycles) consisted of denaturation at 94 uC for

Figure 1 (a) The human p100 protein was efficiently overexpressed in HeLa cells transfected with recombinant plasmids containing p100-Flag. Total cell lysates from parental HeLa cells or stable cells overexpressing p100 (HeLa-p100) were separated on an 8% SDS-PAGE gel and analyzed by western blotting with anti-p100 antibody (upper panel), anti-Flag antibody (middle panel) or anti-b-actin antibody (lower panel). (b) Smad1, Smad2, Smad3, Smad4, Smurf2, TGFb1I1 and TGIF1 mRNA levels were measured by real-time PCR in parental HeLa cells and HeLa-p100 stable cells. (c) The p100 protein was efficiently overexpressed in MDA-MB-231 cells transfected with recombinant plasmid containing p100-Flag. Equal amounts of total cell lysates from pSG5-p100-Flag transfected or untransfected parental MB- 231 cell line were separated on an 8% SDS-PAGE gel followed by immunoblotting with anti-Flag antibody (upper panel), anti-p100 antibody (middle panel) or anti-b- actin antibody as a control (lower panel). (d) Smad1, Smad2, Smad3, TGFb1I1, TGIF1, Smurf2 and Smad4 mRNA levels were detected by real-time PCR. (e) The PCR product was detected on a 2% agarose gel. The results shown are representative of three independent experiments. TGFB1I1, transforming growth factor beta 1- induced transcript 1; TGIF1, transforming growth factor beta 1-induced factor homeobox 1; SDS-PAGE, SDS–polyacrylamide gel electrophoresis.

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Table 1 List of transcription factors and regulators with promoters exhibiting high p100 binding affinity in the TGF-b signaling pathway Nucleotide accession ID Ratio P value Name Description

NM_022739 Amine#10693 4.790 0.04 SMURF2 SMAD-specific E3 ubiquitin protein ligase 2 NM_006022 Amine#18644 4.578 0.03 TGFB1I4 TSC22 domain family, member 1 NM_015927 Amine#18772 4.168 0.03 TGFB1I1 TGF-b1-induced transcript 1 NM_005902 Amine#18626 3.829 0.05 SMAD3 SMAD, mothers against DPP homolog 3 (Drosophila) NM_173211 Amine#13690 3.701 0.05 TGIF1 TGFB-induced factor 1 NM_020429 Amine#10170 3.688 0.05 SMURF1 SMAD-specific E3 ubiquitin protein ligase 1 NM_005359 Amine#18590 2.909 0.05 SMAD4 SMAD, mothers against DPP homolog 4 (Drosophila) NM_005901 Amine#06888 2.675 0.05 SMAD2 SMAD, mothers against DPP homolog 2 (Drosophila) NM_005900 Amine#06887 2.497 0.05 SMAD1 SMAD, mothers against DPP homolog 1 (Drosophila)

Abbreviations: DPP, decapentaplegic protein; TGF, transforming growth factor.

30 s, annealing at 57.5 uC for 30 s (Smad1, Smad2 and Smad3) or suggested that p100 might be a transcriptional coactivator involved 58.1 uC (Smad4 and Smurf2) or 61.2 uC (transforming growth factor in the TGF-b signaling pathway (Table 1). To verify the effects of p100 beta 1-induced transcript 1, TGFb1I1 and transforming growth factor on the expression of the related members of the TGF-b signaling beta 1-induced factor homeobox 1, TGIF1) and elongation at 72 uC for pathway, we performed real-time PCR to detect the mRNA expression 20 s. The PCR samples were detected on 2% agarose gels. in parental HeLa cells and HeLa-p100 stable cells. As shown in Figure 1b, p100 overexpression enhanced Smad1, Smad4, Smurf2 Western blot analysis and TGIF1 mRNA expression, which is consistent with the ChIP- Total cell lysates were collected using ice-cold RIPA lysis buffer (1% GLAS results. However, there was no significant difference in NP-40, 0.1% SDS, 300 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1 mM Smad2, Smad3 and TGFb1I1 mRNA expression. It was recently reported that p100 expression level is higher in breast EDTA, 20% glycerol and 0.1 mM sodium orthovanadate). Proteins 10 were separated on an 8% sodium dodecyl sulfate–polyacrylamide gel cancer cells than in normal breast tissue. Therefore, we used the electrophoresis gel and analyzed by western blotting with p100 and b- breast cancer cell line MDA-MB-231 to further investigate the effect actin mouse monoclonal antibody (1 : 5000 dilution; Sigma) or anti- of p100 on the expression of related target genes in the TGF-b path- Flag monoclonal antibody (1 : 1000 dilution; Sigma). The results were way. As shown in Figure 1d and 1e, while Smad3 mRNA expression visualized using chemiluminescent substrate kit (SuperSignal West level was only slightly increased, Smad1, Smad2, TGIF1, Smad4, Pico Trial Kit; Pierce Biochemicals). TGFb1I1 and Smurf2 mRNA expression levels were increased remark- ably in MDA-MB-231 cells with ectopically expressed p100 compared with parental 231 cells. These data are consistent with the ChIP-GLAS RESULTS AND DISCUSSION results and indicate that ectopic p100 expression enhanced the Setting up stable cells overexpressing p100 protein expression of certain TGF-b signaling pathway members in the breast The eukaryotic expression plasmid containing p100 tagged with Flag cancer cell line MDA-MB-231. Thus, p100 is a potential regulator in peptide was co-transfected with plasmid containing the hygromycin B the TGF-b signaling pathway. Considering the roles of Smad1-4, resistance gene into HeLa cells and then selected with media contain- Smurf2, TGFb1I1 and TGIF1 in the TGF-b signaling pathway and ing hygromycin B. recent studies demonstrating that p100 upregulation is related to Western blot was performed to confirm Flag-tagged p100 expres- breast cancer,10 prostate cancer 11 and colon cancer,12 further under- sion from p100-stable HeLa cells. Figure 1a shows that while the standing of p100 in the regulation of the TGF-b pathway may help parental HeLa cells did not express Flag-tagged p100, the stable cell elucidate the underlying mechanisms of tumorigenesis. line overexpressed Flag-tagged p100 protein detected by anti-Flag antibody. ACKNOWLEDGEMENTS We thank H. Zhang for his kind technical assistance. This work was supported by grants from the National Basic Research Program (973 Program, Transforming growth factor beta (TGF-b) signal pathway target 2009CB918903), 863 Project of the Ministry of Science and Technology of genes identified by ChIP-GLAS China (2007AA02Z115), NSFC (90919032, 30970562, 30670441, 30811130394 We used microarrays containing 20 000 putative human promoters to and 30870562), Tianjin Municipal Science and Technology Commission detect the promoters with high binding affinity to p100. The complete (08ZCGHHZ01900 and 08JCYBJC07700), Specialized Fund for the Doctoral ChIP-GLAS statistical data analysis is shown in the supplementary Program of Higher Education (20091202110001) and Tianjin Educational material. To eliminate background noise, we only considered those Committee Foundation (2008ZD01). gene promoters showing a more than twofold relative binding value. 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