FEBS Letters 583 (2009) 638–642 journal homepage: www.FEBSLetters.org Proteomic and genomic analysis of PITX2 interacting and regulating networks Yue Huang a, Kan Huang a, Goran Boskovic d, Yulia Dementieva e, James Denvir f, Donald A. Primerano a,d, Guo-Zhang Zhu a,b,c,* a Biomedical Science Graduate Program, Marshall University, Huntington, WV 25755, USA b Department of Biological Sciences, One John Marshall Drive, Marshall University, Huntington, WV 25755, USA c Cell Differentiation and Development Center, Marshall University, Huntington, WV 25755, USA d Department of Biochemistry and Microbiology, Marshall University, Huntington, WV 25755, USA e Department of Mathematics, Marshall University, Huntington, WV 25755, USA f Department of Statistics, West Virginia University, Morgantown, WV 26506, USA article info abstract Article history: Pituitary homeobox 2 (PITX2) is a homeodomain transcription factor that has a substantial role in Received 18 October 2008 cell proliferation and differentiation in various tissues. In this report, we have conducted a system- Revised 20 December 2008 atic study, using proteomic and genomic approaches, to characterize PITX2-interacting proteins and Accepted 15 January 2009 PITX2-regulating genes. We identified four novel PITX2-associated protein partners Y box binding Available online 25 January 2009 factor-1, heterogeneous ribonucleoprotein K, nucleolin and heterogeneous nuclear ribonucleopro- Edited by Lukas Huber tein U in mass spectrometry analysis. We also found that overexpression of PITX2 upregulated 868 genes (2–25-fold) and downregulated 191 genes (2–15-fold) in DNA microarray analysis. These data provide an insightful perspective for further studying PITX2 function and mechanism of action. Keywords: PITX2 YB-1 Structured summary: b-Catenin MINT-6823857: PITX2 (uniprotkb:Q99697) physically interacts (MI:0218) with hnRNP K (uni- Proliferation protkb:P61978), YB-1 (uniprotkb:P67809), Nucleolin (uniprotkb:P19338) and hnRNP U (uni- Differentiation protkb:Q00839) by anti tag coimmunoprecipitation (MI:0007) MINT-6823877: PITX2 (uniprotkb:Q99697) physically interacts (MI:0218) with beta catenin (uni- protkb:P35222), hnRNP U (uniprotkb:Q00839), Nucleolin (uniprotkb:P19338), YB-1 (uniprotkb:P67809) and hnRNP K (uniprotkb:P61978) by anti tag coimmunoprecipitation (MI:0007) MINT-6823902: YB-1 (uniprotkb:P67809) physically interacts (MI:0218) with PITX2 (uniprotkb:Q99697) by anti bait coimmunoprecipitation (MI:0006) Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction during the development of cardiac outflow tract [3]. Mutations of PITX2 have been identified in several human disorders, such as Transcription factor pituitary homeobox 2 (PITX2) is a member Axenfeld–Rieger syndrome, iridogoniodysgenesis syndrome and of the homeobox gene family. A number of studies have demon- sporadic Peter syndrome [6,7]. Pitx2-deficient mice are embryonic strated that PITX2 has a diverse role in cell proliferation, differen- lethal and show severe defects in heart, eye, pituitary gland and tiation, hematopoiesis and organogenesis [1–4]. During early tooth organogenesis [3]. Previous studies have shown that PITX2 embryogenesis, PITX2 is a key regulator in the establishment of cooperates with b-catenin and LEF/TCF and thus regulates cell embryo left-right asymmetry [5]. In response to Wnt and other proliferation by directly activating transcription of cyclin Ds and growth factors, PITX2 regulates cell-type specific cell proliferation c-myc [3,8,9]. Besides b-catenin and LEF/TCF, other functional binding partners of PITX2, such as nuclear factor-1 (NF-1), high mobility group-17 (HMG-17), MEF2A, Pit-1 and glial cells missing Abbreviations: PITX2, pituitary homeobox 2; YB-1, Y box binding factor-1; hnRNP U, heterogeneous nuclear ribonucleoprotein U; hnRNP K, heterogeneous a (GcMa), have also been reported [1,10–13]. ribonucleoprotein K; NF-1, nuclear factor-1; HMG-17, high mobility group-17; In this study, we analyzed the co-immunoprecipitated protein GcMa, glial cells missing a; PLOD, procollagen lysyl hydroxylase complex of PITX2 by mass spectrometry and successfully identified * Corresponding author. Address: Department of Biological Sciences, One John four proteins, Y box binding factor-1 (YB-1), nucleolin, heteroge- Marshall Drive, Marshall University, Huntington, WV 25755, USA. Fax: +1 304 696 7136. neous ribonucleoprotein K (hnRNP K) and heterogeneous nuclear E-mail address: [email protected] (G.-Z. Zhu). ribonucleoprotein U (hnRNP U), as novel PITX2-interacting 0014-5793/$34.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.01.028 Y. Huang et al. / FEBS Letters 583 (2009) 638–642 639 partners. We also investigated the regulatory effects of PITX2 by mum missed cleavages: 2; peptide mass tolerance: ±1–2 Da; frag- examining gene expression profile of HEK293 cells with transient ment mass tolerance: ±0.8–1 Da) on the non-redundant fasta overexpression of PITX2. Our result indicated that 868 genes and database obtained from the National Center for Biotechnology 191 genes were upregulated and downregulated more than Information (NCBI) and Swissprot database. The peptides and twofold, respectively. Many of these regulated genes have previ- proteins with significant Mowse score (P < 0.05), except trypsins, ously been linked to cell proliferation, cell differentiation, and were reported. organogenesis of muscle and eye. Taken together, our findings pro- vide an insightful perspective on PITX2 function and related molec- 2.4. RNA preparation ular mechanism. HEK293 cells were transiently transfected with pEGFP-NFLAG- PITX2c or pEGFP-N1. Forty eight hours after transfection, cells were 2. Materials and methods harvested for total RNA extraction by using TRI Reagent (Ambion). RNA quality was assessed by electrophoretic analysis on an Agilent 2.1. Cell culture Model 2100 Bioanalyzer. All RNA samples used in this study had RNA Integrity Numbers greater than 8.0. HEK293 cells were obtained from American Type Culture Col- lection (ATCC, Rockville, MD) and cultured in Dulbecco’s modified 2.5. Microarray analysis Eagle’s medium with 4.5 g/l glucose, 10% fetal bovine serum, and penicillin-streptomycin (100 IU/ml) at 37 °C in a humidified atmo- We employed a balanced block design with dye swap using four sphere with 5% CO2. biological replicates from HEK293 cells transfected with either pEGFP-N1 or pEGFP-NFLAG-PITX2c. For microarray probes, total RNA (250 ng) was used as the template for synthesis of internally 2.2. Immunoprecipitation and immunoblotting labeled cRNAs using Agilent QuickAmp Labeling kit and cyanine 3-CTP and cyanine 5-CTP (Perkin–Elmer, Waltham, MA) and a The open reading frame of human PITX2c, along with a FLAG modified QuickAmp protocol [14]. A total of 825 ng of cyanine 3- epitope inserted between Met1 and Asn2, was amplified by PCR with and cyanine5-labeled cRNAs was combined and hybridized onto 0 the following primers: forward primer (5 -ACTGaagcttgccaccATG- Agilent Whole Human Genome 4 Â 44 K microarrays at 65 °C for GATTACAAGGATGACGACGATAAGAACTGCATGAAAGGCCCGCTTCA- 17 h and then washed according to the manufacturer’s protocol. 0 C-3 , HindIII and Kozak site in lowercase) and reverse primer Slides were scanned on Agilent DNA Microarray Scanner. (50-AGCTggtacctcaCACGGGCCGGTCCACTG-30, KpnI site and stop co- don in lowercase). The PCR product was cloned into the HindIII/KpnI 2.6. Statistical analysis of microarray data sites of the vector pEGFP-N1 (Clontech). The resulting construct pEGFP-NFLAG-PITX2c, along with the control vector pEGFP-N1, Data were lowess-normalized and extracted from arrays using was transfected into HEK293 cells using FugeneHD transfection re- Agilent Feature Extraction v9.5. Features for which both channels agent (Roche). The cells were observed 24 and 48 h post-transfec- had low signal were excluded. Log2 expression ratios of PITX2- tion, and no significant difference of cell growth and cell viability overexpressed to control samples were imported to the Multiple was noticed between the test and control groups. Cells were then Experiment Viewer (MeV) v4.0 to perform statistical analysis lysed in immunoprecipitation buffer (50 mM Tris–HCl pH 7.4, [15]. Values were compared for significant deviation from zero 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) after 48 h of initial using one-class Significance Analysis of Microarrays (SAM) [16]. transfection. For nuclear protein extraction, cells were incubated Only probes for which at least three replicates passed the low sig- in hypotonic buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, nal filter were included. SAM was performed with the maximum 0.5 mM DTT) for 10 min before adding of 0.5% CA-630. Cell nuclei number of unique permutation, and delta was chosen to give 0% re- were then isolated by centrifugation. Nuclear proteins were ex- ported 90th percentile False Discovery Rate (FDR). Data for signif- tracted by the immunoprecipitation buffer. Cell lysates were pre- icantly differentially expressed genes are shown in Supplementary cleared by protein G beads (Pierce) for 2 h at 4 °C, and then incubated Table 1. Raw data from the microarray experiment are available at with anti-FLAG M2 conjugated-agarose beads (Sigma) or rabbit anti- the NCBI Gene Expression Omnibus with Accession No. GSE13216. YB-1 (Epitomics) for 2 h or overnight at 4 °C. The beads were washed five times using the immunoprecipitation buffer. To eliminate