Journal of Cellular Biochemistry 103:182–194 (2008) Induction of Id2 Expression by Cardiac Transcription Factors GATA4 and Nkx2.5 Joong-Yeon Lim, 1,2 Won Ho Kim, 1 Joon Kim, 2 and Sang Ick Park 1 * 1 Division of Intractable Diseases, Center for Biomedical Sciences, National Institute of Health, 194, Tongillo, Eunpyeong-gu, Seoul 122-701, Korea 2 Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea Abstract Inhibitor of differentiation/DNA binding (Id) proteins function as a regulator of helix-loop-helix proteins participating in cell lineage commitment and differentiation. Here, we observed a marked induction of Id2 during cardiomyocyte differentiation from P19CL6 murine embryonic teratocarcinoma stem cells, prompting us to investigate the upstream regulatory mechanism of Id2 induction. Computer analysis of Id2 promoter and subsequent electrophoretic mobility shift assay revealed several binding sites for GATA4 and Nkx2.5 within the Id2 promoter. By further deletion and mutation analysis of the respective binding site, we identified that two motifs located at 497/502 and 264/270 were functionally important for Id2 promoter activation by GATA4 and Nkx2.5, respectively. Overexpression of GATA4 and/or Nkx2.5 induced not only Id2 promoter activity but also Id2 protein expression. Additionally, Id proteins significantly inhibit the GATA4 and Nkx2.5-dependent transcription, suggesting Id proteins may play a regulatory role in cardiogenesis. Collectively, our results demonstrate that GATA4 and Nkx2.5 could be one of the upstream regulators of Id2. J. Cell. Biochem. 103: 182 – 194, 2008. ß 2007 Wiley-Liss, Inc. Key words: Idb2 protein; cell differentiation; transcription factors; myocytes; gene expression regulation Inhibitor of differentiation/DNA binding (Id) proteins belong to a subfamily group of helix- loop-helix proteins (HLH) that are important regulators of cellular differentiation and pro- liferation. Lacking the basic amino acid resi- dues responsible for DNA binding, Id proteins effectively prevent critical HLH transcription factors from binding to tissue-specific gene promoters, thereby acting as dominant negative regulators of HLH transcription factors [Bene- zra et al., 1990; Norton et al., 1998; Ruzinova and Benezra, 2003]. Four Id proteins (Id1 through Id4) are identified in vertebrates and are differentially regulated depending on cell type or stimuli [Ruzinova and Benezra, 2003]. During cardiac development, each Id transcript is widely expressed with temporal and spatial regulation. Id1, Id2, and Id3, but not Id4, are expressed in endocardial cushion, outflow tract, and valves in the developing heart in over- lapping patterns [Evans and Obrien, 1993; Jen et al., 1996]. Id transcript is also highly expressed in intact heart tissue as well as in freshly isolated cardiac muscle cells from neo- natal and adult rat heart [Springhorn et al., 1992]. Moreover, double or triple-Ids knockout mouse embryos have multiple cardiac abnorm- alities including ventricular septal defects, endocardiac cushion defects, and myocardial wall defects, and die during mid-gestation [Fraidenraich et al., 2004]. These studies sug- gest a pivotal role of Id protein in cardiac development. GATA4 and Nkx2.5 are transcription factors essential for heart development and act as early pre-cardiac cell markers. GATA4 is a member of the zinc finger GATA family (GATA1 through GATA6) and is expressed predominantly in the heart, digestive organs, and the extra-embryo- nic endoderm [Heikinheimo et al., 1994; Laver- riere et al., 1994]. GATA4 binds to the WGATAR ß 2007 Wiley-Liss, Inc. Grant sponsor: Korea National Institute of Health Intra- mural Research Grant; Grant number: 2910-213-207. *Correspondence to: Sang Ick Park, Division of Intractable Diseases, Center for Biomedical Sciences, National Insti- tute of Health, 194, Tongillo, Eunpyeong-gu, Seoul 122-701, Korea. E-mail: parksi@nih.go.kr Received 12 March 2007; Accepted 6 April 2007 DOI 10.1002/jcb.21396