A Cysteine Residue in the Helix-Loop-Helix Domain of Id2 Is Critical for Homodimerization and Function Jian Liu, Wei Shi, and David Warburton 1 Developmental Biology Program and Division of Pediatric Surgery, Childrens Hospital Los Angeles, University of Southern California, Keek School of Medicine, 4650 Sunset Boulevard, Los Angeles, California 90027 Received June 16, 2000 Id proteins are negative regulators of basic helix- loop-helix (bHLH) transcription factors. In this study, we compared the expression of Id2 mRNA in prolifer- ating (fetal) and nonproliferating (adult) alveolar ep- ithelial cells (AECs). The expression of Id2 was higher in adult AECs than in the corresponding fetal cells, suggesting that Id2 might play a functional role in developmental regulation of lung epithelial cell prolif- eration. By screening a mouse embryo cDNA library in the yeast two-hybrid system, Id2 was identified as a self-associating protein. Structural analysis by dele- tion and site-directed mutagenesis demonstrated that the HLH domain and a cysteine residue within the HLH domain are essential for Id2 homodimerization. Furthermore, in vitro synthesized Id2 homodimers be- came monomers under reducing conditions, indicat- ing that the formation of an intermolecular disulfide bond is critical for Id2 homodimerization. Transient transfection assays in A549 cells showed that wild- type Id2 down-regulated the activity of the cyclin A promoter by 70%, while mutating the cysteine critical for Id2 homodimerization abolished the inhibitory ef- fect of wild-type Id2. © 2000 Academic Press Helix-loop-helix (HLH) transcription factors play a fundamental role in cell fate determination (1– 4). Most HLH proteins (designated bHLH transcription factors) have a basic, DNA binding region that contacts the E-box (CANNTG) of target genes to regulate their tran- scription. Various HLH family members can interact with each other to form hetero- or homodimers (5, 6). Id proteins contain a HLH domain but lack a basic region for DNA binding. Four members of Id family have been identified that share considerable amino acid sequence homology in the HLH domain (5, 7, 8). Generally, Id genes are highly expressed during embryogenesis and in undifferentiated cells and decrease during tissue differentiation. The product of Id genes preferentially heterodimerize with E proteins to form transcription- ally inactive complexes (5). By competing with tis- sue-specific bHLH proteins for E protein partners, Id proteins globally down-regulate bHLH-mediated tran- scriptional activity. Id2, a member of Id family, has been reported to be expressed in mouse lung, intestine, brain, and thymus (9). It affects the balance between cell growth and differentiation by negatively regulating the function of bHLH transcription factors and multiple tumor sup- pressor proteins (10). We examined the expression of Id2 in several lung-specific cell lines and primary cul- ture of alveolar epithelial cells. In order to identify putative partners of Id2, we screened a mouse cDNA library in the yeast two-hybrid system. From this study, Id2 was identified as a self-associating protein. Mutagenesis analysis and in vitro dimerization studies demonstrated that the HLH domain and C42 (a cys- teine residue in the HLH domain) in Id2 protein were crucial for homodimerization, suggesting that a disul- fide bond was involved in this protein–protein interac- tion. To understand the structural consequences of Id2 homodimerization, we cotransfected plasmids express- ing wild type and mutant Id2 together with a cyclin A promoter/luciferase into A549 lung epithelial cells. Our results point to a previously unrecognized struc- tural parameter C42 that governs the function of Id2 protein. EXPERIMENTAL PROCEDURES Yeast strains and plasmids. The yeast strain PCY2 and all par- ent vectors for the yeast two-hybrid analysis were kindly provided by the laboratory of Dr. Pierre Chevray (11). The mouse Id2 cDNA was kindly provided by Dr. Xiao-Hong Sun (7). The GAL4 DBD (DNA binding domain)-Id2 bait plasmid consists of a PCR-generated Sal I and Not I fragment of Id2, (amino acids 9 –125), ligated in frame into Abbreviations used: Id2, inhibitor 2 of DNA binding; bHLH, basic helix-loop-helix; AEC, alveolar epithelial cell; pRB, retinoblastoma protein; DBD, DNA binding domain; TAD, transcriptional activation domain; WT, wild type; SDS, sodium dodecyl sulfate; PAGE, poly- acrylamide gel electrophoresis. 1 To whom correspondence should be addressed. Fax: (323) 671- 3613. E-mail: dwarburton@chla.usc.edu. Biochemical and Biophysical Research Communications 273, 1042–1047 (2000) doi:10.1006/bbrc.2000.3055, available online at http://www.idealibrary.com on 1042 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.