RATIONALEThe gene encoding the helix-loop-helix transcription factor Id3 (inhibitor of differentiation-3) is located within atherosclerosis susceptibility loci of both mice and humans, yet its influence on atherosclerosis is not known.OBJECTIVEThe present study sought to determine whether polymorphisms in the ID3 gene were associated with indices of atherosclerosis in humans and if loss of Id3 function modulated atherogenesis in mice.METHODS AND RESULTSSix tagging single-nucleotide polymorphisms (SNPs) (tagSNPs) in the human ID3 gene were assessed in participants of the Diabetes Heart Study. One tagSNP, rs11574, was independently associated with carotid intima-media thickness (IMT). The human ID3 variant at rs11574 results in an alanine to threonine substitution in the C terminus. To determine the effect of this polymorphism on the basic function of Id3, site-directed mutagenesis of the human ID3 gene at rs11574 was performed. Results demonstrated a significant reduction in coimmunoprecipitation of the known E-protein partner, E12, with Id3 when it contains the sequence encoded by the risk allele (Id3105T). Further, Id3105T had an attenuated ability to modulate E12-mediated transcriptional activation compared to Id3 containing the ancestral allele (Id3105A). Microarray analysis of vascular smooth muscle cells from WT and Id3(-/-) mice revealed significant modulation of multiple gene pathways implicated in atherogenesis. Moreover, Id3(-/-)ApoE(-/-) mice developed significantly more atherosclerosis in response to 32 weeks of Chow or Western diet feeding than Id3(+/+)ApoE(-/-) mice.CONCLUSIONSTaken together, results provide novel evidence that Id3 is an atheroprotective factor and link a common SNP in the human ID3 gene to loss of Id3 function and increased IMT.