Use of qChIP to identify genes targeted by the Ikaros tumor suppressor

The IKAROS gene encodes a DNA binding zinc finger protein that is essential for hematopoietic differentiation and acts as a tumor suppressor in leukemia. The Ikaros protein is known to both activate and repress target gene expression and to participate in chromatin remodeling. The specific mechanisms by which Ikaros exerts it tumor suppressor activities are unknown. We are using Chromatin Immunoprecipitation (ChIP) assay combined with quantitative PCR (qPCR), called qChIP, to identify genes whose expression is directly regulated by Ikaros. Using this technique we calculate the difference in Ikaros binding to the promoter region of the target genes versus nonspecific binding (control). qChIP assays show that Ikaros binds to the promoter regions of the MYC oncogene, a known Ikaros target, and two other novel target genes. The promoter regions of MYC and the two novel target genes were cloned into luciferase reporter constructs and used to transiently transfect HEK cells that were co‐transfected with Ikaros or empty vector. Luciferase reporter assays showed that Ikaros suppresses the promoter activity of MYC and the two novel target genes. Our data confirm that the MYC oncogene is an Ikaros target and identify two novel Ikaros target genes. These data suggest that Ikaros exerts its tumor suppression function by inhibition of target gene expression in leukemia cells.Supported by R01 HL095120, a St. Baldrick's Foundation Career Development Award, the Four Diamonds Fund of Pennsylvania State University, College of Medicine (to SD), the NIDDK Short‐term Education Program for Under‐Represented Persons (to NFO) and a La Sierra University Dept. of Chemistry and Biochemistry stipend (to JLP).