Identification of GAS2 as a potential ICSBP target gene. (A) ICSBP binds the GAS2 5′ flank in vivo. Chromatin that coimmunoprecipitated (IP) from U937 cells with an antibody to ICSBP was amplified with primers flanking the proximal 1.0 kb of the GAS2 5′ flank. Nonprecipitated input chromatin was a positive control, and chromatin that coprecipitated with preimmune serum was a negative control for these studies. (B) The GAS2 5′ flank includes DNA-binding consensus sequences for IRF protein binding. The proximal 500 bp of the GAS2 5′ flank was analyzed for ICSBP-binding consensus sequences. The human sequence is shown in black, and the murine sequence in blue. Conserved, IRF-binding consensus sequences are indicated in red. The ICSBP-binding cis element is identified by a red bracket. (C) ICSBP represses GAS2 transcription in undifferentiated myeloid cells. U937 cells were cotransfected with a GAS2 promoter/reporter vector (1.0 kb of 5′ flank) and a vector to overexpress ICSBP or the vector control. Because ICSBP tyrosine phosphorylation influences the activation or repression of other target genes, some cells were cotransfected with a vector to overexpress ICSBP with all tyrosine residues mutated to phenylalanine (Y-mut ICSBP). The transfectants were assayed for reporter activity with or without IFN-γ-induced differentiation or with or without trichostatin A (TSA) treatment. *, statistically significant decrease in reporter expression in ICSBP-overexpressing transfectants in comparison to the level of expression in the control (P < 0.001, n = 5); ** or ***, statistically significant increase in GAS2 promoter activity in TSA-treated transfectants without or with ICSBP overexpression, respectively (P < 0.001, n = 5). The lack of a statistically significant difference in reporter activity in IFN-γ-differentiated transfectants is indicated by the bracket (P = 0.2, n = 3). (D) ICSBP represses GAS2 transcription via a cis element between bp −110 and −130 in the GAS2 promoter. U937 cells were cotransfected with a series of reporter constructs with truncations of the GAS2 5′ flank, as indicated, and a vector to overexpress ICSBP or empty vector control. *, **, or ***, statistically significant decrease in activity of the 1.0-kb, 250-bp, or 130-bp reporter constructs, respectively, with ICSBP overexpression (P < 0.01, n = 4). The lack of a statistically significant difference in the activity of the 100-bp GAS2 promoter construct with versus without ICSBP overexpression is indicated by the bracket (P > 0.2, n = 4). Error bars indicate standard errors.