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. 2010 Aug 2;30(19):4575–4594. doi: 10.1128/MCB.01595-09

FIG. 2.

FIG. 2.

ICSBP cooperates with Tel and HDAC3 to repress a GAS2 cis element found between bp −100 and −130 in the promoter. (A) ICSBP repression of the GAS2 cis element is increased by Tel or HDAC3. U937 cells were transfected with an artificial reporter construct with three copies of the GAS2 cis element linked to a minimal promoter and reporter (GAS2-sv40-GL3) or the empty vector control (sv40-GL3) and a vector to express ICSBP, a dose titration of HDAC3 or Tel, ICSBP with a dose titration of HDAC3 or Tel, an shRNA to HDAC3 or Tel, ICSBP with an shRNA to HDAC3 or Tel, or ICSBP with Tel and HDAC3, and reporter gene assays were performed. *, **, or #, statistically significant decrease in reporter activity in ICSBP-, Tel-, or HDAC3-overexpressing cells, respectively, in comparison to control cells (P < 0.01, n = 3); *** or ##, statistically significant difference in reporter expression in cells expressing shTel or shHDAC3, respectively (P < 0.01, n = 3); ###, statistically significant difference with ICSBP with Tel and HDAC3; & or &&, statistically significant difference in reporter expression in cells overexpressing ICSBP with shTel or Tel overexpression (P < 0.01, n = 3); α or αα, statistically significant difference in reporter expression in ICSBP-overexpressing cells with shHDAC3 or HDAC3 overexpression, respectively (P < 0.01, n = 3). (B) ICSBP binds to the bp −100 to −125 GAS2 promoter sequence in vitro. Nuclear proteins were isolated from U937 cells with or without IFN-γ-induced differentiation. Proteins were purified by affinity to a biotin-labeled, double-stranded oligonucleotide probe with the bp −102 to −127 or the bp −75 to −102 sequence from the GAS2 5′ flank. Affinity-purified proteins were analyzed by probing Western blots (WB) with an ICSBP antibody. Lanes with 1/10 the amount of affinity-purified protein were used as a loading control for ICSBP abundance. (C) In vivo ICSBP binding to the bp −100 to −130 GAS2 promoter sequence is decreased by differentiation or Bcr/abl expression. Chromatin coimmunoprecipitation (IP) was performed using U937 cells with or without IFN-γ (IFNg)-induced differentiation. Chromatin was coimmunoprecipitated with antibody to ICSBP, trimethylated lysine 27 histone 3 (H3 K27 tri-methyl), or irrelevant control antibody. Other U937 cells were stably transfected with a vector to express Bcr/abl, and chromatin was coimmunoprecipitated with anti-ICSBP or irrelevant control antibody. Precipitated chromatin was amplified by real-time PCR with primers flanking the bp −100 to −130 GAS2 cis element. The results were normalized to those for input (nonprecipitated) chromatin. * or **, statistically significant decrease in ICSBP binding to the GAS2 cis element in IFN-γ-differentiated or Bcr/abl+ cells, respectively (P < 0.001, n = 3); #, statistically significant increase in binding of H3 trimethyl K27 to the bp −100 to −130 GAS2 cis element in differentiated U937 cells (P < 0.001, n = 3). Error bars indicate standard errors.