ATF-2 and CREB bind the G-CRE in vitro and in vivo. (A) K562 cells were treated with NaB (2 mM) or TSA (0.5 μM) for 24 and 48 hours. Nuclear extracts were prepared and analyzed by EMSA using the G-CRE probe. A single DNA-protein complex (B1) was established with the G-CRE probe and untreated extract (0) which increased in intensity as the duration of HDACI treatment increased up to 48 hours. (B) The EMSA gel is shown for antibody studies with untreated K562 cell extract in the absence (UT) or presence (UT⁺) of G-CRE cold competitor or nuclear extracts isolated from K562 cells treated with NaB and TSA for 48 hours. Studies were completed with a p-CREB1 (Ser 133) (c1), p-ATF-2 (Thr 71) (a2), and IgG antibodies. B1^S (c1/a2)-supershift band obtained with c1 or a2 antibody. (C) EMSA studies with histidine-tagged CREB1 (HisCREB1) and HisATF-2-purified protein. Binding studies were performed with the G-CRE probe and t-CREB1 antibody. In lane 5, HisCREB1 was added first followed by HisATF-2 protein to show binding of a heterodimeric complex. (D) ChIP assay to determine in vivo protein binding. Shown in the minigel are the PCR fragments obtained for the input DNA and different antibodies used to analyze the G-CRE and TATA box regions (see “Materials and methods”) in untreated and K562 cells induced with NaB (2 mM) and TSA (0.5 μM). (E) Chromatin quantifications obtained for qPCR analyses are shown in the graph. Immunoprecipitations were performed with IgG, p-CREB1 (Ser 133), p-ATF-2 (Thr 71), ac-H4, and TFIID antibodies along with a no antibody control. Data are presented as the mean fold increase in chromatin enrichment ± SEM. The bracket and star above the graph represent significant differences (P < .05) for the values obtained with the no antibody (No AB) control samples versus the level of chromatin enrichment obtained for immunoprecipitation experiments with the antibody indicated at the bottom of the graph.