Figure 3

ChIP analysis of the enhancer/promoter region in Gax provirus. A. Schematic representation of the 5'-LTR in the R3Gaxbsr reporter gene. The three 21 -bp enhancer sequences (boxes), the TATA sequence, and the transcription start site (+1) are shown. Primers for PCR are indicated by arrows. The 5'- and 3'-ends of amplified DNA are denoted as the nucleotide positions relative to the transcription start site. Primers #1 and #2 amplify the enhancer region, and primers #3 and #4 amplify the promoter region of Gax-5'-LTR. B. Binding of CREB, phosphor-CREB and CBP to the Gax enhancer region was constant in EL4-Gax cells in vitro (a) and in vivo (b)(left), but the enhancer binding of Gax was reduced when EL4-Gax cells were grown in vivo (right). C. Expression of CREB protein in EL4-Gax cells. Anti-CREB1, anti-phosphor-CREB antibodies were used to detect proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo. Equivalent protein loading was confirmed by stripping and re-probing the blot with an anti-β-actin antibody. D. Binding of acetylated histone 3 at Lys-9, 14 (H3), acetylated histone 4 at Lys- 5, 8, 12, 16 (H4) and trimethylated histone 3 at Lys-4 (H3K4tri) to the Gax enhancer region was not changed in EL4-Gax cells either in vitro (a) or in vivo (b), but the promoter binding of the basic transcription factor TFIIB and of RNA polymerase II (Pol-II) was reduced when EL4-Gax cells were grown in vivo. Factors binding to promoters of EF-1a and β-globin are presented as positive and negative controls, respectively.