The influence of EBNA 2 and 3C on chromosome looping at the <i>WEE1</i> locus.

<p>(A) Diagram (not to scale) showing the <i>Eco</i>R1 restriction fragments at the <i>WEE1</i> locus that encompass the promoter (P), two downstream enhancers (E1 and E2) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2), EBNA 3C knock-out EBV (3CKO-3) or EBNA 3C revertant EBV (3Crev-4) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter and enhancers. (C) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2) or EBNA 2 KO EBV. (D) Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (E) Model for the control of chromatin looping by EBNA 2 and 3 proteins at <i>WEE1</i>. (F) Re-ChIP analysis in Mutu III cells using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2 or EBNA 3A, 3B or 3C antibodies. Primers at peak 5 in enhancer 2 were used for analysis. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (G) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.