Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
Figure 5
EBNA 2 and EBNA 3 protein binding at the WEE1 locus in EBV infected cells.
(A) EBNA 2 (green) and EBNA 3 (red) sequencing reads at the WEE1 locus (displayed as described in Figure 3). Panels B–E show ChIP-QPCR carried out in Mutu III cells and panels F–I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding sites (sets B, D, F, H and J) or regions adjacent to the binding sites (sets A, C, E, G and I). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent ChIP experiments. (J) Q-PCR analysis of WEE1 transcript levels using cDNA from BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2 and 3), EBNA 3C knock-out EBV (3C KO-3 and 6) or EBNA 3C revertant EBV (3Crev-2 and 4). Transcript levels were normalised to GAPDH levels and expressed relative to the level in parental BL31 cells. * indicates a p-value of <0.01 (students t-test) compared to the wtBac-2 cell line. (K) Q-PCR analysis of WEE1 transcript levels using cDNA from 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). Cells were incubated in β-estradiol-free media for 4 days prior to re-addition of β-estradiol for 6 or 17 hrs. Transcript levels were normalised to GAPDH levels and expressed relative to the level in the absence of β-estradiol. All cDNA results (J–L) show the mean −/+ standard deviation of three independent QPCR analyses from two independent cDNA preparations.