Fig 1.
WAPL negatively regulates LMP1 and LMP2A expression.
(A) Schematic diagram of EBV latency programs. (B) Schematic of WAPL antagonism of cohesin-mediated DNA loop formation. WAPL releases cohesin to promote dissolution of chromatin loops. Upon WAPL KO, cohesin occupancy on chromatin increases, resulting in larger DNA loops. (C, E) Volcano plots of RNA-seq analysis visualizing -log10(adj. p-value) vs. log2(fold change of EBV mRNA abundance) from (C) Cas9+ MUTU I Burkitt lymphoma cells and (E) Cas9+ GM12878 LCLs expressing WAPL vs. control sgRNAs, from n = 3 independent biological replicates. (D, F) Immunoblot analysis of whole cell lysates (WCL) from (D) MUTU I cells and (F) GM12878 LCLs expressing control or WAPL sgRNAs, as indicated, representative of n = 3 biological replicates. Shown below are densitometry values that were normalized to GAPDH loading control, with control levels normalized to 1. ND indicates not detected.
Fig 2.
Subcellular distribution of LMP1 and LMP2A de-repressed by WAPL KO.
(A) Representative immunofluorescence images from n = 3 biological replicates of anti-LMP1 (green) vs. nuclear DAPI (blue) staining of Cas9+ MUTU I cells that expressed control or WAPL sgRNAs, as indicated. Shown at right are zoomed images of a representative cell (indicated by the white box). (B) Mean ± standard deviation (SD) percentage of LMP1+ cells per field of view, from n = 3 fields of view from each of three biological replicates. P-values shown as calculated by one-way ANOVA. (C) Representative immunofluorescence images from n = 3 biological replicates of anti-LMP2A (green) vs. nuclear DAPI (blue) staining of Cas9+ MUTU I cells that expressed control or WAPL sgRNAs with zoomed images presented to the right, as in (A). (D) Mean ± SD percentage of LMP2A+ cells per field of view, from n = 3 fields of view from each of three biological replicates. P-values shown as calculated by one-way ANOVA.
Fig 3.
WAPL KO alters higher order latency I Burkitt EBV genome conformation.
(A) Schematic of Hi-C workflow and output. Exposed DNA ends were biotinylated and then ligated to capture close DNA contacts. Ligated DNA was sheared, and biotinylated DNA was captured via streptavidin. EBV DNA was captured to enhance viral DNA Hi-C signal. (B) Hi-C maps of EBV genomic loops that were enriched in Cas9+ MUTU I cells expressing WAPL vs. control sgRNAs, from n = 2 biological replicates. LMPp and oriLyt regions are indicated. (C) Hi-C maps of EBV genomic loops that were depleted in Cas9+ MUTU I cells expressing WAPL vs. control sgRNAs, from n = 2 biological replicates, as in (B).
Fig 4.
WAPL KO alters latency I Burkitt EBV genomic activated chromatin loops and represses LMP promoter epigenetic marks.
(A) Schematic of H3K27ac HiChIP sample preparation and output. Chromatin was formaldehyde crosslinked and fragmented. Exposed DNA ends were biotinylated and then ligated to capture close DNA contacts. Ligated DNA was sheared, DNA was immunopurified by α-H3K27ac antibody, and biotinylated DNA was captured via streptavidin. (B) EBV genomic H3K27ac HiChIP map depicting loops enriched (red) or depleted (blue) in Cas9+ MUTU I cells expressing WAPL vs. control sgRNAs, from n = 3 biological replicates. (C-D) Normalized (C) LMP region-oriLytR loop and (D) LMP region-oriLytL loop read counts from n = 3 replicates, as in (B). EBV genome kilobase coordinates for each looping region are indicated at top. * P ≤ 0.05, ** P ≤ 0.01, as calculated by a two-tailed Student’s t-test. (E-F) ChIP-qPCR analysis of H3K9me3 and H3K27me3 abundances at the (E) LMP1 promoter and (F) LMP2A promoter in Cas9+ MUTU I cells expressing control or WAPL sgRNAs. Shown are the mean fold change of ChIP-qPCR values relative to input values ± SD from n = 3 biological replicates. Values from sgControl expressing cells were normalized to 1. ** P ≤ 0.01, *** P ≤ 0.001, as calculated by a two-tailed Welch’s t-test. (G) Model of WAPL effects on EBV genomic architecture. When present, WAPL releases cohesin (latency I), which enables the accumulation of repressive histone marks and inhibits LMP expression. In the absence of WAPL antagonism, a loop forms between the LMP promoter region and oriLyt regions. Juxtaposition of the oriLyt enhancer reduces repressive H3K9me3 and H3K27me3 marks and supports LMP1 and LMP2A co-expression in the absence of EBNA2 (latency II). For reference, in latency III, an alternative loop forms between the oriP and the Cp to drive expression of all the EBNA genes.