Figure 4. MVM NS1 colocalizes to sites of cellular DNA damage along with MVM genome.

(A) Representative quantile normalized ChIP-seq plots of MVM-NS1 (purple) and BRCA1 (pink) binding to the cellular genome on Chromosome 17 (top) and Chromosome 19 (bottom), with γ-H2AX and V3C at 16 hpi. Red rectangle denotes VAD sites, and y-axis values for ChIP-seq peaks have been restricted from 0 to 5 reads per million. (B) The enrichment of NS1 (left), BRCA1 (middle) and γ-H2AX (right) around MVM-associated regions were calculated and plotted as heatmaps using DeepTools on the Galaxy server (Ramírez et al., 2016). (C) The fraction of NS1 and DDR-positive genomic regions that colocalized with V3C at 16 hpi were calculated using BEDTools, and presented as VAD-positive sites. A library of randomly generated ChIP-seq peaks on the mouse genome with the same fragment size as the called peaks was used as control and also intersected with the MVM-VADs.

Figure 4—figure supplement 1. Called peaks from DDR ChIP-seq in HU-treated primary mouse splenocytes were compared with DDR ChIP-seq in MVM infected murine A9 fibroblasts at 16 hpi.

The comparisons are presented in the form of heatmaps for (A) BRCA1 binding (green) and (B) γ-H2AX modification (blue). (C) The spatial interaction of MVM with common fragile sites were assayed in parasynchronized human NB-324K cells infected with MVM for 16 hr. Focused 3C-qPCR was performed with Taqman probes on the MVM genome, and assayed for interaction with the prototypical human fragile site FRA3B, as well as two other sites FRA5H and FRA11F. Data is presented as mean ± SEM of three independent experiments.

Figure 4—figure supplement 2. Comparisons of MVM-associated VAD sites with topological structure of the mouse nucleome.

Hi-C contact maps in murine CH12 cells were visualized in comparison with MVM-V3C in A9 cells at 16 hpi (grey tracks on top) using the Juicebox data visualization platform (presented as red heatmaps, [Durand et al., 2016; Rao et al., 2014]). Representative contact maps are shown for Chromosomes: (A) 17, (B) 19, (C) 3 and (D) 7. Blue squares indicate robust VAD regions that coincide with Hi-C contact domains. (E) MVM interaction sites on the mouse genome identified by V3Cseq at 16 hpi was also overlayed with published chromatin marks (Kraushaar et al., 2013) for Type A chromatin (Rao et al., 2014) including tri-methylated histone H3 at lysine 36 (H3K36me3), acetylated histone H3 at lysine 27 (H3K27ac), mono-methylated histone H3 at lysine 4 (H3K4me1) and acetylated histone H3 at lysine 9 (H3K9ac) in Aphidicolin-induced NIH-3T3 cells (Kraushaar et al., 2013). Reads-per- million (rpm) values of the ChIP-seq pulldowns are indicated to the right of each corresponding histogram track.