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. 2019 May 7;8:e47098. doi: 10.7554/eLife.47098

Figure 4. ATAC-seq reveals HSV1 DNA is much more accessible than chromatin.

(A) Schematic of the Oligopaint targets for DNA FISH. Separate probe sets target regions in the Unique Long (UL) arm and the Unique Short (US) arm. B) Representative images of DNA FISH of cells four hpi, infected in the presence (PAA, left) or absence (4hpi, right) of the replication inhibitor PAA. Pixel intensity values are the same for the two images. Scale bars are 10 µm. (C) Fluorescence intensity of DNA FISH signal in RCs after infection. 5–95% intervals are shown, with inner quartiles and median. Data are normalized to the median intensity value of PAA-treated infected cells. Medians: PAA = 1.0, 3 hpi = 0.8, 4 hpi = 4.8, 5 hpi = 31.1, 6 hpi = 47.0. (D) Mean fraction bound for Pol II in infected cells with and without PAA. Error bars are the standard deviation of the mean, calculated as described in Materials and methods. (E) H2B-Halo cells show histone H2B is not incorporated into RCs. Innumofluorescence against ICP4 marks RCs. (F) Fragment length distribution of ATAC-seq data for cells 4 hpi. Lengths corresponding to intra-nucleosomal DNA (50–100 bp) and mononucleosomal DNA (180–250 bp) are marked as a reference. (G) ATAC-seq read density plotted across HSV1 genomic coordinates. (H) ATAC-seq analysis of intra-nucleosomal DNA (50–100 bp) and mononucleosomal DNA (180–250 bp). Global analysis of all human Pol II-transcribed genes, centered at the transcription start site (TSS). (I) The same analysis as in (G), but centered at the TSS of HSV1 genes.

Figure 4.

Figure 4—figure supplement 1. Quantification of DNA content and chromatin state in HSV1 RCs.

Figure 4—figure supplement 1.

(A) Representative full fields of view from DNA FISH hybridization. Dashed red boxes indicate the regions displayed in Figure 4B. (B) Dot plot showing the individual RC values from Figure 4C; normalized to the median of cells infected in the presence of PAA. Medians are indicated by a solid line (PAA = 1, 3 hpi = 0.83, 4 hpi = 4.77, 5 hpi = 31.13, 6 hpi = 46.95). (C) Fragment length distributions of all conditions tested after HSV1 infection, for two individual replicates as well as for the pooled data. The green line indicates the lengths of fragments mapping to the viral genome, the gray line indicates lengths of fragments mapping to the human genome. All data are normalized to the total number of mapped reads to the respective genome, per condition.