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, 15 (11), e1008076
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Chromatin Dynamics and the Transcriptional Competence of HSV-1 Genomes During Lytic Infections

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Chromatin Dynamics and the Transcriptional Competence of HSV-1 Genomes During Lytic Infections

MiYao Hu et al. PLoS Pathog.

Abstract

During latent infections with herpes simplex virus 1 (HSV-1), viral transcription is restricted and the genomes are mostly maintained in silenced chromatin, whereas in lytically infected cells all viral genes are transcribed and the genomes are dynamically chromatinized. Histones in the viral chromatin bear markers of silenced chromatin at early times in lytic infection or of active transcription at later times. The virion protein VP16 activates transcription of the immediate-early (IE) genes by recruiting transcription activators and chromatin remodelers to their promoters. Two IE proteins, ICP0 and ICP4 which modulate chromatin epigenetics, then activate transcription of early and late genes. Although chromatin is involved in the mechanism of activation of HSV- transcription, its precise role is not entirely understood. In the cellular genome, chromatin dynamics often modulate transcription competence whereas promoter-specific transcription factors determine transcription activity. Here, biophysical fractionation of serially digested HSV-1 chromatin followed by short-read deep sequencing indicates that nuclear HSV-1 DNA has different biophysical properties than protein-free or encapsidated HSV-1 DNA. The entire HSV-1 genomes in infected cells were equally accessible. The accessibility of transcribed or non-transcribed genes under any given condition did not differ, and each gene was entirely sampled in both the most and least accessible chromatin. However, HSV-1 genomes fractionated differently under conditions of generalized or restricted transcription. Approximately 1/3 of the HSV-1 DNA including fully sampled genes resolved to the most accessible chromatin when HSV-1 transcription was active, but such enrichment was reduced to only 3% under conditions of restricted HSV-1 transcription. Short sequences of restricted accessibility separated genes with different transcription levels. Chromatin dynamics thus provide a first level of regulation on HSV-1 transcription, dictating the transcriptional competency of the genomes during lytic infections, whereas the transcription of individual genes is then most likely activated by specific transcription factors. Moreover, genes transcribed to different levels are separated by short sequences with limited accessibility.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3.
Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.
Fig 2
Fig 2. Intracellular HSV-1 DNA is differentially protected from MCN digestion than deproteinized or encapsidated DNA.
(A) Cartoon presenting the different models proposed for the HSV-1 chromatin and the expected fractionation patterns after serial digestion and sucrose gradients of: (i) mostly non-chromatinized HSV-1 DNA, (ii) HSV-1 DNA associated with nucleosomes only sporadically, (iii) fully (stably or unstably) chromatinized HSV-1 DNA. (B) DNA from each fraction was resolved in 2% agarose gel electrophoresis and stained with EtBr. (C) Western blot for core histones (H2A, H2B, H3 and H4) in all fractions, each resolved by SDS-PAGE and probed with corresponding antibodies. (D) Bar graph showing the number of HSV-1 genome copy equivalents in each fraction. Chromatin of HSV-1 infected cells was digested and fractionated. DNA in each fraction was subjected to deep sequencing. Ins, insoluble chromatin; GCE, genome copy equivalents. Results from one experiment representative of three.
Fig 3
Fig 3. Fractionation of the major HSV-1 DNA binding proteins.
(A) Western blots of VP5, ICP4 and ICP8 in the insoluble and soluble chromatin fractions. Results representative of two independent repeats. (B) Fractionation of ICP4, ICP8, and HSV-1 DNA after serial MCN digestion and sucrose gradient centrifugation. Western blots of ICP4 and ICP8, and line graph presenting the percentage of ICP4, ICP8 and HSV-1 DNA in each fraction. VP5 was below sensitivity levels in all fractions. Ins, insoluble chromatin fraction; sol, soluble chromatin fraction. Average of three (ICP8) or two (ICP4) independent experiments.
Fig 4
Fig 4. All HSV-1 genes resolved together to the least and most accessible chromatin fractions.
(A) Cluster analyses of the number of DNA reads for IE, E or L genes in each fraction. Cluster analysis was performed with Cluster 3.0 from Stanford University, visualized with Java Treeview. Color key on the left indicates ratio of reads in fraction over reads in the total DNA (ranges are in log2). 7, 7 hpi, untreated infections; C, CHX- treated infections (7 hpi); R, roscovitine-treated infections (7 hpi). (B) Gene sampling of each HSV-1 gene in each fraction. Sampling of each HSV-1 gene was calculated and normalized to the sampling of the same gene in the undigested and unfractionated chromatin. Each gene is color coded according to kinetic class. Blue, IE genes; black, E genes; dark purple, unclassified L genes; light purple, early L genes; brown, true L genes; grey, unclassified genes. Ins, insoluble chromatin fraction. Results from one experiment representative of three.
Fig 5
Fig 5. The most and least accessible chromatin enriched in fully sampled HSV-1 genes contained more HSV-1 DNA when transcription was active.
(A) Bar graphs showing average sampling of all genes in each HSV-1 gene kinetic groups in each fraction. Sampling of each HSV-1 gene was calculated and normalized to the sampling of the same gene in the undigested and unfractionated chromatin. The normalized sampling of all genes in each group was then averaged. Blue, IE genes; black, E genes; dark purple, unclassified L genes; light purple, early L genes; brown, true L genes; grey, unclassified genes. (B) Area graphs and pie charts presenting relative enrichment in gene sampling and percentage of HSV-1 DNA in each fraction. Gene sampling in each fraction is expressed as ratio to the average sampling to present the relative enrichment in fully sampled genes in each fraction. The distribution of the cellular chromatin is indicated by the dotted black line. Pie charts indicating the percentages of the HSV-1 DNA in the most accessible chromatin fractions containing completely sampled HSV-1 genes (brown), in the intermediate accessible chromatin fractions containing random samplings of each gene (grey), or in the least accessible chromatin fractions containing completely sampled HSV-1 genes (purple). Ins, insoluble chromatin fraction. Results from one experiment representative of three.
Fig 6
Fig 6. Differential fractionation of HSV-1 DNA throughout infection.
Bar graphs showing HSV-1 genome copy equivalents in each fraction. (*) Data from Fig 2 presented again for comparison. Ins, insoluble chromatin fraction; GCE, genome copy equivalent. Results from one experiment representative of two.
Fig 7
Fig 7. All HSV-1 genes resolved together to the least and most accessible chromatin fractions as infection progressed.
(A) Cluster analyses of the number of DNA reads from IE, E and L genes in each fraction. Cluster analysis was performed using Cluster 3.0 from Stanford University, visualized with Java Treeview. (B) Bar graphs showing sampling of each HSV-1 genes at 2h, 4h, and 15h after infections. Sampling of each HSV-1 gene was calculated and normalized to the sampling of the gene in the undigested and unfractionated chromatin. The sampling of each gene was plotted according to kinetic class. Blue, IE genes; black, E genes; dark purple, unclassified L genes; light purple, early L genes; brown, true L genes; grey, unclassified genes. Ins, insoluble chromatin fraction. Results from one experiment representative of two.
Fig 8
Fig 8. The most and least accessible chromatin enriched in completely sampled HSV-1 genes contained more HSV-1 DNA as the infection progressed.
(A) Bar graphs showing average sampling of the genes in each HSV-1 gene kinetic class in each fraction at 2, 4 and 15 hours after infection. Sampling of each HSV-1 gene was calculated and normalized to the sampling of the gene in the undigested and unfractionated chromatin. The normalized sampling of all genes in each group was then averaged. Blue, IE genes; black, E genes; dark purple, unclassified L genes; light purple, early L genes; brown, true L genes; grey, unclassified genes. (B) Area graphs showing relative enrichment in completely sampled genes and percentage of HSV-1 DNA in each fraction. Gene sampling in each fraction is expressed as ratio to the average sampling (relative enrichment). The distribution of the cellular chromatin is indicated by the dotted black line. (*) Data from Fig 5 presented again for comparison. Pie charts showing percentage of HSV-1 DNA in the most accessible chromatin fractions containing completely sampled HSV-1 genes (brown), in the intermediate accessible chromatin fractions containing random sampling of each gene (grey), or in the least accessible chromatin fractions containing completely sampled HSV-1 genes (purple). Ins, insoluble chromatin fraction. Results from one experiment representative of two.
Fig 9
Fig 9. No HSV-1 loci fractionated differently regardless of transcription levels, but short overrepresented sequences flanked transcribed genes when transcription was restricted.
Line graphs showing HSV-1 number of genome copy equivalents (GCE) in each genome position in untreated infections, or in infections treated with CHX, Rosco, or PAA at 7 hpi, normalized to the number of HSV-1 genome copy equivalents in the respective position of undigested and unfractionated chromatin (blue line). Orange line graphs, HSV-1 RNA reads. X-axes, genome position (cartoon on top). Black downward empty arrows, CTCF binding sites in strain 17; black solid circles, chromatin insulator-like elements in strain 17; black dots, other overrepresented sequences; purple bars underneath genome plots, IE genes; dark green bars underneath genome plots, LAT; light green bar, stable LAT; GCE, genome copy equivalent. Results from one experiment representative of three.
Fig 10
Fig 10. Enlargement of the repeats region, from position 100kbp to 152kbp, of the plots presented in Fig 9.
Orange lines graphs, HSV-1 RNA reads. X-axes, genome position (cartoon on top). Blue lines, HSV-1 number of genome copy equivalents (GCE) in each genome position. Black downward empty arrows, CTCF binding sites in strain 17; black solid circles, chromatin insulator-like elements; black dots, other overrepresented short sequences; purple bars underneath genome plots, IE genes; dark green bars underneath genome plots, LAT; light green bar, stable LAT; GCE, genome copy equivalent. Results from one experiment representative of three. ++ sequences co-immunoprecipitated with CTCF by ChIP in two independent experiments; + sequences co-immunoprecipitated with CTCF by ChIP in one of two independent experiments;—sequences not co-immunoprecipitated with CTCF by ChIP in either of the two independent experiments.
Fig 11
Fig 11. The number of overrepresented short sequences decreases as infection progresses.
Line graphs showing number of HSV-1 genome copy equivalents in each genome position at 2, 4, 7 (*), or 15hpi, normalized to the number of HSV-1 genome copy equivalents in the same position of the respective undigested and unfractionated chromatin. X-axes, genome position (cartoon on top). *: data from Fig 9 presented again for comparison. Black downward empty arrows, CTCF binding sites; black solid circles, chromatin insulator-like elements; black downward solid arrows, CTCF binding- and chromatin insulator-like elements; black dots, peaks of the DNA plots; black diamonds underneath the plots, the seven overrepresented sequences that do not contain the AT-rich motifs; purple bars underneath genome plots, IE genes; dark green bars underneath genome plots, LAT; light green bar, stable LAT; GCE, genome copy equivalent. (A) Presentation of relative HSV-1 DNA plots through the entire genome; (B) Enlargement of the relative HSV-1 DNA plots of the repeat region, from position 100kbp to 152kbp. Results from one experiment representative of two.

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