Cell culture and infections
Human Fetal Foreskin Fibroblasts (HFFF, purchased from ECACC), Telomerase-Immortalized Human Foreskin Fibroblasts (T-HF) (48), Baby Hamster Kidney cell line (BHK, obtained from ATCC), Human Bone Osteosarcoma Epithelial Cells (U2OS, kindly provided by Stacey Efstathiou), ICP27-complementing Vero 2-2 cell line (kindly provided by Prof. Dr. Beate Sodeik) and Human embryonic kidney 293T cells (HEK-293T, obtained from ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher #41966052) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Biochrom #S0115), 1x MEM Non-Essential Amino Acids (ThermoFisher #11140050) and 1% penicillin/streptomycin. All cells were incubated at 37 °C in a 5% (v/v) CO2-enriched incubator. HFFFs were utilized from passage 11 to 17 for all high-throughput experiments.
This study was performed using wild-type (WT) HSV-1 strain 17, BAC-derived HSV-1 strain 17 (kindly provided by Beate Sodeik) (49), wild-type HSV-1 strain F, and mutant viruses R325 (ΔICP22 C-terminal 220 amino acids, strain F (50)), wild-type KOS1.1 (kindly provided by Steven Rice) (51), vhs-inactivated mutant (Δvhs, strain 17, (52)), ICP27-null mutant (ΔICP27, strain KOS, (53)) and ICP0-null mutant (ΔICP0, strain 17, (54)). Virus stocks were produced in BHK cells as described (3) except for viruses mentioned below. Stocks of the ICP27-null mutant were produced on complementing Vero 2–2 cells (55) and ICP0-null mutant in U2OS cells. All produced viruses were Ficoll-gradient purified.
For all experiments, cells were infected using a multiplicity of infection of 10 (MOI) in fresh media 24 h after the last split. Subsequently, the inoculum was removed, and conditioned media was applied to the cells. The time at which inoculum was replaced with growth media was marked as the 0h time point. To block viral DNA replication, phosphonoacetic acid (PAA, 350 μg/ml) was added in conditioned media to cultured cells after the inoculum was removed. The number of biological replicates that were performed for each experiment is indicated in the main text.
Cell line manipulation and generation
Artificial miRNAs (amiRNAs) against SSRP1 and SPT6 were selected as described (35), cloned into a doxycycline-inducible lentiviral vector (see below) and utilized to generate T-HF cells that enable efficient, dox-inducible knock-down of the respective host proteins. Primer sequences are listed in Supplementary table 1. Transduced T-HF cells were maintained in 5 μg/ml Puromycin. Knock-down was induced by 1 μg/ml doxycycline for 72h with fresh Dox added at 48 h.
HA-ICP22 and HA-ICP22 + V5-ICP27 cells were generated as follows. Lentiviral vectors encoding N-terminal 3xFLAG and V5-tagged (tandem tag) UL54 ORF under control of the doxycycline-inducible pTRE-Tight promoter were produced by cloning the corresponding ORF from the HSV-1 genome (strain 17) via intermediate vectors into pW-TH3. The pW-TH3 vector was derived from pCW57.1 by sequential insertion of a synthetic multi-cloning site (prW64/65) and three stop codons (prW110/111) between the NheI and AgeI restriction sites. pCW57.1 was a gift from David Root (Addgene plasmid #41393; http://n2t.net/addgene:41393). pW-TH7 (3xFlag-V5-NT1) was created by amplifying the N-terminal part of NT1 from the V5-NT1 vector (56) by using primers prW196/197 and inserted back between the BamHI and EcoRI of the same V5-NT1 vector. The 3xFlag-V5-NT1 ORF was excised with EcoRI and XbaI and inserted between the EcoRI and NheI sites of pW-TH3 (now designated pW-TH9). The UL54 ORF was amplified from the HSV-1 genome by PCR using primers prW365/366. The PCR product was digested with BamHI and BglII and inserted into BamHI cut pW-TH9 (now designated pW-TH57). To generate the doxycycline-inducible vector with HA-tagged US1 ORF (designated as LDJ5), the vector YC1 was used as backbone. YC1 was generated to carry blasticidin resistance gene instead of puromycin by restriction digestion of pW-TH3 vector with XbaI and AgeI and insertion of a hPGK.blast construct (purchased from GeneArt) via infusion cloning. The US1 ORF was amplified from the HSV-1 genome by PCR using primers prW1656/1657 and the extracted band was cloned via infusion cloning into YC1, linearized by restriction digest with MluI and NheI. Primer sequences used for generating HA-ICP22 and V5-ICP27 cell lines are listed in Supplementary table 2.
To generate V5-ICP27 and HA-ICP22 doxycycline-inducible cell lines, HEK-293T cells were transfected with pW-TH57 and LDJ5, respectively. Transduced T-HF cells were kept in selection with 5 µg/ml puromycin and 5 µg/ml blasticidin, respectively. To generate V5-ICP27 + HA-ICP22 doxycycline-inducible cell line, V5-ICP27 cells were lentivirally transduced with LDJ5. Transduced T-HF cells were kept in selection with both 5 µg/ml puromycin and 5 µg/ml blasticidin. Expression of proteins was induced by 5 μg/ml doxycycline for 48h.
Samples were harvested at the indicated time points by removal of growth media, followed by 1x wash with phosphate buffered saline (PBS, Sigma-Aldrich #D8537) and lysis in 1x Laemmli buffer containing 5% (v/v) β-mercaptoethanol. Samples were sonicated and heated for 5 min at 95 °C before loading onto a Novex WedgeWell 4–20% Tris-Glycine Gel (ThermoFisher #XP04200BOX). Proteins were transferred to 0.2 µm nitrocellulose membranes (Sigma-Aldrich #GE10600001 ), blocked for 1 h at room temperature (RT) in 1x PBS with 0.2% Tween (PBS-T) containing 5% (w/v) milk (Carl Roth #T145.3), and probed using anti-V5 (Cell Signaling #13202), anti-HA clone 11 (Biolegend #16B12), anti-FLAG (Sigma-Aldrich # F3165) anti-α-Tubulin (Cell Signaling #2144), anti-β-Actin clone C-4 (Santa Cruz Biotechnology #sc-47778), anti-GAPDH (Cell Signaling #2118), anti-SPT6 (Novus Biologicals #NB100-2582), anti-SSRP1 (Biolegend #609710), anti-ICP8 clone 11E2 (Santa Cruz Biotechnology #sc-53330), anti-gD clone DL6 (Santa Cruz Biotechnology #sc-21719) overnight at 4 °C at a 1:1,000 diluation unless otherwise specified. Before addition of each antibody, blots were washed with 3x PBS-T. After incubation with either anti-rabbit–horseradish peroxidase (HRP, Sigma-Aldrich #A0545), anti-mouse–HRP (Sigma-Aldrich #A9044) at a 1:10,000 dilution, anti-rat-HRP (Sigma-Aldrich #SAB3700539) at a 1:5,000 diluation, or IRDye 680RD goat-anti-rabbit IgG (Licor #926-68071) and IRDye 800CW donkey anti-mouse IgG secondary antibody (Licor #926-32212) at 1:5,000 dilution, bands were visualized using the LI-COR Odyssey FC Imaging System.
105 HA-ICP22 and HA-ICP22+V5-ICP27 cells were plated in 12 well-dishes with addition of 5 μg/mL of doxycycline (Merck #AMBH2D6FB132). At 48h post-induction, cells were fixed with 4% formaldehyde (PFA) in PBS for 15 min at RT, washed three times in PBS, and either stored at 4 °C overnight in PBS or processed immediately as follows. Cells were permeabilized in 0.5% Triton X-100 in PBS for 5-10 minutes and blocked in blocking buffer (10% FBS, 0.25M glycine, 1x PBS) for 1h at RT. Anti-HA antibody clone F-7 (Santa Cruz Biotechnology #sc-7392) or anti-V5 antibody (Cell Signaling #13202) were incubated in 10% FBS and 1x PBS for 1h at RT at dilution of 1:500. The secondary anti-mouse IgG, Alexa Fluor 488 (ThermoFisher #A11017) or anti-rabbit IgG, Alexa Flour 568 (abcam #ab175471) were incubated in 10% FBS in 1x PBS for 1h at RT with 0.5μg/mL 4’,6-diamidino-2-phenylindole (DAPI). All steps were followed by three 5 min washes in 1x PBS after which the images were taken on Leica DMi8 fluorescence microscope.
ATAC-seq and Omni-ATAC-seq
ATAC-seq was performed according to the original protocol starting with 1x105 cells per condition (57). An improved ATAC-seq protocol, Omni-ATAC-seq was performed according to the original protocol starting with 1x105 cells per condition (33). For each experiment biological duplicates were carried out.
Sequencing libraries for ATAC samples were prepared as specified using the Nextera DNA Library prep kit (Illumina #15028212) or in combination with primers made by IDT based on Illumina primers with unique dual (UD) index adapters for both i_5 and i_7.
Sequencing libraries for Omni-ATAC samples were prepared as 50 µL reactions containing: 12,5 µL DNA, 6,25 µL i_5_x and i_7_x (10 µM) and 25 µL 2x NEBNext Ultra II Q5 Master Mix (NEB #M0544). The number of cycles necessary for the library amplification was determined from the pre-amplification of transposed fragments.
Both ATAC-seq and Omni-ATAC-seq libraries were quantified by Agilent Bioanalyser and sequenced by NextSeq 500 (Ilumina) at the Core Unit Systemmedizin, Würzburg, Germany (35 bp paired-end reads). All samples were sequenced at equimolar ratios.
To confirm the presence of read-through transcription, on the day of ATAC/Omni-ATAC-seq experiment, total RNA was collected. Biological duplicates were carried out. For total RNA, cells were collected in 500 μl TRI reagent and total RNA was isolated with Quick-RNA Microprep Kit (Zymo Research #R1050) according to manufacturer’s instructions. Following steps were performed by the Core Unit Systemmedizin, Würzburg, Germany. For the total RNA libraries, both cytoplasmic and mitochondrial rRNA species were depleted. No rRNA depletion was performed for 4sU-RNA samples as rRNA only contributes about 40–50% of reads in 4sU-RNA samples. Library preparation for sequencing was performed using the stranded TruSeq RNA-seq protocol (Illumina, San Diego, USA). Libraries were sequenced on NextSeq 500 (Ilumina). 4sU-seq data for Figure 1c-d and Supp. Fig. 1c were taken from previous work (3, 5) and data for Figure 2e-f and Supp. Fig. 2i-l from (4).
ChIPmentation, library preparation and sequencing
Two days prior to infection, two million HFFF cells were seeded in 15 cm dishes. At the day of infection, cells had expanded to ~80% confluency. Cells were infected with the respective viruses as described in results section (n=2 for all conditions except for H3K36me3 in WT strain 17 without PAA (n=3)). PAA (350 μg/mL) was added to the conditioned cell culture media that was supplied to the cells after removal of virus inoculum. At 8 p.i., cells were fixed by adding ChIP Cross-link Gold according to manufacturer’s instructions (Diagenode #C01019027) and subsequently with 1% PBS-buffered formaldehyde. Cells were scraped in 1mL of ice-cold 1x PBS containing protease inhibitor cocktail (1x) (Roche #11836153001) with an additional 1mM phenylmethylsulfonyl fluoride (PMSF). Cells were pelleted at 1500 rpm for 20 min at 4 °C. Supernatant was aspirated and cell pellets were frozen in liquid N2.
Cell pellets were resuspended in 1.5 mL 0.25% [w/v] SDS sonication buffer (10 mM Tris pH=8.0, 0.25% [w/v] SDS, 2 mM EDTA) with 1x protease inhibitors and 1 mM additional PMSF and incubated on ice for 10 minutes. Cells were sonicated in fifteen 1 minute intervals, 25% amplitude, with Branson Ultrasonics SonifierTM S-450 until most fragments were in the range of 200-700 bp as determined by agarose gel electrophoresis. Two million cells used for the preparation of the ChIPmentation libraries were diluted 1:1.5 with equilibration buffer (10 mM Tris, 233 mM NaCl, 1.66% [v/v] Triton X-100, 0.166% [w/v] sodium deoxycholate, 1 mM EDTA, protease inhibitors) and spun at 14,000x g for 10 minutes at 4 °C to pellet insoluble material. Supernatant was transferred to a new 1.5 mL screw-cap tube and topped up with RIPA-LS (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.1% [w/v] SDS, 0.1% [w/v] sodium deoxycholate, 1% [v/v] Triton X-100, protease inhibitors) to 200 μL. Input and gel samples were preserved. Lysates were incubated with 1:100/IP of anti-H1 antibody (Invitrogen #PA5-30055), 1:50/IP of anti-H3 antibody (Invitrogen, PA5-16183), 1:50/IP of anti-H4 antibody (Cell Signaling, #14149S), 1 μg/IP of anti-H3K27me3 (Diagenode, #C15410195) and 1 μg/IP of anti-H3K36me3 (Diagenode, #C15410192) on a rotator overnight at 4 °C. Dependent on the added amount of antibody, the amount of Protein A magnetic beads (ThermoFisher Scientific #10001D) was adjusted (e.g. for 1-2 μg of antibody/IP = 15 μL of beads) and blocked overnight with 0.1% [w/v] bovine serum albumin in RIPA buffer. On the following day, beads were added to the IP samples for 2 h on a rotator at 4 °C to capture the antibody-bound fragments. The immunoprecipitated chromatin was subsequently washed twice with 150 μL each of ice-cold buffers RIPA-LS, RIPA-HS (10 mM Tris-HCl pH 8.0, 50 0mM NaCl, 1 mM EDTA pH 8.0, 0.1% [w/v] SDS, 0.1% [v/v] sodium deoxycholate, 1% [v/v] Triton X-100), RIPA-LiCl (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% [w/v] sodium deoxycholate, 0.5% [v/v] Nonidet P-40) and 10 mM Tris pH 8.0 containing protease inhibitors. Beads were washed once more with ice-cold 10 mM Tris pH 8.0 lacking inhibitors and transferred into new tubes.
Beads were resuspended in 25 μL of the tagmentation reaction mix (Nextera DNA Sample Prep Kit, Illumina) containing 5 μL of 5x Tagmentation buffer, 1 μL of Tagment DNA enzyme, topped up with H2O to the final volume and incubated at 37 °C for 10 minutes in a thermocycler. Beads were mixed after 5 minutes by gentle pipetting. To inactivate the Tn5 enzyme, 150 μL of ice cold RIPA-LS was added to the tagmentation reaction. Beads were washed twice with 150 μL of RIPA-LS and 1x Tris-EDTA and subjected to de-crosslinking by adding 100 uL ChIPmentation elution buffer (160 mM NaCl, 40 μg/mL RNase A (Sigma-Aldrich #R4642), 1x Tris-EDTA (Sigma #T9285) and incubating for 1h at 37 °C followed by overnight shaking at 65 °C. The next day, 4 mM EDTA and 200 μg/mL Proteinase K (Roche, #03115828001) were added, and samples incubated for another 2h at 45 °C with 1000 rpm shaking. Supernatant was transferred into a new tube and another 100 μL of ChIPmentation elution buffer was added for another hour at 45 °C with 1000 rpm shaking. DNA was isolated with MinElute PCR Purification Kit (Qiagen #28004) and eluted in 21 μL of H2O.
DNA for the final library was prepared with 25 μL NEBNext Ultra II Q5 Master Mix, 3.75 μL IDT custom primer i5_n_x (10 μM); 3.75 μl IDT custom primer i7_n_x (10 μM) (see Supplementary Table 3); 3.75 μL H20 and 13.75 μL ChIPmentation DNA. The Cq value obtained from the library quantification, rounded up to the nearest integer plus one additional cycle, was used to amplify the rest of the ChIPmentation DNA. Library qualities were verified by High Sensitivity DNA Analysis on the Bioanalyzer 2100 (Agilent) before performing sequencing on NextSeq 500 (paired-end 35bp reads) at the Core Unit Systemmedizin, Würzburg, Germany (samples without PAA) or DNBSEQ-G400 2x100bp in BGI, Hong Kong, China (samples with PAA). All samples were sequenced at equimolar ratios.
Sequencing reads for ATAC-seq, RNA-seq, 4sU-seq and ChIPmentation were mapped against (i) the human genome (GRCh37/hg19), (ii) human rRNA sequences and (iii) the HSV-1 genome (HSV-1 strain 17, GenBank accession code: JN555585, only for HSV-1 infection data) using ContextMap v2.7.9 (58) (using BWA as short read aligner (59) and allowing a maximum indel size of 3 and at most 5 mismatches). For the two repeat regions in the HSV-1 genome, only one copy each was retained, excluding nucleotides 1–9,213 and 145,590–152,222.
Analysis of open chromatin regions
For ATAC-seq data, BAM files with mapped reads were converted to BED format using BEDTools (60) and OCRs were determined from these BED files using F-Seq with default parameters (61). No filtering of OCRs was performed. dOCR length for a gene was calculated as previously described (5). In brief, downstream OCRs were assigned to each gene in the following way. First, all OCRs overlapping with the 10kb downstream of a gene were assigned to this gene. Second, OCRs starting at most 5kb downstream of the so far most downstream OCR of a gene were also assigned to this gene. This was performed iteratively, until no more OCRs could be assigned. Here, individual OCRs could be assigned to multiple genes. dOCR length of a gene was then calculated as the total genomic length downstream of this gene covered by OCRs assigned to the gene. Similarly, OCR length in gene bodies was calculated as the total genomic length of the gene bodies covered by OCRs.
Quantification of downstream transcriptional activity and read-through
Number of read fragments per gene or in downstream regions were determined from the mapped RNA-seq or 4sU-seq reads in a strand-specific manner using featureCounts (62) and gene annotations from Ensembl (version 87 for GRCh37). For genes, all read pairs (= fragments) overlapping exonic regions on the corresponding strand by ≥25bp were counted for the corresponding gene. For downstream regions, all fragments overlapping the 5kb downstream of the gene 3’end were counted. Gene expression and downstream transcriptional activity were quantified in terms of fragments per kilobase of exons per million mapped reads (FPKM) and averaged between replicates. Only reads mapped to the human genome were counted for the total number of mapped reads for FPKM calculation. Percentage of read-through was calculated as previously described (5). In brief: First, the percentage of transcription downstream a gene was calculated separately for each replicate as: Percentage of downstream transcription = 100 x (FPKM in 5kb downstream of gene)/(gene FPKM). Percentage of downstream was averaged between replicates and transcription percentage of read-through was then calculated as percentage of downstream transcription in infected– percentage of downstream transcription in uninfected or untreated cells. Negative values were set to 0.
Metagene analyses were performed as previously described (63) using the software developed for this publication. For each gene, the regions −3 kb to +3 kb of the TSS were divided into 250 bp bins, the regions -5 kb to +100 kb of the TTS into 500bp bins and the remainder of the gene body (+3 kb of TSS to −5 kb of TTS) into 100 bins of variable length in order to compare genes with different lengths. For each bin, the average coverage per genome position was calculated and normalized to a total sum of 1. Metagene curves for each replicate were created by averaging results for corresponding bins across all genes considered and metagene plots show the average metagene curves across replicates. To determine statistical significance of differences between average metagene curves for two conditions, paired Wilcoxon signed rank tests were performed for each bin comparing normalized coverage values for each gene for this bin between the two conditions. P-values were adjusted for multiple testing with the Bonferroni method across all bins within each subfigure and are color-coded in the bottom track of subfigures: red = adj. P-value ≤ 10-5; orange = adj. P-value ≤ 10-3; yellow: adj. P-value ≤ 0.05.