Fig 1.
ChIP-exo in sorted-refractory cells to map genome-wide SZF1-binding sites.
(A) Experimental design for isolation of EBV-positive refractory and lytic cells for ChIP (chromatin immunoprecipitation)-exo protocol. (B) Pre-sort and post-sort analysis of FACS separation of refractory and lytic cells. EBV-positive HH514-16 BL cells were treated with NaB for 24 hours and harvested for flow sorting. (B, top) Reference EBV-seropositive serum was used to demarcate lytic cells and EBV-seronegative serum was used as negative control for gating purposes. (B, bottom) Post-sort analysis was performed to confirm purity and efficacy of sort. (C) Illustration of ChIP-exo protocol for SZF1 in sorted cells. DNA immunoprecipitated by anti-SZF1 antibody is treated with a 5’-to-3’ exonuclease (Exo) while still in the immunoprecipitate. The 5’ ends of digested DNA are concentrated at a fixed distance from the sites of crosslinking (i.e. footprint) and are detected by deep sequencing.
Fig 2.
Effects of EBV genome-derived candidate SZF1-binding sites on extrachromosomal gene expression.
(A) Candidate SZF1-binding site fragments from the EBV genome were cloned into pEGFP-N1 vector. SZF1-binding site pEGFP-N1 vectors or control pEGPFn1 vector (positive control) were transfected into HEK293T cells and assayed for relative GFP expression by flow cytometry. In addition, Cy5-non-targeting siRNA was co-transfected to monitor transfection efficiency between samples. Empty vector and non-fluorescent, non-targeting control siRNA-transfected 293T cells were used as negative control and for gating purposes. Percent cells expressing GFP are labeled in green. (B) Sequences of candidate SZF1-binding sites identified via ChIP-exo and tested in panel A. Positions on the EBV genome and with respect to nearby “target” genes are also shown; Pro, promoter; CDS, coding sequence. (C) Knockdown of SZF1 using two separate siRNAs or a control non-targeting siRNA was performed in HH514-16 BL cells. After 24 hours, NaB was added to activate EBV lytic cycle. After another 24 hours, cells were harvested for RNA extraction and RT-qPCR analysis was performed for relative EBV lytic transcripts in control non-targeting siRNA-transfected cells (white bar) versus two distinct siRNAs targeting SZF1 (black and grey bars). Data represent averages of three independent experiments; error bars, SEM; *, p ≤ 0.05. (D) Twenty-four hours after transfection, cells from (C) were harvested for immunoblot to validate SZF1 knockdown.
Fig 3.
Mutations in candidate SZF1-binding sites derepress EBV lytic genes.
(A) Synonymous point mutations were made in candidate SZF1-binding sites on the p2089 BACmid via red recombineering. Mutant residues are shown in red. (B) After transfection and hygromycin selection (~ 2 weeks later), 293-BAC cells harboring wild-type p2089 BACmid or BACmids with mutant SZF1-binding sites were harvested for RNA extraction and RT-qPCR analysis for relative expression of EBV lytic genes (from each kinetic class: BZLF1, immediate early; BGLF4, early; BDLF2, late), compared to wild-type BAC sample. (C) Supernatants from 293-BAC cells harboring p2089-BACs bearing BZLF1 promoter, BGLF4 coding sequence, or BDLF2 coding sequence SZF1-binding site mutations were harvested and analyzed via qPCR for relative amounts of released DNase-resistant virus compared to the wild-type 293-BAC sample. (D) 293-BAC cells harboring wild-type p2089-BAC or p2089 BACmids that underwent reversion (r) mutations for their respective SZF1-binding sites were tested by RT-qPCR of lytic genes BZLF1, BGLF4, and BDLF2 relative to wild-type 293-BAC. (E-G) SZF1-ChIP was performed on wild-type 293-BAC samples or 293-BACs harboring SZF1-binding site mutations; precipitated chromatin was analyzed via qPCR using primers to amplify PCR products flanking the putative BZLF1 promoter sequence (E), the BGLF4 coding sequence site (F), or the BDLF2 coding sequence site (G). ChIP-PCR results were analyzed relative to 1% input and displayed as percent input. Data represent averages of three independent experiments; error bars, SEM; *, p ≤ 0.05.
Fig 4.
Viruses harboring mutated SZF1-binding sites spontaneously express lytic genes and demonstrate defects in B cell transformation.
(A) Peripheral blood mononuclear cells (PBMC) from three healthy donors were infected with wild-type or SZF1-binding site mutant p2089 viruses in the presence of FK506. Cells were counted using Trypan Blue at indicated time points and absolute live cells were plotted. Results were averaged between three donors. (B-D) PBMC from 3 donors infected as in (A) were harvested after 48 hours for RT-qPCR analysis of EBV lytic genes BZLF1, BGFL4, and BDLF2, relative to wild-type p2089-infected PBMC. Data represent averages from three independent experiments; error bars, SEM; *, p ≤ 0.05.
Fig 5.
Binding sites used by SZF1 to silence EBV lytic genes are not used to repress host genes during latency.
SZF1 was depleted using a validated siRNA or a control non-targeting siRNA (siScram) in HH514-16 BL cells. After 24 hours, NaB was added to activate EBV lytic cycle. After another 24 hours, cells were harvested for RNA extraction and RT-qPCR analysis of host genes harboring the BZLF1p binding site (A-C) or the BDLF2 binding site (D-I). Data represent averages of two independent experiments; error bars, SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 6.
Evaluation of SZF1-footprint derived motifs on the B cell genome.
(A) Informatic workflow for SZF1 motif discovery. (B) Informatically-derived putative SZF1-binding motif consensus sequences were cloned into a pEGFP-N1 vector and transfected into HEK293T cells. GFP expression was assessed relative to control pEGFP-N1 vector lacking putative SZF1 motif consensus sequences; Cy5-non-targeting siRNA was co-transfected to monitor transfection efficiency. Percent GFP+ cells from three of 27 motif-consensus-sequence-bearing cassettes are displayed in green; only consensus sequences derived from motifs 1 and 2 repressed GFP expression, and motif 3 is a consensus sequence representative of the remaining 24 motifs. (C) SZF1 motifs derived from Meme-suite. Motif logos of the two motifs capable of repressing GFP in (B) were aligned and overlap demarcated by a box. Percent GFP knockdown in (B) and statistical significance of motif determined using Meme-suite are shown.
Fig 7.
SZF1 binding motif 1 on viral versus host genomes.
(A) A sequence matching the consensus of motif 1 identified within the BcLF1 gene of the EBV genome was mutated using red recombineering. This mutant and wild-type p2089 BAC were transfected into HEK293T cells. Cells were harvested for RT-qPCR analysis of EBV lytic genes BZLF1 and BcLF1. Data represent averages of three independent experiments; error bars, SEM. (B) Circos plot showing SZF1-footprints across the human genome and the locations of SZF1 binding motif 1. Height of bars represent indexed read counts; read counts are presented in Log10, sigma (s) = 5, exclusion zone = 10 and allowing no singleton. The spikes represent 176 peak-pair midpoints that contributed to the generation of SZF1 binding motif 1. Scales next to chromosomes indicates length of chromosomes. (C) Validated targeting siSZF1-2 from Fig 2 or a control non-targeting siRNA (siScram) was introduced into HH514-16 BL cells. After 24 hours, cells were harvested and RT-qPCR analysis performed for relative levels of ANKRD26P1 transcript. Data represent averages of two independent experiments with three technical repeats each; error bars, SEM; **, p < 0.01; ***, p < 0.001.
Fig 8.
SZF1 is enriched at oriP on the lytic genome but is not associated with actively replicating viral DNA.
A. Read coverage at EBV oriP. Plots show the read distributions at oriP under lytic and refractory conditions. Coverage of the reads from lytic and refractory genomes were determined with Bedtools software (v2.30.0) and plotted in R. Lytic reads were normalized to refractory EBV genome copy number, Rightward and leftward strands are indicated by blue and red, respectively. Genome position numbers corresponding to the reference genome NC_007605 are indicated. B-D. EBV-positive cells were untreated or exposed to NaB for 36 hours (or 24 hours in C) and subjected to (B) ChIP with anti-SZF1 antibody or control antibody followed by qPCR amplification of oriP and BZLF1p and analysis of data by normalizing to 2% input and IgG control, (C) isolation of intracellular DNA and qPCR amplification of BALF5 gene, or (D) isolation of proteins on nascent DNA (iPOND) followed by western blotting with indicated antibodies. Data in B and C represent averages of two independent experiments; error bars, SEM; ***, p < 0.001; iPOND was performed twice.
Table 1.
siRNAs targeting SZF1.
Table 2.
Primers used to generate mutations in the EBV BACmid p2089.
Table 3.
Sequences of primers used for RT-qPCR.