CTCF and Sp1 Interact with the Murine Gammaherpesvirus 68 Internal Repeat Elements

Virus Genes DOI 10.1007/s11262-012-0769-y

CTCF and Sp1 interact with the Murine gammaherpesvirus 68 internal repeat elements

Hannah C. Stevens • Kevin S-W Cham • David J. Hughes • Ren Sun • Jeffery T. Sample • Vivien J. Bubb • James P. Stewart • John P. Quinn

Received: 2 March 2012 / Accepted: 29 May 2012 Ó Springer Science+Business Media, LLC 2012

Abstract Herpesviruses maintain a dynamic balance [CTCF] and Sp1) of the two internal repeat elements in the between latency and productive infection. This is a com- viral genome during latency and reactivation using chro- plex process regulated by viral and cellular factors. We matin immunoprecipitation. Our results show that CTCF have developed a Murine gammaherpesvirus 68 (MHV-68) can bind to the 40-bp and the 100-bp repeat sequences model system in which to study mechanisms underlying during latency, whereas binding is reduced upon reactiva- balance between latency and lytic infection. We have tion. In contrast, Sp1 only bound to the 100-bp repeat after generated an epithelial cell line that carries MHV-68 in a reactivation. Our results indicate that the large internal tightly latent form by using a bacterial artiﬁcial chromo- repeat sequences in MHV-68 have different functions. We some clone of the virus genome with a mutation in the hypothesise that the 40-bp repeat may be involved in reg- MHV-68 major lytic R transactivator gene. Complemen- ulation of gene expression during the maintenance of tation of this defect in trans by transfection with a plasmid latency, while the 100-bp repeat domain may be involved encoding R transactivator initiated and restored the pro- in regulation of the lytic cycle. ductive cycle. This cell line model was used to investigate transcription factor occupancy (CCCTC binding factor Keywords Herpesvirus Latency Reactivation Transcriptional control Mouse model CCCTC binding factor Sp1 H. C. Stevens V. J. Bubb J. P. Quinn Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GE, UK Introduction

& K. S.-W. Cham D. J. Hughes J. P. Stewart ( ) The human c-herpesviruses, Epstein-Barr virus (EBV) and Department of Infection Biology, University of Liverpool, Liverpool L69 3GA, UK Kaposi’s sarcoma-associated herpesvirus (KSHV; alterna- e-mail: [email protected] tively human herpesvirus 8 [HHV-8]), cause significant human disease, most of which are associated with persis- Present Address: tence of these viruses in the host. However, strict host D. J. Hughes Institute of Molecular and Cellular Biology, Faculty of preferences of EBV and KSHV limit assessment of the Biological Sciences, University of Leeds, Leeds LS2 9JT, UK mechanisms that contribute to their persistence and pathogenesis. Consequently, there has been considerable effort R. Sun to develop an amenable small animal model for human Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, c-herpesviruses. Murine c-herpesvirus 68 (MHV-68 or CA 90095-1735, USA cHV68; officially Murid herpesvirus 4 [MuHV-4]) is an endogenous pathogen of free-living rodents of the Apode- J. T. Sample mus genus, e.g. wood mice [1–6]. Experimental infection Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, of laboratory mice with MHV-68 has therefore been Hershey, PA 17033, USA developed and utilised to good effect as a model of 123 Virus Genes c-herpesvirus infection [7–25]. Following intranasal inoc- Fig. 1 MHV-68 internal repeats contain a high density of tandem c ulation of mice with MHV-68, a productive infection CTCF and Sp1 clusters. A diagrammatic representation of the MHV- 68 genome is shown at the top. Unique regions are indicated by occurs in the lung [26]. This is cleared around day 10–14 shaded boxes and repetitive elements by open boxes. The genome is ? post-infection (p.i.) by CD8 T cells [27], though the virus bounded by multiple copies of a terminal repeat element (TR). The persists in a latent or non-productive form in epithelial cells positions of the regions amplified by PCR in the ChIP assay are at this site [28]. MHV-68 spreads to the spleen, where it shown by solid bars. Beneath this, the sequences of the 40- and 100-bp repeats (forward strand) are expanded showing the location of also becomes latent, predominantly not only within B the clustered CTCF and Sp1 motifs as indicated. The sequence of the lymphocytes, but also in macrophages and dendritic cells repeats is shown in upper case and the surrounding sequence in lower [29–32]. case. Motifs for CTCF and Sp1 are highlighted in either red (forward Latency and reactivation from latency are central to the strand)orblue (reverse strand) where motifs overlap are highlighted in green. The sequence of the PCR primers used are shown in purple pathogenesis of c-herpesviruses [33]. Infection of mice (Color figure online) with MHV-68 has enabled many aspects of c-herpesvirus biology to be elucidated. Likewise, MHV-68 readily infects and undergoes productive replication in a range of activator activity. Described originally as binding to the cell lines in vitro, enabling the study of the productive sequence CCCTC, it has now been shown that CTCF has a cycle. However, there is no ideal system with which to much larger 20-bp consensus binding sequence [51]. The study latency and the reactivation from latency in vitro. presence of a single CTCF DNA-binding motif is sufficient The S11 B cell tumour cell line [34] which is predomi- for binding. However, clustering of these consensus nantly latently infected is relevant and has been used to sequences leads to a higher CTCF-binding affinity. CTCF define aspects of MHV-68 latency [35]. However, it has a is associated with several distinct activities, including measurable rate of spontaneous reactivation making the transcriptional activation/repression, the formation of discrimination of latency and reactivation hard. Likewise, chromatin insulators, imprinting and X chromosome inac- latent infection of A20 mouse B cells with a selectable tivation [52]. MHV-68 has value but still suffers from spontaneous Sp1 is a zinc finger-containing DNA binding protein that reactivation [36]. There is therefore a need for a cell culture is ubiquitously expressed and can either activate or repress system that supports MHV-68 in a tightly latent form with transcription. It binds to GC-rich motifs (such as 50-G/T- which to study latency and reactivation. GGGCGG-G/A-G/A-C/T-30 or 50-G/T-G/A-GGCG-G/T-G/ The R transactivator homologue (RTA) encoded by A-G/A-C/T-30). Sp1 has also been linked to chromatin MHV-68 open reading frame 50 (ORF50) is an immediate- remodelling via interactions with chromatin-modifying early gene product that is conserved among all character- factors such as p300 and histone deacetylases (HDACs) ised c-herpesviruses and is a critical regulator of lytic [53]. Sp1 and CTCF have been demonstrated to bind to a replication and reactivation from latency [6, 37–44]. MHV- GC-rich triplet domain in the c-myc gene and proposed to 68 RTA is responsible for transactivating its own promoter alter transcription start site usage of c-myc [54, 55]. in addition to other virus promoters to reactivate latent Both CTCF and Sp1 have been shown to play a role in virus [38, 45]. It has previously been shown that an RTA- herpesvirus gene regulation. CTCF binds during latency to null mutant is incapable of viral protein synthesis, viral clusters of consensus binding motifs that are present in the DNA replication or virion production. This phenotype can repetitive regions of the HSV-1 genome. CTCF could be rescued by expressing RTA in trans [46]. therefore be important in the maintenance of herpes virus The MHV-68 genome contains two internal repetitive latency [56]. A number of promoters for HSV-1 immedi- sequences known as the 40-bp and 100-bp repeats (Fig. 1) ate-early genes (e.g. ICP4) have consensus binding sites [39]. The 40-bp repeat is located between nt 26778-28191 that have been shown to interact with Sp1. This factor in the genome, and the 100-bp repeat is between nt therefore has an important role during reactivation and the 98981-101170 (Genbank AF105037). The 40-bp repeat has lytic cycle [57–59]. been identified as an enhancer for latency and is important The aim of this study was to analyse the potential role of for the expression of mK3 (ORF12) and ORF72 in the Ag8 CTCF and Sp1 in MHV-68 gene expression during the cell line that is derived from B cells [47]. Moreover, a switch from latency to reactivation. We describe the con- region of both the 40- and 100-bp repeat sequence is struction of an epithelial cell line model for MHV-68 that essential for the lytic origins of replication to function [48– was capable of analysing latency and reactivation. We 50]. We surmised, therefore, regulation of these loci may describe the clustering of CTCF and Sp1 DNA-binding be important for maintenance of latency and reactivation. motifs in the two internal repetitive sequences and go on to CCCTC-binding factor (CTCF) is a zinc finger DNA- use the cell line model to analyse transcription factor binding protein that is highly conserved in vertebrates. It is occupancy and the epigenetics of MHV-68 during latency expressed in most cell types and has transcriptional and after reactivation. 123 Virus Genes

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Table 1 PCR primers used in Target Sequence MHV-68 genome the ChIP coordinate

40 bp repeat 50 flank 50-TATTCCCCCTGTTTTGGAGA-30 (F) 26,606–26,757 50-GGTCGAGGAACAAAACCAAA-30 (R) 40 bp repeat 30 flank 50-TTCGCAAAAGAGCTGCTGTA-30 (F) 28,338–28,521 50-ACCCACGGTGGTAGAAAGTG-30 (R) 100 bp repeat 50 flank 50-CATACCGGCCGATAATGTCT-30 (F) 98,828–98,936 50-TTGCTGAGAAAGACGAGATACAA-30 (R) 100 bp repeat 30 flank 50-CAGGGTGAACCCAACCAT-30 (F) 101,425–101,574 50-CGTAAAAGGTAGGGTGTGGA-30 (R)

Materials and methods cover slips and transfected as above with varying concentrations of pFLAG-CMV1-ORF50 or GFP (pEGFP-N1, Generation of the C127DRTA cell line and reactivation Clontech). The secondary antibody was anti-rabbit IgG from latency conjugated to Texas Red (Jackson Immunoresearch Labo- ratories). The cells were visualised on a Zeiss Axioscop 40 The C127 epithelial cell line (ATCC CRL 1616; mouse microscope, and images were captured using Axiovision mammary epithelial cells) was grown in Dulbecco’s software. Modified Eagles Medium (DMEM) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 70 lg/ml qRT-PCR for viral gp150 transcripts penicillin and 10 lg/ml streptomycin. The BAC clone containing the MHV-68 genome with a transposon-inser- Total RNA was extracted, treated with DNase and checked tion mutation rendering ORF50 non-functional (BAC- for quality, followed by reverse transcription using a MHV68DORF50) has been described previously [60]. This Reverse Transcription System (Promega). The qRT-PCR BAC also contains a functional puromycin cassette was carried out using the iQ SYBR Green supermix (Bio- enabling selection in mammalian cells. Rad) with 0.18 lM primers and 30 ng of each cDNA sam- BACMHV68DORF50 DNA was transfected into the ple. Primer sequences were as follows: RPL8 (forward, C127 cell line by the calcium phosphate method as 50-CAGTGAATATCGGCAATGTTTTG-30; reverse, 50-TT described previously [61, 62]. Three days post-transfection, CACTCGAGTCTTCTTGGTCTC-30) and gp150 (forward, cells were transferred into culture medium containing 50-GAACCTCCCACCTCCAATGC -30; reverse, 50-TTGT puromycin (3 lg/ml). Surviving cell colonies containing GGGGGTGTCTCATGGTTCG-30). PCR was performed in MHV-68 genomes were isolated by ring cloning [63]. This an iQ5 cycler (BioRad) under the following conditions: was repeated three times to obtain pure cell clones. Colo- 95 °C for 10 min, and then 45 cycles at 94 °C for 30 s and nies were screened by PCR analysis for the presence of 60–61 °C for 40 s. Melting curve analysis was carried out to MHV-68 genomes using primers specific for gp150 as confirm the specificity of the products between 65 and 95 °C described previously [28]. One clone that grew and at 0.2 °C increments. Data were analysed using Bio-Rad iQ5 stably maintained the MHV-68 genome was selected optical system software. (C127DRTA) for further analysis. To reactivate virus from latency, C127DRTA cells ChIP, PCR and densitometry were transfected with a mammalian expression vector (pFLAG-CMV1, Sigma) containing ORF50 (pFLAG- ChIP was performed using the ChIP-IT Express kit (Active CMV1-ORF50) using 1.5 lg plasmid per cm2 cells with Motif) as per the manufacturer’s instructions. C127DRTA Lipofectamine 2000 (Invitrogen) according to the manu- cells were fixed with 1 % formaldehyde for 10 min and facturer’s protocol. then washed and lysed, and the DNA sheared by sonication to produce DNA fragments in the range of 500–1500 bp. Immunostaining The chromatin/DNA complexes were incubated with anti- bodies to Sp1 (Santa Cruz Biotechnology), CTCF (BD The percentage of cells reactivating MHV-68 was assessed Transduction Laboratories), or non-specific IgG (Active by immunofluorescence using a polyclonal rabbit antise- Motif) for 16 h at 4 °C. The immune complexes were then rum to MHV-68 structural antigens [6] as described pre- precipitated, washed, eluted, reverse cross-linked and viously [64, 65]. C127DRTA cells were plated onto glass treated with proteinase K. The resulting DNA fragments

123 Virus Genes were amplified with primers that adjoined the repeats as MHV-68, had a minimal level of spontaneous reactivation described in Table 1. (Genbank NC001826). and that was capable of being reactivated into the lytic Each PCR reaction contained 35 ng DNA template, the cycle. All of the cell lines available (e.g. S11) had a rela- appropriate concentration of MgCl2 (2.5-4 mM), 0.2 mM of tively high level of spontaneous reactivation. Thus, to each dNTP (except dGTP where 0.1 mM of deoxy-7-deaz- generate a cell line that contained MHV-68 in a tightly aguanosine triphosphate and 0.1 mM of dGTP was used), latent form, a bacterial artificial chromosome (BAC) con- 0.2 pM of each primer, 19 Diamond buffer, 1.5 U Diamond taining the MHV-68 genome with an insertional (transpo- DNA polymerase (Bioline) and 0.5 M betaine. The PCR son) mutation of ORF50 (that encodes RTA) was used to conditions were 96 °C for 3 min, followed by 36 cycles of stably transfect C127 epithelial cells. Epithelial cells are a 94 °C(1min),58°C (30 s) and 72 °C (20 s). PCR products relevant cell type to study since MHV-68 both replicates were analysed by agarose gel electrophoresis in the presence and becomes latent in epithelial cells in vivo [28]; fur- of ethidium bromide, followed by visualisation using a UV thermore, C127 is a mouse epithelial cell line that supports transilluminator. Signal intensities from PCR reactions MHV-68 productive replication [69]. ORF50/RTA is crit- obtained from ChIP assays or from whole cell lysates (control ical for the initiation of the MHV-68 lytic cycle, and pro- for PCR) were quantified from the TIFF images with ImageJ ductive gene expression and replication should not occur in software (National Institutes of Health; http://rsbweb.nih. its absence. One cell clone (C127DRTA) that contained gov/ij/). Area, fraction and mean gray values were taken. MHV-68 DNA as determined by PCR analysis was selec- Mean gray values were used as a measure of relative number ted for further analysis. of pixels. Fold enrichment = specific antibody/IgG was cal- The C127DRTA cell line was examined over several culated for each independent experiment. passages in culture and did not develop viral plaques and thus, appeared to be latently infected. To confirm that the lytic cycle was not occurring, and that RTA provided in Results and discussion trans could induce reactivation of virus, C127DRTA cells were analysed for lytic cycle antigens and transcripts before The MHV-68 internal repeat elements contain clusters and after transient transfection with an expression plasmid of binding motifs for the cellular proteins CTCF that encodes RTA (pFLAG-CMV1-ORF50). The expression and Sp1 of MHV-68 structural antigens was analysed in cells by immunofluorescence staining using a polyclonal antibody to To determine the location of consensus CTCF and Sp1 MHV-68 [6]. Transfection with a control plasmid encoding binding sites within the MHV-68 genome, the DNA GFP (pEGFP-N1) revealed highly efficient transfection sequence was scanned using open-source prediction algo- levels with[90 % of C127DRTA cells expressing GFP (not rithms. CTCF motifs were predicted using the CTCF binding shown). There was no evidence of the expression of struc- site prediction tool [66](http://insulatordb.uthsc.edu/ tural antigens in untreated cells or after transfection with the storm.php) and Sp1 motifs were predicted using Consite empty expression vector pFLAG-CMV1 (Fig. 2a, b). [67](http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/) However, after transient transfection of C127DRTA with which utilises the JASPAR database [68]. Initial scans pFLAG-CMV1-ORF50 (supplying RTA), structural anti- revealed that the 40-bp and 100-bp tandem direct repeat gens were detected (Fig. 2c–h). The dose- and time-depen- sequences contained a high density of consensus binding dent effect of RTA on reactivation was assessed using sequences for both CTCF and Sp1 (Fig. 1). These motifs concentrations from 2 to 4 lg of pFLAG-CMV1-ORF50, were only rarely found throughout the unique regions of the assayed at both 24 and 48 h post-transfection. Reactivation genome (not shown). The 40-bp internal repeat contains 26 occurred at all concentrations; however, it occurred in the high-fidelity CTCF and 23 Sp1 consensus binding sequen- highest proportion of cells when 4 lg of pFLAG-CMV1- ces, whereas the 100-bp repeat contains 28 CTCF and 50 Sp1 ORF50 was used and fluorescence analysed at 48 h post- consensus binding sequences. The number and clustering of transfection (Fig. 2h), suggesting a dose dependant effect. these sequences were highly striking and suggested a sig- To quantify the time-course of reactivation, we analysed nificant role for these repeat regions in controlling virus gene the expression of transcripts specific for the major MHV-68 expression. glycoprotein gp150 that is known to be expressed with late kinetics [64]. Expression was quantified by qRT-PCR. The Generation and validation of a cell line containing results (Fig. 3) showed that expression of gp150 mRNA was latent MHV-68 not detectable after transfection with pFLAG-CMV1 and was not significantly increased 3 h after transfection with Analysis of factor binding during viral latency and reacti- pFLAG-CMV1-ORF50. However, expression thereafter vation required a cell line that was latently infected with increased rapidly to plateau at around 12 h post-transfection. 123 Virus Genes

A 2Ab° only B Mock

C 2 µg D 3 µg E 4 µg

24 h

F G H

48 h

Fig. 2 Reactivation of MHV-68 occurs after transfection of DAPI. The cells were visualised on a Zeiss Axioscop 40 microscope; C127DRTA cells with RTA. C127DRTA cells were cultured on images were captured using Axiovision software. Scale bars represent glass cover slips and reactivation was assessed at 24 and 48 h post- 20 lm. a Texas red conjugated secondary antibody only; b Transfec- transfection with varying concentrations of the RTA expression tion with pFLAG-CMV1 backbone alone; c 2 lg of pFLAG-CMV1- plasmid pFLAG-CMV1-ORF50, as indicated. Reactivation was ORF50, 24 h; d 3 lg of pFLAG-CMV1-ORF50, 24 h; e 4 lgof assessed by indirect immunoﬂuorescence for expression of MHV-68 pFLAG-CMV1-ORF50, 24 h; f 2 lg of pFLAG-CMV1-ORF50, structural antigens using a rabbit polyclonal anti-MHV68 and Texas 48 h; g 3 lg of pFLAG-CMV1-ORF50, 48 h; h 4 lg of pFLAG- red anti-rabbit secondary. Nuclei were counter-stained (blue) with CMV1-ORF50, 48 h (Color ﬁgure online)

Levels were then maintained through 36 h post-transfection, Thus, the C127DRTA cell line contains a tightly latent and declined slightly by 48 h. The kinetics of gp150 MHV-68 genome that can be made to reactivate by sup- expression observed was similar to that seen after infection plying RTA in trans by transfection. While this manuscript of cells with MHV-68 [64], implying a rapid and efﬁcient was in preparation, a similar approach to generating a cell induction of the productive cycle in C127DRTA cells that line containing latent MHV-68 was described [70]. This mirrors natural infection. utilised a doxycycline-inducible promoter to control RTA

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** association with the repeat sequence [56]. Furthermore, we * analysed sequences flanking both sides of the repeat to 1.0 * * more accurately assess factor binding. We first addressed by ChIP whether the 40-bp tandem repeat sequence could associate with CTCF using an anti- CTCF antibody. The analysis was performed on C127DRTA 0.5 cells both before and after reactivation of MHV-68 from latency by transfection with pFLAG-CMV1-ORF50. We demonstrated CTCF-binding to the 40-bp repeat flanking Cq normalised to RPL8 regions during latency as illustrated by an enriched number 0 0 0.0 of PCR amplicons both 5 and 3 to the 40 bp tandem repeat 0 10 20 30 40 50 region, whereas after reactivation there was a reduction in Hours post transfection CTCF-binding to both flanking regions (Fig. 4a). Likewise, analysis of the flanking regions of the 100-bp repeat dem- Fig. 3 qRT-PCR analysis of gp150 mRNA after transfection of C127DRTA cells with pFLAG-CMV1-ORF50. RNA was extracted onstrated CTCF-binding during latency, but after reactiva- from C127DRTA cells at the indicated times after either transfection tion this was also reduced (Fig. 4a). with pFLAG-CMV1 (filled triangle) or the RTA-expressing vector Thus, it appears CTCF can bind to the 40-bp and the pFLAG-CMV1-ORF50 (filled circle). The fold change in gp150 100-bp repeat sequences during latency, whereas binding is mRNA was normalised to the levels of the cellular ribosomal protein L8 mRNA and expressed as relative to the 0 h control as described reduced upon reactivation. CTCF is important for regula- elsewhere [74]. The quantitative cycle (Cq) values of test samples tion at the KSHV major latency control region, at which it spanning the time-course were plotted. Error bars depict the standard can associate with three repeated consensus sequences; at error of the means for 6 replicates. Statistical analysis was performed these sites CTCF may be important for the creation of by two-way ANOVA with Bonferroni post-tests. Points where a significant (P \ 0.001) difference in gp150 mRNA levels were seen intragenomic domains important for transcription during are indicated by asterisks latency [71, 72]. Thus, this might point to a conserved mechanism between herpesviruses that uses CTCF to segregate active from inactive genes during latency. Fur- expression. While this system is roughly equivalent to the ther, our observations fit with reverse genetic analysis one described here, the use of fibroblasts may limit its showing that the MHV-68 40-bp repeat is important for usefulness, as MHV-68 does not become latent in this cell latency amplification [47]. type in vivo. We next addressed whether the repeats could associate with Sp1, repeating ChIP with an anti-Sp1 antibody. Sig- CTCF and SP1 associate with the 40 bp and 100 bp nificant Sp1 binding was not detected at either the 40- or repeat sequences 100-bp repeat domains prior to reactivation (Fig. 4b). However, after reactivation, while Sp1 still was not sig- Since our bioinformatic analyses showed that the MHV-68 nificantly enriched at the 40-bp repeat (Fig. 4b), there was internal tandem repeat sequences contained clustered enrichment at both sides of the 100-bp repeat. Specifically, CTCF and Sp1 motifs (Fig. 1), we determined whether we observed a more than threefold enrichment at the 50 end these transcription factors bound to the internal repeats and a fivefold enrichment at the 30 end of the 100-bp repeat during latency and reactivation. Chromatin immunopre- (Fig. 4b). cipitation (ChIP) analysis was performed on chromatin During the lytic cycle, DNA is replicated and tran- extracted from the C127DRTA cell line. In initial pilot scription of the ordered cascade of lytic cycle-associated studies, we found that primers directed to the internal viral genes occurs. Interestingly, the 30 end of the 100-bp repeat sequences generated a large number of non-specific repeat is essential for the function of the lytic origin of products, which is likely due to their extremely high GC replication [48]. Therefore, binding of Sp1 at this region content and repetitive nature. We therefore used primers in may be involved in the regulation of concomitant tran- the ChIP assay that amplified flanking sequences directly scription and DNA replication. adjoining the repeats. Random DNA fragments generated In summary, we have developed a tightly latent but by sonication in this protocol were of such a size reactivatable MHV-68-containing cell line which provides (500–1500 bp) that the primers specific for the directly a model to enable analysis of factors regulating the MHV- adjoining flanking regions would still amplify precipitated 68 genome, both during latency and during and after DNA containing internal repeat sequences (Fig. 1). Ana- reactivation. Our investigation of both the 40-bp and lysing for enrichment of a sequence adjacent to a repeat has 100-bp internal repeat domains revealed differences in been validated previously as an indicator of factor factor binding before and after reactivation. This indicates 123 Virus Genes

A CTCF B Sp1 8 8

6 6

4 4 Fold change Fold change 2 2

0 0

40 bp 5' 40 bp 3' 40 bp 5' 40 bp 3' 100 bp 5' 100 bp 3' 100 bp 5' 100 bp 3'

Fig. 4 Chromatin immunoprecipitation (ChIP) analysis of CTCF and non-speciﬁc IgG as a control. PCR products were analysed by agarose Sp1 at the MHV-68 internal tandem repeat sequences. Latently gel electrophoresis and densitometric image analysis of agarose gels. infected C127DRTA cells were assayed either without treatment or The results are expressed as the mean fold enrichment (n = 2) of after reactivation from latency by delivering RTA with pFLAG- transcription factor relative to the level seen for IgG alone from CMV1-ORF50. Proteins were cross-linked in vivo at 36 h after C127DRTA cells either untreated (solid bars) or after reactivation transfection and subjected to ChIP using anti-CTCF, anti-Sp1 or (open bars). a anti-CTCF; b anti-Sp1 that the large internal repeat sequences in MHV-68 have 4. S.D. Becker, M. Bennett, J.P. Stewart, J.L. Hurst, Lab. Anim. 41, different functions. We hypothesise that the 40-bp repeat 229–238 (2007) 5. D.J. Hughes, A. Kipar, G.H. Leeming, E. Bennett, D. Howarth, may be involved in regulation of gene expression during J.A. Cummerson, R. Papoula-Pereira, B.F. Flanagan, J.T. Sample, the maintenance of latency, while the 100-bp repeat J.P. Stewart, PLoS Pathog. 7, e1001321 (2011) domain may be involved in regulation of the lytic cycle. 6. D.J. Hughes, A. Kipar, S.G. Milligan, C. Cunningham, M. Furthermore, this model will be of use in further dissecting Sanders, M.A. Quail, M.A. Rajandream, S. Efstathiou, R.J. Bowden, C. Chastel, M. Bennett, J.T. Sample, B. Barrell, A.J. the regulation of MHV-68 by transcription factors. Davison, J.P. Stewart, J. Gen. Virol. 91, 867–879 (2010) The role of CTCF and Sp1 to regulate an important 7. A.A. Nash, B.M. Dutia, J.P. Stewart, A.J. Davison, Philos. Trans. switch in herpesvirus latency is also consistent with our R. Soc. Lond. B Biol. Sci. 356, 569–579 (2001) data on the regulation of the HSV-1 repeat RE1 adjacent to 8. P.G. Stevenson, S. Efstathiou, Viral Immunol. 18, 445–456 (2005) the LAT region. This domain demonstrates tissue speciﬁc 9. M.A. Blackman, E. Flano, E. Usherwood, D.L. Woodland, Mol. and inducible transcriptional properties which are in part Med. Today 6, 488–490 (2000) regulated by these transcription factors. This may point to 10. S.H. Speck, H.W. Virgin, Curr. Opin. Microbiol. 2, 403–409 the conserved role of this signalling pathway in herpesvirus (1999) 11. E. Flano, I.J. Kim, D.L. Woodland, M.A. Blackman, J. Exp. Med. evolution [73]. 196, 1363–1372 (2002) 12. P.C. Doherty, J.P. Christensen, G.T. Belz, P.G. Stevenson, M.Y. Acknowledgments HCS was funded by the Wellcome Trust Prize Sangster, Philos. Trans. R. Soc. Lond. B Biol. Sci. 356, 581–593 Studentship. JPQ and VJB were funded by the BBSRC (award BB/ (2001) D016754/9). This work was funded in part by U.S. Public Health Ser- 13. J.P. Simas, S. Efstathiou, Trends Microbiol. 6, 276–282 (1998) vice grant CA090208 from the National Cancer Institute and the Penn 14. A.A. Nash, E.J. Usherwood, J.P. Stewart, Semin. Virol. 7, State Hershey Cancer Institute. JPS was funded by a Royal Society 125–130 (1996) (London) University Research Fellowship. The authors would like to 15. S.S. Lok, Y. Haider, D. Howell, J.P. Stewart, P.S. Hasleton, J.J. thank Dr Bahram Ebrahimi for the kind gift of pFLAG-CMV1-ORF50. Egan, Eur. Respir. J. Off. J. Eur. Soc. Clin. Respir. Physiol. 20, 1228–1232 (2002) 16. A.I. Macrae, B.M. Dutia, S. Milligan, D.G. Brownstein, D.J. Allen, J. Mistrikova, A.J. Davison, A.A. Nash, J.P. Stewart, References J. Virol. 75, 5315–5327 (2001) 17. A.I. Macrae, E.J. Usherwood, S.M. Husain, E. Flano, I.J. Kim, 1. B. Ehlers, J. Kuchler, N. Yasmum, G. Dural, S. Voigt, J. Schmidt- D.L. Woodland, A.A. Nash, M.A. Blackman, J.T. Sample, J.P. Chanasit, T. Jakel, F.R. Matuschka, D. Richter, S. Essbauer, D.J. Stewart, J. Virol. 77, 9700–9709 (2003) Hughes, C. Summers, M. Bennett, J.P. Stewart, R.G. Ulrich, 18. J.J. Obar, D.C. Donovan, S.G. Crist, O. Silvia, J.P. Stewart, E.J. J. Virol. 81, 8091–8100 (2007) Usherwood, J. Virol. 78, 10829–10832 (2004) 2. D.J. Hughes, A. Kipar, J.T. Sample, J.P. Stewart, J. Virol. 84, 19. D. Verzijl, C.P. Fitzsimons, M. Van Dijk, J.P. Stewart, H. 3949–3961 (2010) Timmerman, M.J. Smit, R. Leurs, J. Virol. 78, 3343–3351 (2004) 3. K. Blasdell, C. McCracken, A. Morris, A.A. Nash, M. Begon, 20. J.P. Stewart, A. Kipar, H. Cox, C. Payne, S. Vasiliou, J.P. Quinn, M. Bennett, J.P. Stewart, J. Gen. Virol. 84, 111–113 (2003) PLoS ONE 3, e1673 (2008)

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