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

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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 Á Latency Á Reactivation Á Transcriptional control Á Mouse model Á CCCTC binding factor Á Sp1 H. C. Stevens Á V. J. Bubb Á 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 Á D. J. Hughes Á 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 path- ogenesis. 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 123 Virus Genes 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 concen- trations of pFLAG-CMV1-ORF50 or GFP (pEGFP-N1, Generation of the C127DRTA cell line and reactivation Clontech).
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