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CTCF-mediated Genomic Effects of BART Region on Epstein-Barr Virus Chromatin 3D Structure in Gastric Carcinoma Cells

EBV latent infection in gastric carcinoma (GC) cells is characterized by distinct viral gene expression programs. CCCTC-binding factor (CTCF) is a chromatin structural factor that has been involved in coordinated chromatin interactions between multiple loci of Epstein-Barr virus (EBV) genes. Here, we investigate the role of CTCF in regulating EBV gene expression and chromosome conformation in model of EBV-associated gastric carcinoma (EBVaGC). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) against CTCF revealed 16 CTCF binding sites (BS) in EBV genome of EBVaGC, SNU719 cells. Among the CTCF BSs, one site named as BARTp (BamHI A right transcript promoter) CTCF BS is located at upstream of 11.8-kb BART region (EBV genome: 139724-151554) and was not yet defined its biological functions in EBV life cycle. EBV BART encodes a complex miRNA cluster of highly spliced transcripts that is implicated in EBV cancer pathogenesis. This present study investigated the functional role of the CTCF binding site at BARTp (BARTp CTCF BS) in regulating EBV gene transcription and EBV three-dimensional (3D) genome structure as DNA loop maker. Circular chromatin confirmation capture (4C)-seq and chromatin confirmation capture (3C)-semi-quantitative(sq)PCR assays using SNU719 cells revealed that BARTp CTCF BS interacts with CTCF BSs of LMP1/2, Cp/OriP, and Qp in EBV genome. We generated mutations in BARTp CTCF BS (S13) in bacmids with (BART+) or without (BART−) the 11.8-kb BART transcript unit (B(+/−)). ChIP-qPCR assay demonstrated that CTCF binding was ablated from BARTp in EBV B(+/−) S13− genomes (mutant S13), elevated at several other sites such as LMP1, OriP, and Cp in EBV B(-) (BART−) S13− genome, and decreased at the same sites in EBV B(+) S13− genome. Infection assay showed that BARTp CTCF BS mutation reduced infectivity, while BART transcript deletion has no detectable effects. Gene expression tests showed that EBNA1 was highly downregulated in B(+/−) S13− EBVs related to B(+/−) S13+ EBVs (wild-type S13). LMP1 and BZLF1 were more downregulated in B(-) S13− EBV than B(+) S13− EBV. Taken together, these findings suggest that the CTCF binding and BART region contribute to EBV 3D genome structure via a cluster of DNA loops formed by BARTp CTCF BS (S13) and are important for coordinated viral gene expression and EBV infectivity.

. Approximately 10% GC has been diagnosed as EBV associated gastric carcinoma 68 (EBVaGC), estimating more than 70,000 cases worldwide per year [7][8][9]. EBVaGC appears 69 lymphoepithelioma-like carcinoma whose definition is an undifferentiated carcinoma with 70 lymphocytic infiltrate, histologically similar to NPC [2,7]. EBV of EBVaGC maintains the 71 type I latency phase and express the narrowest group of EBV latent genes such as EBNA1, 72 EBER, BARTs and sometimes LMP2A. These genes are implicated with the EBVaGC 73 oncogenesis [10]. 74 The EBV genome contains two miRNA cluster that encoded by BamHI fragment H 75 rightward open reading frame 1 (BHRF1) and BamHI A right transcripts (BARTs) [11][12][13][14]. 76 EBV BARTs are a complex miRNA cluster of highly spliced transcripts initially found in 77 NPC EBV strain [15,16]. Some lymphotropic BL EBV strain, like B95-8, have a deletion 78 overlapping the 11.8-kb BART region (139724-151554), while EBV strains derived from GC, 79 such as GC1 and YCCEL1, contain the full BART region [17,18]. BART miRNAs are 80 substantially expressed in EBV infected epithelial cells such as NPC and EBVaGC [19][20][21]. 81 The BART miRNAs are highly implicated in EBV-mediated epithelial malignancies but 82 sometimes dispensable in EBV-mediated lymphoma. Thus, their function in EBV life cycle 83 is only partially elucidated [22]. 84 The maintenance of chromatin structure is also largely dependent on cellular 85 mechanisms that regulate several chromatin interactions exemplified an interaction between 86 enhancer and promoter [23][24][25][26]. The CCCTC-binding factor, also referred to CTCF, is a 87 transcription factor that contains DNA binding domain and 11 zinc fingers. CTCF is involved 88 in other functions such as epigenetic insulator, gene boundary factor and DNA looping maker 89 [27][28][29]. In particular, CTCF is highly associated with regulating long range chromatin 5 90 interaction by chromatin loop organization [30]. Cohesin composed of SMC1, SMC3, and 91 non-SMC components including RAD21, SA1, and SA3 are known to assist in CTCF-92 mediated stabilization of EBV genome structure [31][32][33]. Cohesion binds at multiple control 93 regions of EBV genes and involves in maintain EBV genome structure to regulate EBV gene 94 expression, along with CTCF [34][35][36]. 95 Here, we have identified 16 CTCF binding sites (BS) in the EBV genome in EBVaGC 96 using ChIP-seq analysis against CTCF. Among them, one site (BARTp CTCF BS) located in 97 close proximity to the transcription start site of the 11.8-kb BART region that has not yet been 98 defined for its biological function in EBVaGC. This CTCF binding site, referred to here as 99 the BARTp CTCF BS (S13), exits in most EBV genomes regardless of the existence of 11.8-100 kb BART. Here, we test the hypothesis that the BARTp CTCF BS (S13) is important for 3D 101 conformation of EBV genome, and that the BART transcripts affect this conformation. We 102 further test whether BARTp CTCF BS (S13) regulates EBV gene expression. We find that 7 133 was prepared as followed: 5 ul 4X EMSA buffer (400 mM KCl, 80 mM HEPES, 0.8 mM 134 EDTA, 80% glycerol, pH 8.0) freshly added 1 mM DTT, target DNA (125 nM), 1 ul 500 135 ng/ul sonicated salmon sperm DNA, 2.5 ul ligand protein (CTCF, 3.12 μM, 6.25 μM), and 136 8.5 ul sterile water. Afterward, each mixture was incubated 30 min on ice and subjected to 137 Nanotemper Monolith NT.115 (Munich, Germany) as recommended by manufacturer. To confirm BART region in EBV genome, PCR was performed at BART region 141 using each EBV BART + (B(+)) and EBV BART -(B(-)) S13bacmids. One directional primers 142 were used to avoid the amplication for self-ligation products; primers for PCR products of 143 BART region from EBV B(+) Wt bacmid were used the forward primer. Each 25μl reaction 144 contained 5 μl of EBV B(+) and B(-) Wt bacmid templates, 5 μl of 5× reaction mix 145 (NanoHelix, Korea), 5 μl of 5× TuneUp solution (NanoHelix, Korea), 1 μl of Taq-plus 146 polymerase (NanoHelix, Korea), and 2.5 μl of 10 μM forward/reverse primer. The following 147 cycle conditions were used: 95°C for 3 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 148 72°C for 30 s; followed by 72°C for 10 min. The reactions were performed using a TaKaRa 149 PCR Thermal Cycler (Otsu, Japan) and then run on a 1.5% agarose/TBE gel. following cycles thermal conditions were used: 95°C for 10 min; 45 cycles of 95°C for 10 159 sec, 55°C for 10 sec, and 72°C for 10 sec; 95°C for 5 sec; followed by 65°C for 1 min. In case 160 of necessity, semi-quantitative PCR (sqPCR) was also conducted as previously described [37].

161
PCR products were amplified in a 25 µL reaction solution containing 5 µL of 5× reaction mix, 162 5 µL of 5× TuneUp solution, 1 µL of Taq-plus polymerase, and 2.5 µL of 10 pmol 163 forward/reverse primer. The following cycle conditions were used: 95•C for 3 min; 30 cycles 164 of 95 •C for 10 s, Tm (specific to primer sets) for 30 s, and 72 •C for 30 s; followed by 72 •C 165 for 10 min. The reactions were performed using a TaKaRa PCR Thermal Cycler (TaKaRa,166 Kyoto, Japan) and then run on a 1.2% agarose/TBE gel.

319
These peaks were located in control regions of EBV genes such as upstream or downstream 320 of their transcriptional start sites. One peak, S13 is located close to the BART promoter region S5, and S16 in Fig.1A were characterized in previous studies and implicated in forming 16 338 looping structure [41,43,44]. However, S13 (BARTp CTCF BS) was not previously 339 characterized. To understand the role of CTCF binding at S13, we first validated that S13 340 bound directly to a CTCF protein from Sf9 cells using baculovirus CTCF expression system 341 (Fig. 2B). To validate the specific sequence recognition site for CTCF, we used site-directed 342 mutation to introduce mutations predicted to disrupt CTCF binding to S13 ( Fig. 2A). We 343 conducted microscale thermophoresis (MST) assay to confirm CTCF binding to wild-type 344 (Wt) S13 and mutated (Mt) S13 DNA fragments (Fig. 2C). We observed strong binding of 345 CTCF to Wt S13 DNA fragments that were significantly compromised for binding to Mt S13 346 DNA fragments (Fig. 2C lower panel). As control, total proteins isolated from Sf9 cells 347 showed no strong selectivity in binding to Wt S13 or Mt S13 DNA fragments ( Fig. 2C

352
To examine if BARTp CTCF BS (S13) is required for the formation of DNA 353 looping structure, 4C-seq was conducted at around BARTp CTCF BS in SNU719 cells.

394
As BARTp CTCF BS is located in RPMS1 intron region, an introduction of site-directed 395 mutation in BARTp CTCF BS would not affect the RPMS1 expression (Supplemental Fig.   396 1B). Resultant EBV B(+) S13bacmid from red-recombination was confirmed the site-397 directed mutation in BARTp CTCF BS by Sanger DNA sequencing (Supplemental Fig. 1C).

487
Secondly, we found that functional BARTp CTCF BS (S13) is required to make a stable DNA 488 loop between OriP and LMP1/2. Thirdly, we showed that BARTp CTCF BS (S13) is an 489 important site of transcriptional regulation of EBV genes such as EBNA1 and BZLF1. Finally,

490
we found that functional BARTp CTCF BS (S13) is necessary for a full capacity of EBV transcriptional enhancer of Cp and LMP1 promoter whose mechanism was to use the CTCF-497 mediated DNA loop structure [48]. In the present study, we examined the role of CTCF at the 498 BARTp (S13) using EBV-associated gastric carcinoma cells and EBV infected HEK293 cells. 499 We found that deletion of the BARTp CTCF BS (S13) in B(-) EBV genome resulted in more 500 enrichment of CTCF at CTCF BSs in OriP and LMP1/2, while the deletion in B(+) EBV 501 caused to deprive almost all the CTCF from CTCF BSs in OriP and LMP1/2. In similar 502 context, the chromatin interactions of OriP and LMP1/2 was more severely defected in EBV 503 B(+) S13genome than EBV B(-) S13 -EBV genome due to the differential distribution of 504 CTCF. These results suggested that 11.8-kb BART region could make spatial effects on 505 forming CTCF-mediated DNA loops of OriP and LMP1/2 loci.

506
Taken together, these results suggested that BARTp CTCF BS (S13) can play a 507 complex role in regulating epigenetic modifications at both BART region and its surrounding 508 regions such LMP1/2 locus and OriP locus. our findings indicate that CTCF-mediated chromatin interaction is likely to account for most 539 DNA loops within EBV genomes. It is also possible that EBV association with host 540 chromosome may also contribute to some aspects of EBV chromosome conformation.

541
Although our high-resolution 4C analysis did reveal extensive conformational structure of 25 542 EBV genome during EBV latency in EBVaGC, further studies will be required to resolve 543 some of these more complicated functions of CTCF and to better understand how EBV has 544 exploited CTCF binding sites to confer coordinate gene regulation and genome propagation 545 in latent infection.  ChIP-seq assay were confirmed through the CTCF ChIP-qPCR assay. The ChIP-qPCR was 584 assayed with SNU719 cells using antibody for CTCF. EBV 38,173 primer set was used as 585 negative control. C) Like CTCF ChIP-qPCR assay, cohesin subunits such as SMC1 and 586 SMC3 were also tested their enrichment at CTCF BSs by the cohesin ChIP-qPCR assays. labeled 49-mer primer whose sequence was mutated (Mt S13 primer sets) or not (Wt S13 590 primer sets  using 147K (S14) region as a view primer. EBV genome in SNU719 cells were cut by XhoI 607 restriction enzyme, ligated by T4 DNA ligase, and then purified as described previously.