The CCCTC-binding factor CTCF represses hepatitis B virus enhancer I and regulates viral transcription

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein, CCCTC-binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or epi-somal copies of the viral genome. siRNA knock-down of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.

The HBV genome is transcribed by the host RNA polymerase II (RNA pol II) complex from four promoters (basal core promoter, BCP, Sp1, Sp2 and Xp) (Hong et al., 2017) that results in six major viral RNAs of increasing length with heterogeneous 5 0 ends and a common polyadenylation signal (Stadelmayer et al., 2020). These RNAs include: pre-core (preC) that encodes e antigen (HBeAg); pre-genomic (pgRNA) that is translated to yield core protein (HBcAg) and polymerase; preS1, preS2 and S RNAs encoding the surface envelope glycoproteins and X transcript for the multi-functional x protein (HBx). Two viral enhancers regulate viral transcription: Enhancer I (EnhI) is located upstream of and partially overlaps the X promoter (Xp) and directs BCP activity (Hu & Siddiqui, 1991), which stimulates the production of both preC and pgRNAs. Enhancer II (EnhII) overlaps a large portion of BCP and stimulates activity of the distal Sp1 and Sp2 promoters, as well as the BCP and Xp (Yuh & Ting, 1990). The BCP encodes a negative regulatory element (NRE) that overlaps with Enh II (Sun et al., 2001) and has been reported to repress promoter activity.
Encapsidated pgRNA is reverse-transcribed by the viral polymerase to generate new DNA genomes that can be re-imported to the nucleus to maintain the cccDNA pool or are enveloped and secreted as infectious particles (Urban, Schulze, Dandri, & Petersen, 2010), highlighting an essential role for the BCP in regulating viral replication.
HBV cccDNA is assembled into nucleosomes by cellular histones to form episomal chromatin . The viral DNA is enriched with active epigenetic histone modifications including trimethylation of lysine 4 (H3K4Me3) and acetylation of lysine 27 on histone 3 (H3K27Ac) but devoid of repressive marks, such as trimethylation of lysine 27 on histone 3 (H3K27Me3) (Flecken et al., 2019;Tropberger et al., 2015). The overlap of active histone marks with RNA pol II occupancy suggests that viral transcription is regulated by epigenetic modification. In support of this, treating de novo infected primary human hepatocytes with inhibitors of the histone acetyltransferase p300/CBP reduces HBV RNA levels (Tropberger et al., 2015). Although the mechanisms underlying the epigenetic regulation of HBV cccDNA are not fully understood, several epigenetic modifiers are recruited to HBV cccDNA by HBx. As such, HBx behaves as a transcriptional regulator of both viral and cellular promoters (Guerrieri et al., 2017), and although HBx cannot bind to DNA directly, it can associate with components of the basal transcription machinery, transcription factors and transcriptional coactivators (Tang, Oishi, Kaneko, & Murakami, 2006). HBx coordinates the recruitment of the CBP/p300 and PCAF histone acetyl transferases (HAT) to cccDNA while facilitating the exclusion of histone deacetylases (HDACs) HDAC1 and Sirtuin 1 (Sirt1), resulting in hyperacetylation of cccDNA Chong et al., 2020). HBV transcription is dependent on an array of ubiquitous and liver-specific cellular transcription factors, including the liver-specific hepatocyte nuclear factors, 1 and 4 (HNF-1/4), and ubiquitously expressed octamer-binding protein 1 and specificity protein 1 (reviewed in Oropeza et al., 2020;Turton, Meier-Stephenson, Badmalia, Coffin, & Patel, 2020).
The genomes of metazoans are organised into megabase-sized regions termed topologically associated domains (TADs) that provide regulatory segmentation required for appropriate gene expression and replication. TADs are separated by regions enriched in binding sites of the ubiquitously expressed CCCTC-binding factor (CTCF), which stabilises chromatin loops by anchoring cohesin rings at the base of the loops (Rowley & Corces, 2018). Such spatial organisation can create epigenetic boundaries that separate transcriptionally active and inactive chromatin domains and control cis-regulatory elements, such as transcriptional enhancers. CTCF binds to tens of thousands of either ubiquitous or cell type specific consensus binding sites within the human genome, regulating both tissue-specific and developmental changes in gene expression (Chen, Tian, Shu, Bo, & Wang, 2012).
The occupancy of specific CTCF binding sites is dictated by chromatin accessibility and local epigenetic status (Kumar & Bucher, 2016). In addition to the organisation of chromatin domains, CTCF can function as a transcriptional repressor, or activator, by direct association with promoter proximal elements. CTCF was shown to act as a transcriptional repressor of the c-myc oncogene by creating a roadblock to RNA pol II (Filippova et al., 1996). Conversely, CTCF can physically associate with transcriptional regulators, such as the general transcription factor, TFII-I, to promote recruitment of the cyclin-dependent kinase 8, resulting in stimulation of RNA pol II activity (Pena-Hernandez et al., 2015). CTCF regulates the transcription (up or down) of evolutionarily distinct DNA viruses  including: Kaposi sarcoma-associated herpesvirus; Epstein-Barr virus and herpes simplex virus (Chen et al., 2014;Kang, Wiedmer, Yuan, Robertson, & Lieberman, 2011;Lang et al., 2017;Washington, Musarrat, Ertel, Backes, & Neumann, 2018). We have demonstrated that CTCF recruitment to the human papillomavirus (HPV) genome negatively regulates early promoter usage via host cell differentiation-specific stabilisation of an epigenetically repressed chromatin loop (Paris et al., 2015;Pentland et al., 2018 (Hayer et al., 2013) identified two CTCF binding sites (BS) between nucleotides 1194-1209 in EnhI (CTCF BS1) and 1275-1291 in the Xp (CTCF BS2). Importantly, these binding sites are conserved among all HBV genotypes (Figure 1a). The Hepadnaviridae family includes a number of related viruses that infect other species, including birds, mammals, fish, reptiles and amphibians. Inspection of reference sequences from distinct Hepadnaviridae showed that both consensus CTCF binding sites are conserved in viruses infecting primates and the majority of mammals and bats but are absent from viruses infecting birds, fish or amphibians, demonstrating evolutionary conservation of both CTCF binding sites (Figure 1b).
To assess whether these putative motifs can bind CTCF, we selected two independent lines, HepG2.2.15 (Sells et al., 1987) and HepAD38 (Ladner et al., 1997), that carry integrated copies of HBV genomes, maintain cccDNA and generate infectious virus. We isolated chromatin from nuclear fractions to limit contamination of cytoplasmic rcDNA and performed anti-CTCF chromatin immunoprecipitation (ChIP) followed by quantitative PCR (ChIP-qPCR). Primers were selected to amplify 100-200 base pair regions of the viral genome to provisionally identify CTCF binding sites. We show low-level CTCF binding above the control IgG across the viral DNA with a significant enrichment in the Xp in both cell lines (Figure 2a), consistent with our motif scanning results. These data are in line with earlier reports for CTCF binding cellular target genes . Analysing histone modifications of HBV chromatin purified from HepG2.2.15 cells showed minimal evidence for the repressive H3K27Me3, which is in agreement with previous reports (Flecken et al., 2019;Tropberger et al., 2015) (Figure 2b). In contrast, ChIP for histone marks associating with active transcription, including H4Ac and H3K4Me3, identified these epigenetic marks throughout the viral genome, with an enrichment in the BCP and Xp regions (Figure 2b).
Since both HepG2.2.15 and HepAD38 cell lines carry integrated viral genomes and cccDNA, we are unable to discriminate CTCF binding between these forms of viral DNA. We studied HepG2 cells expressing an episomal copy of HBV DNA (HepG2-HBV-Epi) (Lucifora et al., 2014) to evaluate whether CTCF can bind episomal non- HepG2.2.15 or HepAD38 cells, which may reflect differences in the epigenetic status of the viral DNA in these model systems. Our observation that CTCF binds EnhI, the major transcriptional regulatory element of the BCP and Xp, suggests that CTCF regulates its activity.

| CTCF represses HBV enhancer I
To analyse the role of CTCF in regulating HBV enhancer activity, we used promoter constructs encoding Firefly luciferase under the control of EnhI and Xp (nt 900-1358) or the BCP (nt 900-1859) ( Figure 3a) (Ko, Lee, Windisch, & Ryu, 2014). We silenced CTCF in HepG2-NTCP using an siRNA Smartpool ( Figure 3b) and transfected the viral promoter plasmids along with a Renilla luciferase control plasmid and measured the activity after 72 hr. Transient knock-down of CTCF protein significantly increased HBV EnhI activity ( Figure 3c). To assess whether the putative CTCF directly regulated EnhI, we introduced silent mutations into the pEnhI-Luc to abrogate CTCF binding (Schmidt et al., 2012) suggesting that CTCF represses EnhI but this effect may be blunted in the presence of an NRE and overall reduced activity in the full transcriptional reporter construct ( Figure 3c). Together, these data suggest that CTCF binds to both motifs within EnhI to directly repress its activity.

| Silencing CTCF increases HBV preC/pgRNA levels
To determine the effect of CTCF depletion on viral transcripts, we selected to use the HepG2-HBV-Epi cells as we previously demonstrated CTCF binding to the viral genome in these cells. We confirmed effective knock-down of CTCF at the protein and RNA level 72 hr post-siRNA transfection ( Figure 4a). We observed a significant increase in total HBV transcripts and preC/pgRNA levels after CTCF depletion (Figure 4b,c). To determine whether the observed increase in preC/pgRNA levels was due to an alteration of the HBV epigenome after CTCF depletion, we measured H4Ac modification of viral DNA as this was previously reported to associate with HBV transcription (Pollicino et al., 2006). Silencing of CTCF in HepG2-HBV-Epi cells

| Mutation of CTCF binding sites within HBV enhancer I increases transcription
To demonstrate a direct role for CTCF binding to, and regulating, cccDNA transcription, we utilised the HBV minicircle (mcHBV) F I G U R E 3 CTCF represses HBV Enhancer I activity. (a) Depiction of HBV genome regions cloned upstream of Firefly luciferase in transcriptional reporter plasmids and mutagenesis strategy of CTCF BS1 and BS2 showing viral enhancers, Xp and BCP, and CTCF BS 1 (blue) and CTCF BS 2 (green). (b) Western blot showing depletion of CTCF after siRNA transfection in pEnhI-Luc and pBCP-Luc transfected HepG2-NTCP cells. (c) Firefly luciferase activity normalised to Renilla luciferase expression in HepG2-NTCP cells co-transfected with pGL3-basic, pEnhI-Luc or pBCP-Luc and either scrambled (Scr) or CTCF-specific siRNA duplexes. Normalised luciferase activity in HepG2-NTCP cells transfected pEnhI-Luc containing mutations in CTCF binding site 1 (BS1m) or 2 (BS2m) or a combination of both (BS1/2m). Data shown are the mean ± SEM of three independent repetitions. p values were determined by the Sidak's ANOVA multiple comparisons test. ***denotes p < .001 technology, as a model to study cccDNA transcription and replication (Yan et al., 2017). We mutated CTCF BS1 and BS2 alone or in combination in the mcHBV as described in Figure 3a

| DISCUSSION
In this study, we identified two CTCF-binding motifs within transcription regulatory elements, EnhI and Xp, of the HBV genome. We demonstrate CTCF binding to HBV DNA by ChIP-qPCR in the region of these binding sites using various model systems that bear both integrated genomes and a cccDNA pool, or cells exclusively expressing F I G U R E 4 CTCF represses preC/ pgRNA transcription from HBV cccDNA. (a) HepG2-HBV-Epi cells were untransfected (UT) or transfected with scrambled (Scr) or CTCF-specific siRNA duplexes and incubated for 72 hr. CTCF depletion was assessed by western blotting and quantification in three independent experiments shown. (b) Total viral RNA and (c) preC/pgRNA levels were quantified by qPCR as previously described . Data are the mean ± SD of two independent experiments performed in triplicate. p values were determined by the Kruskal-Wallis ANOVA multiple group comparison. (d) Enrichment of H4Ac marks was assessed by ChIP-qPCR and shown as % Input recovery. p values were determined using a paired t test. *denotes p < .05, **denotes p < .01, ***denotes p < .001 episomal copies of viral DNA. Our sonication method sheared cccDNA-like episomes and demonstrated CTCF binding to EnhI, albeit slightly upstream of the peak enrichment of CTCF binding in the integrant lines. This altered location of CTCF binding could reflect differential usage of CTCF binding sites in the different model systems or could reflect less efficient shearing of cccDNA-like molecules compared to integrated HBV DNA. Nonetheless, our ChIP-qPCR experiments allowed provisional mapping of CTCF binding sites that were confirmed by mutagenesis studies using promoter reporter constructs and mcHBV DNA. Importantly, these CTCF binding sites are conserved among all HBV genotypes and across the wider Hepadnaviridae family, consistent with an evolutionary conserved role in the replication of these viruses. Finally, we show a role for CTCF to repress HBV transcription.
Using several complementary HBV replication models, we show that siRNA depletion of CTCF and mutation of CTCF binding sites significantly increased preC/pgRNA levels, consistent with a role for CTCF in repressing viral transcription. To understand the mechanism of CTCF action, we used transcriptional reporter assays and found that silencing CTCF significantly increased EnhI activity.
Furthermore, mutating the CTCF BS within EnhI attenuated this phenotype, confirming a direct role for CTCF in regulating EnhI.
However, analysis of the full BCP, containing both EnhI and EnhII, revealed that the phenotype of CTCF silencing was lost. It is likely that the attenuation of BCP activity after CTCF silencing is explained by the dominant repressive effects of the NRE within EnhII, highlighting the context-dependent activity of CTCF in regulating HBV. However, increased activity of the BCP is observed after CTCF silencing in cells containing the full viral episome, which may reflect differential chromatinization and epigenetic modification of the transcriptional reporters as compared to the full viral episome. Alternatively, the transcriptional elements in isolation are no longer subject to regulation by distal elements contained within the intact episome. While the transcriptional reporters used in this study provide a useful tool in the initial characterisation of CTCF function in HBV EnhI modulation, the results obtained in the context of a chromatinised viral episome may better reflect the role of CTCF in the HBV infection cycle.
To confirm a direct role of CTCF in repressing HBV transcription, we transfected HepG2-NTCP cells with mcHBV mutated in the CTCF BSs. Although the extent to which we could mutate CTCF BS was limited, to maintain the amino acid sequence of the polymerase, we observed a significant reduction of CTCF binding to mcHBV, lacking either BS1 or BS2, or both sites mutated in combination. These studies identify CTCF BSs within the viral genome and confirm CTCF association with HBV DNA. Consistent with the increased preC/pgRNA levels observed in two HBV replication model systems after CTCF depletion, we observed a significant increase in preC/ pgRNA when CTCF BS1 was mutated. A similar increase in preC/ pgRNA was observed when CTCF BS2 was mutated, although this did not reach statistical significance. While the mutation of both BS showed a significant increase in preC/pgRNA abundance, suggesting F I G U R E 5 CTCF represses HBV preC/pgRNA transcription in de novo infected HepG2-NTCP cells. (a) HBV infected HepG2-NTCP were transfected with scrambled (Scr) or CTCF-specific siRNA duplexes and cultured for 72 hr and CTCF depletion assessed by western blotting. (b) Total HBV transcript abundance, (c) preC/pgRNA levels and (d) the relative proportion of individual HBV transcripts were analysed by PCR as previously described . Data are the mean ± SD of two independent experiments performed in triplicate. p values were determined using the Mann-Whitney U test (two group comparisons). *denotes p < .05, **denotes p < .01 these sites do not function in a synergistic manner within this model system.
Aberrant reverse transcription of pgRNA can generate doublestranded linear DNA that can integrate into the host genome (Tu, Budzinska, Shackel, & Urban, 2017). This integration step is not part of the productive HBV life cycle and occurs at a low frequency (<1 copy per diploid host genome in infected tissues) (Podlaha et al., 2019). Since integrated copies of HBV DNA generally lack a functional basal core promoter and associated CTCF binding sites, we would anticipate a minimal role for CTCF in directly regulating integrant derived transcripts. HBV integration can cause host genomic instability leading to tumour progression through tumour suppressor gene inactivation and/or oncogene activation (Zhao et al., 2016). Oncogenic integration events are thought to provide a growth advantage to cells, inducing tumourigenesis. HBV integration occurs at random sites, although a preference for integration within regions of open chromatin has been reported (Furuta et al., 2018). It will be interesting to determine whether integration of HBV DNA into the host results in an alteration of local chromatin interactions and host cell gene regulation by the insertion of a virally encoded CTCF binding site(s), as reported for the human retrovirus, HTLV-1 (Melamed et al., 2018). Such genomic rearrangements could have an impact on host cell gene expression and contribute to HBV-driven carcinogenesis.
Analysing the epigenetic status of HBV DNA in HepG2.2.15 hepatoma cells revealed a lack of the repressive H3K27Me3 and enrichment of epigenetic marks associated with active transcription in the BCP and Xp regions, downstream of the CTCF binding sites. We noted a similar enrichment of H4Ac in episomal DNA in HepG2-HBV-Epi cells. These findings are consistent with previous reports studying the epigenetic status of HBV cccDNA in various model systems and liver biopsy samples (Flecken et al., 2019;Tropberger et al., 2015).
Silencing of CTCF resulted in an increase in H4Ac abundance in HBV cccDNA, which associates with increased HBV preC/pgRNA levels.
CTCF has been reported to directly repress transcription via recruitment of the Sin3/histone deacetylase compressor complex resulting in reduced histone acetylation (Lutz et al., 2000) that may explain these observations. Taken together, these findings suggest that CTCF represses HBV transcription by insulating the BCP from the upstream enhancer element. EnhI is an important regulator of all HBV promoters and is essential for viral transcription (Hu & Siddiqui, 1991).
The repression of EnhI by CTCF is likely to have a significant impact on the virus life cycle and reduce particle genesis and may therefore limit cccDNA pools. To assess whether infection perturbs CTCF expression, we analysed a publically available Affymetrix microarray database from chronic HBV infected patients . We observed comparable CTCF transcript levels in normal and chronic HBV infected samples ( Figure S1) with no evidence for HBV infection to perturb intra-hepatic CTCF transcript levels.
Analysis of the genomic distribution of CTCF BS in the human genome suggests a similar enhancer-blocking activity of CTCF as numerous CTCF binding loci are situated between known transcriptional enhancers and associated promoter elements (Xie et al., 2007). Such enhancer-blocking activity has been extensively characterised at imprinted loci, such as the insulin-like growth factor 2 (IGF2)/H19 locus and in development at the β-globin locus (Bell, West, & Felsenfeld, 1999;Chung, Whiteley, & Felsenfeld, 1993).
CTCF regulates herpes simplex virus differential transcriptional programmes during the lytic and latent phases of the viral life cycle through its enhancer-blocking activity (Washington et al., 2018).
Our previous work with HPV showed that CTCF repressed transcription by stabilising an epigenetically repressed chromatin loop between the viral proximal enhancer and a distal CTCF binding site.
However, this repression was not associated with direct binding of CTCF to the HPV enhancer, suggesting that HBV and HPV have evolved fundamentally different mechanisms of CTCF-dependent transcriptional repression.
F I G U R E 6 Mutation of CTCF binding sites in HBV mcDNA results in increased preC/pgRNA levels. (a) HepG2-NTCP cells were transfected with wild type HBV mcDNA (WT) or mcDNA with CTCF binding 1 (BS1m) or 2 (BS2m) or both sites mutated in combination (BS1/2m). (b) Cells were harvested 72 hr post transfection and CTCF binding analysed by ChIP-qPCR and presented as % of enrichment relative to input chromatin (% input). (c) preC/pgRNA and levels were quantified by qRT-PCR and normalised to cccDNA amount per cell and (d) total HBV DNA levels were quantified by qRT-PCR and normalised to cccDNA amount per cell to determine mcHBV transfection efficiency. Data are the mean ± SEM of at least three independent experiments. p values were determined using the Kruskal-Wallis ANOVA multiple group comparison. *denotes p < .05, **denotes p < .01 4 | EXPERIMENTAL PROCEDURES

| ChIP and quantitative PCR
HepG2.215, HepAD38 cells or HepG2-HBV-Epi cells were fixed with 1% formaldehyde (Sigma Aldrich) for 10 min at room temperature before quenching with 125 mM glycine. The cells were washed with ice cold PBS, containing EDTA-free protease inhibitors (Roche) and 5 mM sodium butyrate, and frozen at −80 C. Pellets were resuspended in ChIP lysis buffer (Active Motif) supplemented with protease inhibitors and incubated on ice for 30 min. Cells were dounced 30 times using the tight pestle to release nuclei and centrifuged at 2500g for 10 min at 4 C. The supernatant was removed and discarded. Nuclei were re-suspended in shearing buffer (Active Motif) pulse sonicated using a Sonics Vibra Cell CV18 sonicator fitted with a micro-probe at 25% amplitude for 15 min on ice using 30 s on/off cycles. Chromatin samples were cleared by centrifugation and stored at −80 C.
Sonication of HBV cccDNA was evaluated by conventional PCR amplification of increasing amplicon size using a constant sense primer and anti-sense primers described in Table 1 For ChIP, sonicated lysates were clarified by centrifugation at 16,000g for 10 min and CTCF or histone complexes immunoprecipitated with 5-8 μg antibody using a ChIP-IT® Express Chromatin Immunoprecipitation kit, including Protein A magnetic beads as per manufacturer's instructions (Active Motif, USA). The input and immunoprecipitated DNA were quantified by real-time PCR using a Stratagene MX3500P PCR System. The values were calculated as % recovery respective to input DNA signals. All oligonucleotide sequences are listed in Table 1.

| siRNA transfection
Cells were trypsinized to reverse transfect with 25 nM of CTCFspecific or scrambled TARGETplus Smartpool siRNAs (Horizon, USA) using DharmaFECT4 (20% of amount recommended by the manufacturer's protocol; ThermoFisher). Cells with no siRNA (un-treated; UT) were also assayed to assess lethality of CTCF depletion.

| SDS-PAGE and western blots
Cells were lysed in urea lysis buffer (8 M urea, 150 mM NaCl, 20 mM Tris, pH 7.5, 0.5 M β-mercaptoethanol) supplemented with protease inhibitor cocktail (Roche) and sonicated for 10 s at 20% amplitude using a Sonics Vibra Cell sonicator fitted with a micro-probe. Following quantification of protein concentration by Bradford assay, samples were diluted in Lameli buffer before incubating at 95 C for 5 min. Proteins were separated on a 10% polyacrylamide gel and transferred to PVDF membranes (Amersham). The membranes were blocked in TBS-T, 5% skimmed milk, and proteins were detected using specific primary (diluted at 1:1000) and HRP-secondary antibodies (ThermoFisher, diluted at 1:10,000). Protein bands were detected using Pierce SuperSignal West Pico chemiluminescent substrate kit (Pierce) and images were collected using a Fusion FX Imaging system (Peqlab).

| HBV de novo infection
Purified HBV was produced from HepAD38 cells as previously reported (Ko et al., 2018). HepG2-NTCP cells were seeded on collagen-coated plasticware and infected with HBV at an MOI of 250 genome equivalents per cell in the presence of 4% PEG 8,000.

| HBV mcDNA purification and transfection into cells
The plasmid, pMC-HBV, contains the 1.0 HBV genome (awy) and has been previously described (Yan et al., 2017). CTCF BS1 and CTCF BS2 were mutated by site-directed PCR mutagenesis using the primers detailed in Table 1 (Testoni et al., 2019). Serial dilutions of a plasmid containing an HBV monomer (pHBV-EcoRI) served as quantification standard for total HBV DNA and cccDNA.
The number of cellular genomes was determined by using the β-globin TaqMan assay Hs00758889_s1 (ThermoFisher). preC/pgRNA was quantified using the following primers and probe: forward inhibitor (Roche Diagnostics) and 1 mM PMSF and pre-cleared for 2 hr at 4 C by adding magnetic Protein G Dynabeads (Life Technologies). Beads were discarded and 1 μg of anti-CTCF antibody (Diagenode #C15410210) or isotype-matched negative control were added to the chromatin. After an overnight incubation at 4 C, magnetic Protein G Dynabeads and samples were incubated for 2 hr at 4 C with agitation. Beads were washed five times with RIPA buffer, once with TE buffer and re-suspended in Elution buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 μg/ml proteinase K). Chromatin was reverse cross-linked by incubation at 68 C for 2 hr and purified by phenol:chloroform:isoamyl alcohol 25:24:1 (Life Technologies) extraction and ethanol precipitation. cccDNA was quantified using the primers and probes listed above (Testoni et al., 2019).