Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Apr 10.
Published in final edited form as: Cell Rep. 2024 Feb 27;43(3):113888. doi: 10.1016/j.celrep.2024.113888

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA

Xiang Ye 1, Lindsey N Guerin 2, Ziche Chen 1, Suba Rajendren 1, William Dunker 1, Yang Zhao 1, Ruilin Zhang 1, Emily Hodges 2,3,4, John Karijolich 1,2,3,5,6,7,*
PMCID: PMC11005752  NIHMSID: NIHMS1980926  PMID: 38416644

SUMMARY

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

Graphical Abstract

graphic file with name nihms-1980926-f0001.jpg

In brief

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus. Ye et al. uncover a role of the KSHV tethering protein LANA in the regulation of cellular gene expression through the manipulation of enhancer biology.

INTRODUCTION

The limiting confines of the cell nucleus necessitate an elaborate and dynamic higher-order structure of the eukaryotic genome. Ultimately, this hierarchical organization proceeds in a manner and scale that functionally relate to gene expression. For example, within this framework operate enhancers, which have primarily been defined by their ability to activate promoters independent of distance and orientation. Indeed, enhancers are frequently separated by large genomic distances from their associated promoters and therefore must be brought into proximity with the target promoter via chromatin looping to influence gene expression. The closest promoter is frequently not the target of an enhancer, and, coupled with the great distances that can exist between functional enhancer-promoter pairs, accurate assignment of these relationships is challenging.1–20 However, application of methods for assaying chromatin interactions, including extensions of the chromosome conformation capture (3C) assay, such as Hi-C/HiChIP, can resolve this.21 Moreover, histone modifications, such as histone H3 lysine 4 monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac), have been proposed to mark enhancers, and H3K27ac HiChIP has been used to assign active enhancer-promoter pairs.22–24

The juxtaposition of two genomic regulatory sequences, such as enhancers and promoters, is reminiscent of the genomic arrangements that occur during latent infection with the human DNA tumor viruses Epstein-Barr virus (EBV), human papillomavirus (HPV), and Kaposi sarcoma-associated herpesvirus (KSHV).25 During latent infection, the viral DNA is maintained as multicopy covalently closed circular episomes that are physically tethered to the host chromatin. This juxtaposition of host and viral DNA is facilitated by virus-encoded proteins that anchor viral DNA to the host genome. For example, HPV E2, EBV EBNA1, and KSHV latency-associated nuclear antigen (LANA), which are structural and functional orthologs, mediate episome maintenance.26–38 Therefore, the ability to bring two distant, in fact physically separate, genomic sequences into proximity with one another is hard wired into the genomes of many viruses. However, whether virus-encoded tethering proteins participate in inter- or intra-host chromosomal looping, such as enhancer-promoter pairing, is not known.

KSHV is the etiological agent of several malignancies, including Kaposi sarcoma and a highly aggressive B cell lymphoma, primary effusion lymphoma (PEL).39–44 KSHV episome tethering is mediated by the multifunctional virus-encoded LANA protein.28 The mechanism of LANA-mediated tethering is still being defined; however, both the N- and C-terminal domains of LANA are important, with the N terminus binding an acidic pocket formed by chromosomal histones H2A/H2B and the C terminus engaging in sequence-specific interactions within the terminal repeats of the viral episome.45–50 In addition, cellular chromatin binding proteins, including BRD2, BRD4, and the ChAHP complex proteins CHD4 and ADNP, have been proposed to participate in tethering.51–53 Interestingly, many of these factors, including LANA, have also been demonstrated to possess a transcriptional regulatory property at various RNA polymerase II (RNAPII) transcribed units.54–58 However, whether this involves integration of the ability of LANA to juxtapose two distinct genomic elements is not known.

Here, by comprehensively profiling the genome-wide occupancy of LANA as well as the epigenomic landscape of KSHV-infected PEL cells, we discover a role of LANA in host intra-chromosomal looping and the regulation of enhancer-target gene expression. LANA binding is significantly enriched on enhancers and is associated with higher H3K27ac levels and enhancer RNA (eRNA) synthesis. LANA HiChIP revealed its participation in 3D genome organization and identified promoter targets of LANA-occupied enhancers to be enriched in genes and pathways important for latency and tumorigenesis. Consequentially, knocking down LANA with CRISPRi reduced RNAPII recruitment at LANA occupied enhancers and target gene promoters and, thus, decreased nascent RNA synthesis of the target genes. These results reveal the ability of a viral tethering protein to participate in chromatin looping and the regulation of enhancer-promoter activity. Moreover, our results suggest means for other episomal viruses to similarly co-opt distal gene-regulatory elements and facilitate their juxtaposition via virus-encoded tethering proteins.

RESULTS

Epigenetic profiling reveals enrichment of LANA on cellular gene-regulatory sequences

LANA interacts with several chromatin and transcription regulatory complexes and has also been demonstrated to impact expression of various RNAPII genes. However, the mechanisms by which LANA regulates gene expression are not well defined. To investigate the mechanism of LANA-mediated transcriptional control, we generated high-resolution genome-wide occupancy maps of LANA in two PEL cell lines, TREx-BCBL1-RTA and BC-3, by cleavage under targets and release using nuclease (CUT&RUN) (Figure 1A).59–61 Although LANA engaged a variable number of target sites in the two cell lines, the genomic distribution of binding was concordant and indicated that ~40% of LANA binding occurred within promoter regions. In addition, In both TREx-BCBL1-RTA and BC-3 cells, we observed a collective ~50% of LANA bound within intronic regions and intergenic space, defined as more than 2 kb upstream of annotated transcription start sites (TSSs) (Figure 1A). Profiling of the accessible chromatin and DNA methylation status by ATACme revealed LANA binding to be highly correlated with open chromatin and a hypomethylated state (Figure 1B).62 The accessibility and hypomethylation coupled with the enrichment of LANA binding in genomic locations known to harbor gene-regulatory sequences supports the notion that, in addition to viral genome tethering, LANA has the potential to regulate host gene expression.

Figure 1. LANA is enriched on cis-regulatory elements.

Figure 1.

Open in a new tab

(A) Distribution of the LANA signal in TREx-BCBL1-RTA and BC-3 cells.

(B) Chromatin accessibility and DNA methylation profiled by ATACme at LANA peaks.

(C) Heatmap of LANA, H3K27ac, H3K4me1, H3K4me3, and H3K27me3 levels at LANA binding sites.

(D) Aggregation of histone modifications at LANA peaks.

(E) Unsupervised clustering of LANA peaks based on the four histone modifications.

(F) Aggregation of LANA and active histone modification CUT&RUN signals at promoter and non-promoter LANA binding sites.

To further define the environment of genome-bound LANA, we profiled chromatin modifications, including H3K4me1 (enhancers), H3K4me3 (promoters), H3K27ac (active gene-regulatory features), and H3K27me3 (silenced or poised chromatin) in TREx-BCBL1-RTA cells. Consistent with previous studies, we observed strong H3K4me3 and H3K27ac signals near LANA peaks, while H3K27me3 was absent (Figures 1C and 1D). Unexpectedly, we also observed a robust H3K4me1 signal near LANA, suggesting its presence at enhancer elements.

As LANA is a multifunctional protein with known roles in the regulation of transcription and episome tethering, we speculated that it may reside in distinct epigenetic environments corresponding to its different functions. To test this, we performed k-means clustering of host LANA peaks using the histone modifications H3K27ac, H3K27me3, H3K4me3, and H3K4me1 (Figure 1E). The clustering results revealed 5 distinct epigenetic signatures associated with LANA binding. Clusters 3–5 were associated with an undistinguishable epigenetic profile, and, thus, no further analyses were performed on them. However, in contrast, both clusters 1 and 2 exhibit a robust H3K27ac signal with segregation of the H3K4me1 and H3K4me3 signals (Figure 1E). The presence of both H3K27ac and H3K4me3 in cluster 1 supports its associations with promoters, while the presence of both H3K27ac and H3K4me1 in cluster 2 suggests active enhancers.

To further investigate the association of LANA with enhancers, we separated LANA peaks based on their genomic annotations into promoter (2 kb up or downstream of a TSS) and non-promoter sites (all other sites). Aggregation analysis of H3K27ac, H3K4me3, and H3K4me1 signals on these two subsets demonstrates that, while H3K27ac levels were similar, the promoter cluster displayed higher H3K4me3 compared with non-promoter sites (Figure 1F). In contrast, the level of H3K4me1 is increased at non-promoter sites. These analyses are consistent with defining cluster 1 as promoters and cluster 2 as enhancers. Collectively, these analyses provide evidence that LANA is bound to active enhancer elements.

LANA-bound enhancers exhibit higher activity compared with enhancers without LANA

To investigate a potential role of LANA in enhancer biology, we next sought to define the enhancer landscape of TREx-BCBL1-RTA and BC-3 cells. We classified active enhancers in TREx-BCBL1-RTA and BC-3 cells as non-promoter regions with both H3K27ac and H3K4me1 peaks (false discovery rate [FDR] < 0.05) and identified 11,386 and 8,507 enhancers, respectively (Figures 2A and 2B). Intersection analysis of the LANA-bound regions with the enhancers revealed that 159 and 390 of the LANA peaks are present at enhancers in the two cell lines. We tested the statistical significance of this overlap using Fisher’s exact test and determined that LANA binding to enhancers was eight and 20 times more than expected, respectively, and was highly significant in both cell lines (p < 1e–100) (Figures 2C and 2D). Interestingly, LANA-bound enhancers are enriched for motifs of transcription factors known to play a role in KSHV biology (Figure S1).

Figure 2. LANA is enriched on enhancers with higher activity.

Figure 2.

Open in a new tab

(A) Heatmap of H3K27ac and H3K4me1 signals on enhancers in TREx-BCBL1-RTA cells.

(B) Heatmap of H3K27ac and H3K4me1 signals on enhancers in BC-3 cells.

(C) Fisher’s exact permutation test of LANA peaks that overlap enhancers in TREx-BCBL1-RTA cells.

(D) Fisher’s exact permutation test of LANA peaks that overlap enhancers in BC-3 cells.

(E) H3K27ac signal intensity in TREx-BCBL1-RTA cells, ranked by ROSE.

While individual enhancers play an important role in gene transcription, they often form clusters generating what are referred to as super-enhancers. Therefore, we investigated LANA binding to super-enhancers. We identified super-enhancers using the ROSE algorithm, where neighboring (distance < 12.5 kb) enhancer elements based on H3K27ac CUT&RUN were merged and ranked according to H3K27ac signal, and top ranked regions were designated as super-enhancers (Figures 2E and 2F). Intersection analyses and Fisher’s exact test demonstrated that LANA binding was significantly enriched on super-enhancers (Figures 2G and 2H).

LANA interacts with multiple chromatin binding proteins that have been implicated in enhancer regulation, including BRD2, BRD4, EZH2, IRF4, and CHD4.51,53,63–69 To test whether LANA occupancy overlaps with their binding, we generated CUT&RUN data in TREx-BCBL1-RTA cells for each factor, with the exception of CHD4, in which we analyzed published chromatin immunoprecipitation sequencing (ChIP-seq) data generated in TREx-BCBL1-RTA cells.51 Aggregation of the EZH2 and BRD2 CUT&RUN signal revealed minimal overlap. In contrast, a robust signal for BRD4, IRF4, and CHD4 was observed at LANA peaks (Figure 2I). We next tested whether these factors were differentially present at enhancers occupied by LANA. Segregation of enhancers by the presence of LANA and aggregation of the BRD4, IRF4, and CHD4 signals within the two clusters demonstrated that LANA-bound enhancers display increased signals of all three factors (Figure 2J). These results demonstrate that chromatin- and enhancer-regulatory factors exhibit increased occupancy at enhancers bound by LANA.

H3K27ac levels and eRNA synthesis have been shown to be predictive of their effect on target gene expression.24,70 To investigate the impact of LANA on enhancer activity, we segregated enhancers into two categories based on LANA occupancy and quantified H3K27ac and H3K4me1 levels. While the level of H3K4me1 was similar between the two groups, H3K27ac levels were significantly higher when LANA was present (Figure 2K). Moreover, quantification of published latent TREx-BCBL1-RTA GRO-seq data revealed increased eRNA synthesis at enhancers bound by LANA compared with non-LANA-bound enhancers (Figure 2L). Together, these analyses demonstrate that the presence of LANA at an enhancer is associated with its increased activity.

Distribution of episome tethering sites proximal to LANA-bound enhancers

Recent work leveraging capture Hi-C to investigate the sites of KSHV tethering have revealed an overlap between CHD4 binding and sites of episome tethering.51 The presence of CHD4 at LANA-bound enhancers prompted us to investigate whether viral episomes tether in proximity to enhancers. KSHV genome tethering is mediated through the viral terminal repeats, which span at least 40 kb in size. Thus, the chimeric reads from the KSHV genome to the host are dispersed across great distances, rending the ability to call discreet sites of tethering obsolete and preventing intersection analyses. Therefore, taking advantage of the capture Hi-C data, we separated LANA peaks into two subtypes, enhancer and non-enhancer sites, and quantified the capture Hi-C KSHV-host chimeric reads within a 500-kb window. Using this approach, we observed a clear signal for episome tethering at both peak subtypes (Figure 3A). However, the signal proximal to enhancers is more intense, suggesting that a greater number of episomes are tethered near enhancers (Figure 3B). However, there is not a strict requirement for LANA to tether episomes proximal to enhancers.

Figure 3. Distribution of episome capture Hi-C signals proximal to LANA-bound enhancers.

Figure 3.

Open in a new tab

(A) Aggregation (top) and heatmap (bottom) of episome capture HiC signals on LANA peaks in TREx-BCBL1-RTA cells.

(B) Quantification of episome capture HiC signals at LANA peaks segregated on enhancer overlap status.

LANA is associated with enhancer-promoter interactions

While the 1D landscape of LANA binding demonstrates its enrichment on enhancer sequences, these data do not inform on whether LANA participates in intra-chromosomal looping events, such enhancer-promoter pairing. To test this, we determined the LANA-centric chromatin interactions by HiChIP (Figure 4A). LANA HiChIP libraries were prepared from latent TREx-BCBL1-RTA and BC-3 cells in triplicate, and we generated more than 100 million paired-end reads for each replicate (Figures 4B and S2A). The libraries exhibited high 1D signal enrichment at enhancers and promoters and globally recapitulated LANA CUT&RUN peaks (Figures 4C and S2B). Moreover, LANA HiChIP anchors were associated with chromatin that displayed high H3K27ac, H3K4me1, and H3K4me3 CUT&RUN signals (Figure 4D).

Figure 4. LANA physically associates with promoter-enhancer interactions.

Figure 4.

Open in a new tab

(A) Schematic of the LANA HiChIP experiment.

(B) Statistics of LANA HiChIP chimeric reads.

(C) Aggregation of LANA CUT&RUN signals on LANA HiChIP loop anchors.

(D) Aggregation of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 signals on LANA HiChIP loop anchors.

(E) Annotation of LANA HiChIP loops. Enhancer and promoter annotations were downloaded from the Encode project (ENCFF535MKS_3.bed and ENCFF379UDA_3.bed). Distal refers to non-enhancer and non-promoter anchors.

(F) Table of top enriched MSigDB Hallmark terms for LANA enhancer looped genes.

(G–J) IGV view of LANA HiChIP loops on CCND2, LEF1, MYC, and IL-10 together with H3K27ac and H3K4me1 CUT&RUN and ATAC signals.

Three types of chimeric reads were observed; namely, virusvirus, host-virus, and host-host (Figures 4B, S2A, S2B, and S3A). Examination of the KSHV genome revealed a similar pattern of LANA-mediated 3D genome organization in both cell lines (Figure S3A). While the host-virus reads could provide insight into the location of KSHV tethering, the number of reads within this class are not sufficient for tethering site analysis as in the capture Hi-C data.

Analysis of host-host chimeric reads identified 618 and 1,063 significant interactions in TREx-BCBL1-RTA and BC-3 cells, respectively (Figures 4E and S2C). Annotation of TREx-BCBL1-RTA LANA HiChIP anchors reveals that ~50% of LANA-centric loops are enhancer-enhancer interactions, with another ~30% percent of the loops describing promoter-enhancer interactions. Thus, more than 80% of the identified LANA HiChIP interactions are among enhancers and promoters in TREx-BCBL1-RTA cells (Figure 4E). Although we identified an additional ~400 loops in BC-3 cells, there were fewer interactions among enhancers and promoters (~30%) (Figure S2C). This suggests that LANA may bind additional chromatin with a different landscape in BC-3 cells relative to TREx-BCBL1-RTA cells. However, importantly, the ontological associations of LANA-bound enhancer target genes were similar in both cell lines and revealed an enrichment of pathways important for latency and PEL cell survival (Figures 4F and S2D). For example, prominent LANA HiChIP contacts are present in genes associated with wnt-β-catenin signaling, including at the MYC, CCND2, LEF1, and interleukin-10 (IL-10) loci (Figures 4G–4J and S2D). These data reveal the ability of LANA to participate in intrachromosomal looping. Moreover, HiChIP revealed the presence of LANA on enhancers that drive the expression of genes important for tumorigenesis.

LANA regulates enhancer-target gene expression

To determine the impact of LANA on enhancer and target gene expression, we identified all enhancer-promoter pairs using published H3K27ac HiChIP data generated in BCBL1 cells and separated the pairs into two categories based on LANA HiChIP signal (Figure 5A).63 We quantified RNA expression of each pair by generating RNA sequencing (RNA-seq) data as well as analyzing published GRO-seq data (Figure 5A).71,72 In all datasets, we observed that LANA enhancer-promoter pairs are associated with higher RNA expression than non-LANA enhancer-promoter pairs (Figure 5A). Moreover, a similar trend was observed in RNA-seq data when we compared gene expression between LANA-bound and non-LANA bound enhancer-promoter pairs with matched H3K27ac levels (Figure S4). However, the difference was not as robust, suggesting that, while H3K27ac levels play a significant role, LANA may impact additional features of enhancer biology, such as enhancer-promoter interactions.

Figure 5. LANA regulates enhancer-target gene expression.

Figure 5.

Open in a new tab

(A) Boxplot of transcriptomic profiling by GRO-seq and RNA-seq for the genes looped with LANA positive and negative enhancers.

(B) Western blot of LANA protein level in LANA knockdown BCBL1 cells.

(C) Aggregation of LANA CUT&RUN coverage in nontarget control (NTC) and LANA-specific CRISRPi BCBL1 cells.

(D) Aggregation of H3K4me1 and H3K27ac CUT&RUN coverage on enhancers with or without LANA knockdown BCBL1 cells.

(F) H3K27ac signal intensity in BC-3 cells, ranked by ROSE.

(G) Fisher’s exact permutation test of super-enhancers in (E) with LANA peaks in TREx-BCBL1-RTA cells.

(H) Fisher’s exact permutation test of super-enhancers in (F) with LANA peaks in BC-3 cells.

(I) Aggregated signal of BRD4, BRD2, EZH2, IRF4, and CHD4 CUT&RUN on LANA peaks in TREx-BCBL1-RTA cells.

(J) Aggregated signals of BRD4, IRF4, and CHD4 on enhancers with or without LANA.

(K) Aggregated signals of H3K27ac and H3K4me1 on LANA with or without LANA occupancy.

(L) Boxplot of quantified GRO-seq signal on enhancers with or without LANA.

The p values for LANA overlapping enhancers were calculated by the Fisher’s exact test function provided in Bedtools to test significance of overlap between two sets of genomic regions.

(E) Aggregation of BRD4 and IRF4 CUT&RUN coverage in NTC and LANA-specific CRISRPi BCBL1 cells.

(F) Boxplot of RNAPII S2p and S5p log-transformed CUT&RUN coverage changes in LANA knockdown cells on enhancers with or without LANA.

(G) Aggregation of RNAPII CTD S2p and S5p CUT&RUN coverage changes around TSS of LANA+/− enhancer looped genes in LANA knockdown BCBL1 cells.

(H) Boxplot quantification of gene expression by RNA-seq after LANA knockdown by CRISPRi in BCBL1 cells.

(I) Quantification of nascent pre-mRNA by RT-qPCR for CCND2 and LEF1. Error bars represent standard error.

(J) Quantification of enhancer-promoter contacts at CCND2 and LEF1 by 3C-qPCR in the indicated cell lines. Error bars represent standard error.

The p values were determined by Wilcoxon rank-sum and signed-rank tests for GRO-seq, RNA-seq, and CUT&RUN data analysis and by Student’s t test for RT-qPCR data analysis. NS, not significant. **p < 0.01, ***p < 0.001.

To test whether the effect of LANA on enhancers can be functionally perturbed, we investigated enhancer activity in previously described BCBL1 cells expressing non-target or LANA-specific CRISPRi guide RNAs and performed CUT&RUN for LANA, H3K27ac, and H3K4me1.73 In LANA knockdown cells, we observed a significant reduction of LANA at all occupied sites; however, as ~25% of LANA is still present, there is residual LANA occupying the genome (Figures 5B, 5C, S5A, and S5B). Importantly, upon LANA knockdown, we did not observe lytic reactivation (Figures S5C–S5F). While the depletion of LANA did not affect the levels of H3K4me1, we observed a reduction of H3K27ac at LANA-bound enhancers, indicating a reduction in their activity (Figure 5D).

In addition to increased H3K27ac signal at LANA-bound enhancers the levels of BRD4, IRF4, and CHD4 are also elevated (Figure 2J). Therefore, we generated CUT&RUN for these factors in non-target and LANA-specific CRISPRi cells. Multiple attempts at CHD4 CUT&RUN proved unsuccessful, thus we analyzed BRD4 and IRF4 levels. While we observed a minor reduction in BRD4 occupancy at LANA-bound enhancers in LANA-CRISPRi cells, the levels of IRF4 were not affected, suggesting that the presence of IRF4 at LANA occupied enhancers is not sufficient for enhanced expression (Figure 5E).

To further define the mechanism by which LANA facilitates increased enhancer-mediated gene expression, we profiled the genome-wide occupancy of RNAPII as well as its C-terminal domain (CTD) phosphorylation status at positions serine-2 (S2p) and serine-5 (S5p), which are associated with transcription elongation and initiation, respectively. Notably, LANA knockdown resulted in the specific reduction of RNAPII S5p occupancy at previously bound LANA enhancers (Figure 5F). Furthermore, consistent with what we observed at enhancers, depletion of LANA resulted in a prominent decrease in RNA polymerase CTD S5p at promoters of LANA-bound enhancer-promoter pairs, whereas S5p was only minimally reduced at non-LANA-bound enhancer-promoter pairs (Figure 5G). The minimal reduction of S5p did not affect expression of non-LANA-bound enhancer-promoter pairs (Figure 5H). The levels of S2p were unaffected (Figure 5G).

The specific reduction of S5p at LANA-bound enhancers and their linked genes suggested that the presence of LANA facilitates increased gene expression that is mediated at or upstream of transcription initiation. To test this, we quantified nascent RNA production using 4-thiouridine (4sU) pulse labeling and purification coupled to RT-qPCR. The expression of CCND2 and LEF1, which are associated with LANA-occupied enhancers, was significantly reduced when LANA was silenced (Figure 5I). Moreover, we observed a significant reduction in enhancer-promoter contacts at CCND2 and LEF1 by 3C-qPCR when LANA expression was reduced (Figure 5J). Nascent RNA synthesis and enhancer-promoter contacts were not affected at enhancer-promoter pairs that lack LANA (Figure S6). Collectively, these data demonstrate that LANA is a critical regulator of gene expression through promoter-enhancer interactions.

DISCUSSION

The 3D organization of the human genome occurs in a manner and scale that functionally relates to gene expression. The juxtaposition of distinct and sometimes physically separate nucleic acids lies at the heart of this organization. A prominent example of this is enhancer-dependent gene expression, where chromatin looping brings an enhancer into proximity with its target promoter. While functionally distinct, many viruses are faced with a situation where they must maintain a non-integrating genome in cells with replicative capacity and accomplish this through encoding a protein that tethers the viral genome to host chromatin. Thus, the ability to bring two physically distinct nucleic acids together is hard wired in the genomes of many viruses. However, whether viral tethering proteins are similarly able to participate in other intra- or inter-chromosomal interactions is unclear. Here, by combining epigenetic and genomic 3D structure profiling, we discover a role of LANA in intra-chromosomal communication through a role in enhancer-dependent gene expression.

Enhancer biology has emerged as an important aspect of viral oncogenesis. EBV is a related human oncogenic gammaherpesvirus, and in the context of EBV-mediated transformation, four virus-encoded proteins, together with components of the nuclear factor kB transcription factor complex, are present at several super-enhancers.74–77 Suggestive of a role for the viral proteins in super-enhancer control, inactivation of one viral protein, EBNA2, results in a decrease in expression of genes involved in B cell growth and survival.78,79

Enhancers have recently been demonstrated to be important during KSHV infection as well. For example, enhancer activity appears to be globally reduced during KSHV lytic reactivation.71 Moreover, H3K27ac HiChiP experiments in PEL revealed its enhancer connectome, and enhancer-promoter modules that are important for PEL cell viability were discovered.63 Interestingly, several genes encoding dependency factors identified by the enhancer connectome study, such as CCND2 and MYC, were also identified here in our LANA connectome. The virus-encoded interferon regulator factor 3 homolog (vIRF3) is also enriched at several of the super-enhancers controlling expression of genes involved in cell survival.69 While these studies have revealed important findings regarding enhancers, the significance of vIRF3 at enhancers is unclear. Intriguingly, in the published vIRF3 ChIP-seq data, while it is clearly at enhancers, there is no signal at the promoters of the target genes, suggesting that it is located outside of the juxtaposed chromatin loop.

Consistent with the recent work by Manzano et al.,69 we observed IRF4 at PEL super-enhancers. While depletion of LANA reduces enhancer activity, we did not observe a reduction in IRF4 occupancy. In contrast, we did observe a reduction in both H3K27ac and BRD4. These data suggest that the presence IRF4 is not sufficient for driving a high level of expression from LANA-bound super-enhancers. LANA and IRF4 have been shown to directly interact; however, this interaction has been shown to inhibit the ability of IRF4 to activate transcription of the major histocompatibility complex (MHC) class II transactivator (CIITA) promoter.68 Given these data, we hypothesize that the interaction between LANA and IRF4 can provide either an activating or inhibitory signal for gene expression and that this may be dependent on the epigenomic status of a locus.

Our observation of LANA enrichment at enhancers adds an additional layer to enhancer-promoter control during oncogenic virus infection. Multiple analyses of gene expression and RNAPII status support the conclusion that LANA occupancy at distal gene-regulatory elements facilitates enhanced expression of LANA enhancer-promoter pairs relative to non-LANA bound pairs. Moreover, in contrast to the previously reported viral factors, LANA CUT&RUN and HiChIP demonstrate its presence at both enhancers and the promoters of target genes. Furthermore, perturbation of LANA expression with CRISPRi demonstrates that the effect of LANA on enhancer-promoter expression is not global but specific to genes connected to LANA-occupied enhancers. This effect is mediated at or upstream of transcription initiation, as we observed reduced RNAPII S5p at enhancers and gene promoters when LANA was depleted.

The role of LANA in gene regulation is an important area of investigation, and, using ChIP-seq, several groups have evaluated LANA occupancy in various cell types. While LANA occupancy at promoters of select cellular genes has emerged as a common theme, the influence of LANA on transcription output has varied. For example, comparing KSHV-infected and uninfected lymphatic endothelial cells (LECs), Mercier et al.80 did not observe differential expression of LANA-occupied loci. However, in contrast, several others have observed both positive and negative regulation of gene expression by LANA.54,81–83 We speculate that these differences arise from both technical and biological sample differences. For example, there is great variability among the number of sites that LANA occupies in various cell types and even within the same cell type in different studies. Along this line, although both are PEL, these cancer lines were derived from separate individuals, and there is variability in LANA HiChIP in TREx-BCBL1-RTA and BC-3 cells. However, the overall ability of LANA to participate in enhancer-promoter gene regulation is conserved. Therefore, we speculate that clustering LANA based on the epigenomic landscape as well as 3D genomic architecture will resolve some of the previous discrepancies.

Capture Hi-C recently provided insight into where KSHV tethers on the human genome.51 Although the distribution of the capture Hi-C chimeric reads precluded comprehensive intersection analyses, we were able to observe episome tethering occurring at LANA CUT&RUN peaks classified as both enhancers and non-enhancers. The tethering signal was stronger near LANA-bound enhancers, which is consistent with the original study revealing episome tethering on chromatin with enhancer features. Whether tethering of an episome near enhancers or promoters results in altered cellular gene expression is not known and a topic of active investigation. Along this line, an emerging concept in cancer and gene expression control is extrachromosomal DNA (ecDNA), in which large regions of the human genome harboring oncogenes and enhancer sequences are present at structures analogous to episomes.84 However, this analysis requires suitable tethering site information, which we were not able to obtain by LANA HiCHIP. Whether cellular enhancers can influence viral gene expression due to their proximity is not known, and future studies should consider the crosstalk between tethering site choice and gene expression control.

The ability of LANA to participate in looping was not restricted to the host genome. LANA HiCHIP analyses of the KSHV genome revealed a conserved LANA-associated 3D organization between TREx-BCBL1-RTA and BC-3 cells (Figure S3A). Recent work by Campbell et al.85 leveraged capture Hi-C to profile the KSHV genomic structure and identified several direct physical, long-range, and dynamic genomic interactions. Intersection of their capture Hi-C data with LANA HiCHIP revealed the participation of LANA in loops between PAN/K9–10 and K9–10/ORF72.

In addition to KSHV, several other viruses establish latency and maintain an episomal genome physically tethered to host chromatin. Moreover, many of the virus-encoded tethering proteins are known to interact with a variety of chromatin interacting proteins. For example, the EBV genome is tethered via EBNA1, while the HPV genome is tethered via E2, and it is well established that both factors interact with gene- and chromatin-regulatory factors.33,86 However, none of these have been demonstrated to engage in chromatin looping. Our LANA HiChIP data therefore reveal a property of viral tethering proteins to participate in intra-chromosomal interactions and enhancer-promoter looping. We speculate that this biological property is conserved in other virus-encoded proteins.

Limitations of the study

One of the limitations of our study is the use of PEL cell lines. As these are already transformed, our data may not accurately reflect a non-transformed KSHV B cell infection. In addition, KSHV can infect multiple cell types and promote transformation of cells with various origins, including endothelial. Therefore, LANA binding as well as its epigenomic landscape may vary in cell types other than those reported here. Moreover, our epigenomics and transcriptomics were all generated on cell populations and thus represent ensemble measurements. Application of single-cell approaches will therefore aid in defining how KSHV alters the enhancer-promoter landscape on an individual-cell basis.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for reagents may be directed to, and will be fulfilled by the lead contact, John Karijolich (john. karijolich@vumc.org).

Materials availability

This study did not generate new unique reagents.

Data and code availability

  • All the CUT&RUN, RNA-seq, ATACme, and HiChIP sequencing data generated in this study have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Original western blot images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
LANA Millipore Clone LN53
GAPDH Proteintech Cat# 60004–1-Ig; RRID: AB_2107436
Phospho-Rpb1 CTD (Ser2) Cell Signaling Technology (CST) Clone E1Z3G; RRID: AB_2798238
Phospho-Rpb1 CTD (Ser5) Cell Signaling Technology (CST) Clone D9N5I; RRID: AB_2798246
Rpb1 Santa Cruz Biotechnology (SCBT) Clone N-20; RRID: AB_632359
H3K27ac Cell Signaling Technology (CST) Clone D5E4; RRID: AB_2904133
H3K27me3 Cell Signaling Technology (CST) Clone C36B11; RRID: AB_2616029
H3K4me1 Cell Signaling Technology (CST) Clone D1A9; RRID: AB_10695148
H3K4me3 Cell Signaling Technology (CST) Clone C42D8; RRID: AB_2616028
BRD2 Cell Signaling Technology (CST) Clone D89B4; RRID: AB_2535181
BRD4 Santa Cruz Cat# ab128874; RRID: AB_11145462
EZH2 Cell Signaling Technology (CST) Clone D2C9; RRID: AB_10694683
IRF4 Cell Signaling Technology (CST) 4964S; RRID: AB_10698467
Chemicals, peptides, and recombinant proteins
Doxycycline Fisher Cat# BP2653–1
Triton X-100 VWR Cat# 0694–1L
Bovine Serum Albumin Rockland Cat# BSA-1000
Tween 20 Fisher Cat# BP337–500
TRIzol Invitrogen Cat# 15596018
Tris Base VWR Cat# 0497–5KG
NaCl Fisher Cat# S271–10
Ribo-lock Thermo Cat# EO0381
M-MLV RT Promega Cat# M170A
PVDF Millipore Cat# IPFL00010
Formaldehyde VWR Cat# M134–500ML
Glycine RPI Cat# G36050–1000
DTT VWR Cat# 0281–5G
SDS VWR Cat# M107–500G
Phenol:chloroform VWR Cat# 0883–400mL
Trypan Blue Corning Cat# 25–900-CI
Dulbecco’s modified Eagle medium (DMEM) Corning Cat# 10–017-CV
RPMI 1640 Medium Thermo Cat# 11875093
Fetal Bovine Serum (FBS) Invitrogen Cat# 26140–079
Penicillin/streptomycin GIBCO Cat# 15140–122
PowerUP SYBR green Applied Biosystems Cat# 100029284
PBS Corning Cat# 21–031-CV
Bromophenol blue Sigma Cat# B0126
Sodium deoxycholate Fisher Cat# BP349
DNase I NEB Cat# M0303S
EDTA RPI Cat# E57020–500
EGTA Sigma Cat# 324626
IGEPAL-630 Sigma Cat# I8896
Phenol:Chloroform, pH8.0 VWR Cat# 0883–100ML
Dynabeads™ MyOne™ Streptavidin C1 Invitrogen Cat# 65002
SureBeads Protein G Magnetic Beads Bio-Rad Cat# 161–4023
cOmplete™, EDTA-free Protease Inhibitor Cocktail Roche Cat# 04693132001
5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) Sigma Cat# D1916
4-Thiouridine (4sU) Sigma Cat# T4509
EZ-Link™ HPDP-Biotin Thermo Cat# A35390
SDS VWR Cat# M107–500G
HindIII NEB Cat# R0104L
T4 DNA Ligase NEB Cat# M0202S
Critical commercial assays
CUT&RUN Assay Kit Cell Signaling Technology (CST) Cat# 86652
truChIP Chromatin Shearing Kit with Formaldehyde Covaris Part# 520154
Arima-HiC + Kit Arima Part# A510008
KAPA Hyper Prep Kit Roche cat#07962363001
DNA Clean and Concentrator-5 Zymo Cat# D4003
DNA Clean and Concentrator-25 Zymo Cat# D4006
Lightning EZ DNA Methylation-Lightning Kit Zymo Cat# D5030
IDT for illumine TruSeq DNA UD indexes IDT Ref# 20021453
NEBNext rRNA Depletion Kit NEB Cat# E6310
NEBNext® Ultra™ II Directional RNA Library Prep NEB Cat# E7760
Deposited data
RNA-seq, CUT&RUN, LANA HiChIP, ATACme This paper GSE231452
RAMPAGE Ye et al.72 GSE129902
GRO-seq Park et al.71 GSE147063
CHD4 CUT&RUN Kumar et al.51 GSE163695
KSHV episome cHiC Campbell85 GSE163695
Original Western blot images https://doi.org/10.17632/dc4fbk8nnm.1 N/A
Experimental models: Cell lines
TREx-BCBL1-RTA Nakamura et al.60 N/A
BCBL1-NTC Brackett et al.73 N/A
BCBL1-LANA-KD Brackett et al.73 N/A
BC-3 ATCC Cat# CRL-3615
Oligonucleotides
See Tables S1 and S2 for PCR oligos for RT-qPCT and 3C-qPCR N/A N/A
Software and algorithms
STAR https://github.com/alexdobin/STAR Version 2.7.3a
featureCounts http://bioinf.wehi.edu.au/featureCounts/ Version 2.0.0
Trimmomatic http://www.usadellab.org/cms/index.php?page=trimmomatic Version 0.39
bowtie2 https://github.com/BenLangmead/bowtie2 Version 2.3.5.1
MACS https://github.com/macs3-project/MACS/wiki/Install-macs2 Version 2.2.7.1
IDR https://github.com/nboley/idr Version 2.0.3
WALT https://github.com/smithlabcode/walt Version 1.0
MethPipe https://github.com/smithlabcode/methpipe Version 5.0.1
HiCUP https://www.bioinformatics.babraham.ac.uk/projects/hicup/ Version 0.8.0
CID http://groups.csail.mit.edu/cgs/gem/cid/ Version 1.0
MICC https://github.com/rakarnik/MICC No release
bedtools https://bedtools.readthedocs.io/en/latest/ Version 2.30.0
samtools http://www.htslib.org/ Version 1.9
deeptools https://deeptools.readthedocs.io/en/latest/ Version 3.3.1
homer http://homer.ucsd.edu/homer/ Version 4.11.1
edgeR https://www.bioconductor.org/packages/release/bioc/html/edgeR.html Version 2.26.5
Open in a new tab

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell lines used in this study have been listed in the key resources table.

Cell culture

TREx-BCBL1-RTA and BC-3 (kindly provided by Dr. Britt Glaunsinger, University of California, Berkeley), BCBL1-NTC and BCBL1-LANA-KD (kind gifts from Dr. Carolina Aria, University of California, Santa Barbara) cells were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 2 mM L-glutamine (Invitrogen). All cells were maintained with 100 U of penicillin/ml and 100 mg of streptomycin/ml (Invitrogen) at 37°C under 5% CO2. Lytic activation of BCBL1-NTC and BCBL1-LANA-KD cells were induced with 2 mg/mL of doxycycline (Dox; Fisher Scientific) for 48 h.

METHOD DETAILS

RT-qPCR

Total RNA was isolated with TRIzol (Invitrogen) in accordance with the manufacturer’s instructions. RNA was DNase I (NEB) treated at 37°C for 20 min, then inactivated with EDTA at 70°C for 10 min. cDNA was synthesized from DNase-treated RNA with random 6-mer (Integrated DNA Technologies) and M-MLV RT (Promega). qPCR was performed using the PowerUp SYBR Green qPCR kit (Thermo Scientific) with appropriate primers (Table S1).

Western blotting

Whole cell lysates were prepared with lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 0.5% NP-40) and quantified by Bradford assay (BioRad). Equivalent amounts of each sample were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred to polyvinylidene difluoride membrane (Millipore), and blotted for LANA (Millipore, clone LN-53), GAPDH (Proteintech, 60004–1-Ig).

RNA-seq

RNA was isolated and DNAse I treated from latent and lytic cells as described above. Following DNAse I treatment, RNA was recovered by extraction with phenol:chloroform:isoamyl alcohol [25:24:1 (v/v)] followed by ethanol precipitation. Isolated RNA was subjected to ribosomal RNA depletion using NEBNext rRNA depletion kits (Human/Mouse/Rat: NEB E6310) according to the manufacturer’s instructions. The rRNA-depleted samples were used as the starting material to generate sequencing libraries using the NEBNext Ultra II directional RNA library prep Kit (NEB E7760) according to the manufacture recommendations. Libraries were then subjected to paired-end sequencing on a NovaSeq-6000 (PE-150) at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).

CUT&RUN

LANA, H3K27ac, H3K27me3, H3K4me1, H3K4me3, BRD2, BRD4, EZH2, polymerase II total, polymerase II CTD S2, and polymerase II CTD S5 CUT&RUN was performed on 200,000 cells using the CUT&RUN kit (Cell Signaling, CUT&RUN Assay Kit, Cat#86652) according to the native version of manufacturer’s instructions. IRF4 (CST, #4964) CUT&RUN was performed on cells fixed with 0.01% formaldehyde at room temperature for 10 min before processing. Following capture of the released DNA fragments, sequencing libraries were prepared using the KAPA HyperPrep Kit (cat#07962363001). Libraries were multiplex sequenced (2 × 150bp, paired-end, 5–10 million reads per sample) on an Illumina Novaseq 6000 sequencing system at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).

LANA HiChIP

LANA HiChIP was performed using the Arima-HiC + kit (Cat# A510008) according to manufactures instructions. Nuclei were sonicated with Covaris LE220 using Covaris truChIP chromatin shearing kit reagents (1x Lysis Buffer B, 1xWash Buffer C, 1xShearing Buffer D3, with proteinase inhibitor) for 4 min in 130 μL. 10 mg anti-LANA IgG (LN-53) was used for the immunoprecipitation. Sequencing libraries were prepared on bead using the Kapa HyperPrep Kit with Library Amplification Module (Roche, Basel, Switzerland). Libraries were multiplex sequenced (2×150bp, paired-end, 200 million mapped reads/mate pairs per sample) on an Illumina Novaseq 6000 sequencing system at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).

ATACme

ATACme was perform as described.62 TREx-BCBL1-RTA and BC-3 cells were centrifuged at 4°C, 500 R.C.F for 5 min and subsequently resuspended in 1mL ice-cold PBS. Cell suspension volume corresponding to 2×105 TREx-BCBL1-RTA or BC-3 cells was pipetted into a 1.5mL eppendorf tube and pelleted at 4°C, 500 R.C.F for 5 min. Supernatant was aspirated and the cell pellet resuspended in 150μL cold ATAC lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL-630). 10μL of pre-assembled Tn5 transposome (containing methylated adaptors) were added to the nuclie and incubated at 37°C, 30 min, 700RPM in an Eppendorf Thermomixer. ATAC-Me reactions were terminated by adding 1mL Zymo DNA binding buffer purified according to manufacturer instructions in a DNA Clean and Concentrator-5 kit (Zymo).

DNA eluate was gap repaired bisulfite converted according to using the Zymo Lightning EZ DNA Methylation-Lightning Kit (cat no D5030). Purification/desulfonation was performed per kit instructions. Eluted ATAC-Me DNA was amplified and barcoded in 50μL PCR reactions. Libraries were cleaned and concentrated with a Zymo DNA Clean and Concentrator-5 column kit and sequenced using 2×150bp paired-end reads on the NovaSeq6000 instruments at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).

4sU-DRB labeling

4sU-DRB assays were performed as previously described.87 Breifly, TREx-BCBL1-RTA Cells were cultured RPMI-1640 with 100 μM DRB (sigma) for 3 h, washing cells twice with 1xPBS, and labeled with RPMI-1640 containing 500 μM 4sU (Sigma) for 10 min prior to isolating RNA with TRIzol, followed by isopropanol precipitation. Total RNA (100 μg) was incubated in biotinylation buffer (10 mM Tris [pH 7.4], 1 mM EDTA) and 200 μg HPDP-biotin (EZ-link HPDP-biotin; Thermo Scientific) with constant rotation at room temperature for 1.5 h. RNA was then phenol-chloroform extracted and precipitated with isopropanol. The pellet was resuspended in DEPCtreated water and mixed with 50 μL Dynabeads MyOne streptavidin C1 (Invitrogen) that had been pre-washed twice with 1× wash buffer (100 mM Tris [pH 7.5], 10 mM EDTA, 1 M NaCl, 0.1% Tween 20). Samples were rotated for 15 min at RT, then washed 3× with 65°C wash buffer and 3× with RT wash buffer. Samples were eluted with 100 μM DTT, and the RNA was precipitated with ethanol prior to RT-qPCR. The respective transcripts measurements by RT-qPCR were first normalized to 18S level and subtract by the DRB positive (with DRB during 4SU treatment) control and then standardized by setting the nascent RNA level in knock down control cells as 1.

Chromatin conformation capture (3C)-PCR

3C was performed essentially as previously described.88 Briefly, PEL cells (3×106) were washed in 1 × PBS and resuspended in 1.0 mL 10% FCS/PBS. Cells were fixed in 10 mL total of 1.0% formaldehyde in 10% FCS/PBS for 10 min at RT before glycine (125 mM final concentration) treatment for 5 min. Cells were then pelleted (500 × g) at 4°C for 5 min, after which they were resuspended in 1.0 mL of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.7; 10 mM NaCl; 5 mM MgCl2; 0.1 mM EGTA; protease inhibitors [Roche]) and incubated on ice for 30 min before nuclei were pelleted (400 × g) for 5 min at 4°C. Next, cell nuclei were resuspended in 500 μL of 1.2X restriction enzyme buffer and 0.3% SDS (final concentration) and incubated at 37°C with shaking at 300 rpm (Eppendorf Thermomixer). After 1 h, 50 μL of 20% Triton X-100 was added and samples were incubated at 37°C for an additional 1 h with shaking (300 rpm). At this point, 10% of each sample was retained as an input control, while 400 U of HindIII was added to each remaining sample and incubated overnight at 37°C with shaking (300 rpm). Samples were then halved and 50% was retained as a digest control. SDS (0.1% final concentration) was added to the remaining half of each sample and then incubated for 25 min at 65°C with shaking (300 rpm). Next, 6.5 mL of 1.15X ligation buffer containing Triton X-100 (1% final concentration) was added, and samples were incubated for 1 h at 37°C with gentle shaking (300 rpm). T4 DNA ligase (200 U) was then added, and samples were incubated for 4 h at 16°C, followed by 30 min at RT. All input controls, digest controls, and ligation samples were then treated with proteinase K for 1.5 h at 65 C, followed by RNase A treatment for 0.5 h at 37 C. DNA was then isolated by phenol:chloroform extraction and alcohol precipitation, and all samples were resuspended in 10 mM Tris-HCl, pH 7.5 for use in PCR. All 3C primers are listed in Table S2.

NGS data analysis

CUT&RUN fastq files were first trimmed with Trimmomatic (version 0.39) to remove adapter and low-quality nucleotides and then mapped to the combined human hg38 (GRCh38) and KSHV (GQ994935.1) genome using bowtie2 (version 2.3.5.1) with options: –local –very-sensitive-local –no-unal –no-mixed –no-discordant –phred33 -k 1 -I 10 -X 700. For mapping signal around gene promoters, the gene annotation track (gencode version 23 known genes track) was downloaded from the UCSC table browser as a bed file and used as reference for heatmaps. MACS2 (version 2.2.7.1) was used for calling peaks with options: “–broad -p 1e-5 -f BAMPE –keep-dup all” for histone modifications and without “–broad” option for transcription factors. IDR (version 2.0.3) was used to get the reproducible peaks between duplicates with FDR <0.05.

ATACme data were processed as described.62 ATACme fastq files were first trimmed with Trimmomatic (version 0.39) to remove adapter and low-quality nucleotides. Then WALT (version 1.0) was used to align the trimmed reads to the combined human hg38 (GRCh38) and KSHV (GQ994935.1) genome. The DNA methylation was called using MethPipe pipeline.

FASTQ files for LANA HiChIP experiments were processed through HiCUP (version 0.8.0) using a combined human hg38 (GRCh38) and KSHV (GQ994935.1) genome. Valid interaction bam files called by HiCUP were first convert to bedpe files with bedtools before further processed using CID pipeline to call the loops. MICC R script was used to assign significance to loops, and loops with FDR < 1×10–10 and backed by more than 10 reads were used as valid loops for the other analyses. The enrichment of LANA peaks on features (enhancer/super-enhancer) was determined using Fisher exact test tools provided in bedtools with command ‘bedtools fisher -a enhancers.bed -b LANA_peaks.bed -g chromosome.size.txt’, and the expected overlapping number was calculated by randomly shuffle the LANA peaks using command ‘bedtools shuffle -i LANA_peaks.bed -g chromosome.sizes’ 100 times and get the rounded average overlapping counts.

For visualization of alignment of signal on the genome, the alignment SAM files were transformed to BAM and BIGWIG files using samtools (version 1.9) and deeptools (version 3.3.1). Data visualization of CUT&RUN data were done with plotHeatmap and plotProfile tools in deeptools (version 3.3.1) or with R package ggplots (version 3.3.5). All sequencing data has been deposited under accession number GEO: GSE231452.

The Capture HiC data (GSE163695) was first download from SRA database and then processed the same way as LANA-HiChIP dataset using HiCUP. The processed chimeric pairs between viral and the host were then selected using custom bash script and aggregated with deeptools.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical parameters are reported in the figures and corresponding figure legends. Student t test was used to determine statistical significance * p % 0.05; ** p % 0.01; *** p % 0.001; ns: not significant. The results were expressed as mean ± SD. qPCR was performed using the QuantStudio 3 (Thermo). Immunoblots were developed using the Odyssey Li-Cor (Li-Cor) and signal intensities were quantified using ImageJ software. NGS was performed using the NovaSeq 6000 (Illumina).

Supplementary Material

1

Highlights.

  • KSHV LANA is enriched on cellular enhancers in PEL cells

  • LANA occupancy is associated with higher enhancer activity and gene expression

  • Loss of LANA reduces enhancer-promoter contacts and gene expression

ACKNOWLEDGMENTS

We thank members of the Karijolich lab for discussions and Britt Glaunsinger (University of California, Berkeley), Carolina Arias (University of California, Santa Barbara), and Ruben Barricarte (Vanderbilt University Medical Center) for kindly sharing cell lines and reagents. The Karijolich laboratory was supported by startup funds from Vanderbilt University Medical Center, National Institutes of Health (NIH) grant R01CA250051 (to J.K.), and American Cancer Society Research Scholar Award RSG MPC - 133907 (to J.K.). J.K. is a Pew Biomedical Scholar.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.113888.

DECLARATION OF INTERESTS

The authors declare no competing interests.

REFERENCES

  • 1.Sanyal A, Lajoie BR, Jain G, and Dekker J (2012). The long-range interaction landscape of gene promoters. Nature 489, 109–113. 10.1038/nature11279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Alexandrov NN, Troukhan ME, Brover VV, Tatarinova T, Flavell RB, and Feldmann KA (2006). Features of Arabidopsis genes and genome discovered using full-length cDNAs. Plant Mol. Biol 60, 69–85. 10.1007/s11103-005-2564-9. [DOI] [PubMed] [Google Scholar]
  • 3.Hillier LW, Marth GT, Quinlan AR, Dooling D, Fewell G, Barnett D, Fox P, Glasscock JI, Hickenbotham M, Huang W, et al. (2008). Whole-genome sequencing and variant discovery in C. elegans. Nat. Methods 5, 183–188. 10.1038/nmeth.1179. [DOI] [PubMed] [Google Scholar]
  • 4.Massouras A, Waszak SM, Albarca-Aguilera M, Hens K, Holcombe W, Ayroles JF, Dermitzakis ET, Stone EA, Jensen JD, Mackay TFC, and Deplancke B (2012). Genomic variation and its impact on gene expression in Drosophila melanogaster. PLoS Genet. 8, e1003055. 10.1371/journal.pgen.1003055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tang R, Noh HJ, Wang D, Sigurdsson S, Swofford R, Perloski M, Duxbury M, Patterson EE, Albright J, Castelhano M, et al. (2014). Candidate genes and functional noncoding variants identified in a canine model of obsessive-compulsive disorder. Genome Biol. 15, R25. 10.1186/gb-2014-15-3-r25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Manolio TA, Brooks LD, and Collins FS (2008). A HapMap harvest of insights into the genetics of common disease. J. Clin. Invest 118, 1590–1605. 10.1172/JCI34772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Gusev A, Lee SH, Trynka G, Finucane H, Vilhjálmsson BJ, Xu H, Zang C, Ripke S, Bulik-Sullivan B, Stahl E, et al. (2014). Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases. Am. J. Hum. Genet 95, 535–552. 10.1016/j.ajhg.2014.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Frazer KA, Murray SS, Schork NJ, and Topol EJ (2009). Human genetic variation and its contribution to complex traits. Nat. Rev. Genet 10, 241–251. 10.1038/nrg2554. [DOI] [PubMed] [Google Scholar]
  • 9.Lindblad-Toh K, Garber M, Zuk O, Lin MF, Parker BJ, Washietl S, Kheradpour P, Ernst J, Jordan G, Mauceli E, et al. (2011). A high-resolution map of human evolutionary constraint using 29 mammals. Nature 478, 476–482. 10.1038/nature10530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.ENCODE Project Consortium (2012). An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74. 10.1038/nature11247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bernstein BE, Stamatoyannopoulos JA, Costello JF, Ren B, Milosavljevic A, Meissner A, Kellis M, Marra MA, Beaudet AL, Ecker JR, et al. (2010). The NIH Roadmap Epigenomics Mapping Consortium. Nat. Biotechnol 28, 1045–1048. 10.1038/nbt1010-1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Celniker SE, Dillon LAL, Gerstein MB, Gunsalus KC, Henikoff S, Karpen GH, Kellis M, Lai EC, Lieb JD, MacAlpine DM, et al. (2009). Unlocking the secrets of the genome. Nature 459, 927–930. 10.1038/459927a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ernst J, Kheradpour P, Mikkelsen TS, Shoresh N, Ward LD, Epstein CB, Zhang X, Wang L, Issner R, Coyne M, et al. (2011). Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49. 10.1038/nature09906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA, Kasowski M, Karczewski KJ, Park J, Hitz BC, Weng S, et al. (2012). Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 22, 1790–1797. 10.1101/gr.137323.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ward LD, and Kellis M (2012). HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 40, D930–D934. 10.1093/nar/gkr917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, and Shendure J (2014). A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet 46, 310–315. 10.1038/ng.2892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gulko B, Hubisz MJ, Gronau I, and Siepel A (2015). A method for calculating probabilities of fitness consequences for point mutations across the human genome. Nat. Genet 47, 276–283. 10.1038/ng.3196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lettice LA, Heaney SJH, Purdie LA, Li L, de Beer P, Oostra BA, Goode D, Elgar G, Hill RE, and de Graaff E (2003). A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum. Mol. Genet 12, 1725–1735. 10.1093/hmg/ddg180. [DOI] [PubMed] [Google Scholar]
  • 19.Lomelin D, Jorgenson E, and Risch N (2010). Human genetic variation recognizes functional elements in noncoding sequence. Genome Res. 20, 311–319. 10.1101/gr.094151.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Schaub MA, Boyle AP, Kundaje A, Batzoglou S, and Snyder M (2012). Linking disease associations with regulatory information in the human genome. Genome Res. 22, 1748–1759. 10.1101/gr.136127.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, and Chang HY (2016). HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat. Methods 13, 919–922. 10.1038/nmeth.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera LO, Van Calcar S, Qu C, Ching KA, et al. (2007). Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet 39, 311–318. 10.1038/ng1966. [DOI] [PubMed] [Google Scholar]
  • 23.Mumbach MR, Satpathy AT, Boyle EA, Dai C, Gowen BG, Cho SW, Nguyen ML, Rubin AJ, Granja JM, Kazane KR, et al. (2017). Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nat. Genet 49, 1602–1612. 10.1038/ng.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, Hanna J, Lodato MA, Frampton GM, Sharp PA, et al. (2010). Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936. 10.1073/pnas.1016071107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.De Leo A, Calderon A, and Lieberman PM (2020). Control of Viral Latency by Episome Maintenance Proteins. Trends Microbiol. 28, 150–162. 10.1016/j.tim.2019.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Poddar A, Reed SC, McPhillips MG, Spindler JE, and McBride AA (2009). The human papillomavirus type 8 E2 tethering protein targets the ribosomal DNA loci of host mitotic chromosomes. J. Virol 83, 640–650. 10.1128/JVI.01936-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Dao LD, Duffy A, Van Tine BA, Wu S-Y, Chiang C-M, Broker TR, and Chow LT (2006). Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus. J. Virol 80, 4792–4800. 10.1128/JVI.80.10.4792-4800.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ballestas ME, Chatis PA, and Kaye KM (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641–644. 10.1126/science.284.5414.641. [DOI] [PubMed] [Google Scholar]
  • 29.Henderson A, Ripley S, Heller M, and Kieff E (1983). Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro. Proc. Natl. Acad. Sci. USA 80, 1987–1991. 10.1073/pnas.80.7.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Harris A, Young BD, and Griffin BE (1985). Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt’s lymphoma-derived cell lines. J. Virol 56, 328–332. 10.1128/JVI.56.1.328-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wang L, Laing J, Yan B, Zhou H, Ke L, Wang C, Narita Y, Zhang Z, Olson MR, Afzali B, et al. (2020). Epstein-Barr Virus Episome Physically Interacts with Active Regions of the Host Genome in Lymphoblastoid Cells. J. Virol 94, 10–1128. 10.1128/JVI.01390-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kim K-D, Tanizawa H, De Leo A, Vladimirova O, Kossenkov A, Lu F, Showe LC, Noma K-I, and Lieberman PM (2020). Epigenetic specifications of host chromosome docking sites for latent Epstein-Barr virus. Nat. Commun 11, 877. 10.1038/s41467-019-14152-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Skiadopoulos MH, and McBride AA (1998). Bovine papillomavirus type 1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin. J. Virol 72, 2079–2088. 10.1128/JVI.72.3.2079-2088.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lehman CW, and Botchan MR (1998). Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc. Natl. Acad. Sci. USA 95, 4338–4343. 10.1073/pnas.95.8.4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Cotter MA, and Robertson ES (1999). The latency-associated nuclear antigen tethers the Kaposi’s sarcoma-associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells. Virology 264, 254–264. 10.1006/viro.1999.9999. [DOI] [PubMed] [Google Scholar]
  • 36.Oliveira JG, Colf LA, and McBride AA (2006). Variations in the association of papillomavirus E2 proteins with mitotic chromosomes. Proc. Natl. Acad. Sci. USA 103, 1047–1052. 10.1073/pnas.0507624103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Cotter MA, Subramanian C, and Robertson ES (2001). The Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen binds to specific sequences at the left end of the viral genome through its carboxy-terminus. Virology 291, 241–259. 10.1006/viro.2001.1202. [DOI] [PubMed] [Google Scholar]
  • 38.McBride AA (2013). The papillomavirus E2 proteins. Virology 445, 57–79. 10.1016/j.virol.2013.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Soulier J, Grollet L, Oksenhendler E, Cacoub P, Cazals-Hatem D, Babinet P, d’Agay MF, Clauvel JP, Raphael M, and Degos L (1995). Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood 86, 1276–1280. [PubMed] [Google Scholar]
  • 40.Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, and Moore PS (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266, 1865–1869. 10.1126/science.7997879. [DOI] [PubMed] [Google Scholar]
  • 41.Cesarman E, Chang Y, Moore PS, Said JW, and Knowles DM (1995). Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med 332, 1186–1191. 10.1056/NEJM199505043321802. [DOI] [PubMed] [Google Scholar]
  • 42.Dupin N, Diss TL, Kellam P, Tulliez M, Du MQ, Sicard D, Weiss RA, Isaacson PG, and Boshoff C (2000). HHV-8 is associated with a plasmablastic variant of Castleman disease that is linked to HHV-8-positive plasmablastic lymphoma. Blood 95, 1406–1412. [PubMed] [Google Scholar]
  • 43.Dupin N, Fisher C, Kellam P, Ariad S, Tulliez M, Franck N, van Marck E, Salmon D, Gorin I, Escande JP, et al. (1999). Distribution of human herpesvirus-8 latently infected cells in Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma. Proc. Natl. Acad. Sci. USA 96, 4546–4551. 10.1073/pnas.96.8.4546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Moore PS, and Chang Y (1995). Detection of herpesvirus-like DNA sequences in Kaposi’s sarcoma in patients with and those without HIV infection. N. Engl. J. Med 332, 1181–1185. 10.1056/NEJM199505043321801. [DOI] [PubMed] [Google Scholar]
  • 45.Barbera AJ, Chodaparambil JV, Kelley-Clarke B, Joukov V, Walter JC, Luger K, and Kaye KM (2006). The nucleosomal surface as a docking station for Kaposi’s sarcoma herpesvirus LANA. Science 311, 856–861. 10.1126/science.1120541. [DOI] [PubMed] [Google Scholar]
  • 46.Barbera AJ, Ballestas ME, and Kaye KM (2004). The Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen 1 N terminus is essential for chromosome association, DNA replication, and episome persistence. J. Virol 78, 294–301. 10.1128/jvi.78.1.294-301.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kelley-Clarke B, Ballestas ME, Komatsu T, and Kaye KM (2007). Kaposi’s sarcoma herpesvirus C-terminal LANA concentrates at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes. Virology 357, 149–157. 10.1016/j.virol.2006.07.052. [DOI] [PubMed] [Google Scholar]
  • 48.Kelley-Clarke B, Ballestas ME, Srinivasan V, Barbera AJ, Komatsu T, Harris T-A, Kazanjian M, and Kaye KM (2007). Determination of Kaposi’s sarcoma-associated herpesvirus C-terminal latency-associated nuclear antigen residues mediating chromosome association and DNA binding. J. Virol 81, 4348–4356. 10.1128/JVI.01289-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Piolot T, Tramier M, Coppey M, Nicolas JC, and Marechal V (2001). Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes. J. Virol 75, 3948–3959. 10.1128/JVI.75.8.3948-3959.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Wong L-Y, Matchett GA, and Wilson AC (2004). Transcriptional activation by the Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen is facilitated by an N-terminal chromatin-binding motif. J. Virol 78, 10074–10085. 10.1128/JVI.78.18.10074-10085.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Kumar A, Lyu Y, Yanagihashi Y, Chantarasrivong C, Majerciak V, Salemi M, Wang K-H, Inagaki T, Chuang F, Davis RR, et al. (2022). KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4. Cell Rep. 39, 110788. 10.1016/j.celrep.2022.110788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Viejo-Borbolla A, Ottinger M, Brüning E, Bürger A, König R, Kati E, Sheldon JA, and Schulz TF (2005). Brd2/RING3 interacts with a chromatin-binding domain in the Kaposi’s Sarcoma-associated herpesvirus latency-associated nuclear antigen 1 (LANA-1) that is required for multiple functions of LANA-1. J. Virol 79, 13618–13629. 10.1128/JVI.79.21.13618-13629.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.You J, Srinivasan V, Denis GV, Harrington WJ, Ballestas ME, Kaye KM, and Howley PM (2006). Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes. J. Virol 80, 8909–8919. 10.1128/JVI.00502-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Hu J, Yang Y, Turner PC, Jain V, McIntyre LM, and Renne R (2014). LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex. PLoS Pathog. 10, e1004240. 10.1371/journal.ppat.1004240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Jeong JH, Orvis J, Kim JW, McMurtrey CP, Renne R, and Dittmer DP (2004). Regulation and autoregulation of the promoter for the latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus. J. Biol. Chem 279, 16822–16831. 10.1074/jbc.M312801200. [DOI] [PubMed] [Google Scholar]
  • 56.Jeong J, Papin J, and Dittmer D (2001). Differential regulation of the overlapping Kaposi’s sarcoma-associated herpesvirus vGCR (orf74) and LANA (orf73) promoters. J. Virol 75, 1798–1807. 10.1128/JVI.75.4.1798-1807.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.An F-Q, Compitello N, Horwitz E, Sramkoski M, Knudsen ES, and Renne R (2005). The latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus modulates cellular gene expression and protects lymphoid cells from p16 INK4A-induced cell cycle arrest. J. Biol. Chem 280, 3862–3874. 10.1074/jbc.M407435200. [DOI] [PubMed] [Google Scholar]
  • 58.Di Bartolo DL, Cannon M, Liu Y-F, Renne R, Chadburn A, Boshoff C, and Cesarman E (2008). KSHV LANA inhibits TGF-beta signaling through epigenetic silencing of the TGF-beta type II receptor. Blood 111, 4731–4740. 10.1182/blood-2007-09-110544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Skene PJ, and Henikoff S (2017). An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, e21856. 10.7554/eLife.21856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Nakamura H, Lu M, Gwack Y, Souvlis J, Zeichner SL, and Jung JU (2003). Global changes in Kaposi’s sarcoma-aociated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator. J. Virol 77, 4205–4220. 10.1128/jvi.77.7.4205-4220.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Arvanitakis L, Mesri EA, Nador RG, Said JW, Asch AS, Knowles DM, and Cesarman E (1996). Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus. Blood 88, 2648–2654. [PubMed] [Google Scholar]
  • 62.Barnett KR, Decato BE, Scott TJ, Hansen TJ, Chen B, Attalla J, Smith AD, and Hodges E (2020). ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions. Mol. Cell 77, 1350–1364.e6. 10.1016/j.molcel.2020.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Wang C, Zhang L, Ke L, Ding W, Jiang S, Li D, Narita Y, Hou I, Liang J, Li S, et al. (2020). Primary effusion lymphoma enhancer connectome links super-enhancers to dependency factors. Nat. Commun 11, 6318. 10.1038/s41467-020-20136-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Platt GM, Simpson GR, Mittnacht S, and Schulz TF (1999). Latent nuclear antigen of Kaposi’s sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene. J. Virol 73, 9789–9795. 10.1128/JVI.73.12.9789-9795.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Mattsson K, Kiss C, Platt GM, Simpson GR, Kashuba E, Klein G, Schulz TF, and Szekely L (2002). Latent nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 induces and relocates RING3 to nuclear heterochromatin regions. J. Gen. Virol 83, 179–188. 10.1099/0022-1317-83-1-179. [DOI] [PubMed] [Google Scholar]
  • 66.Hellert J, Weidner-Glunde M, Krausze J, Richter U, Adler H, Fedorov R, Pietrek M, Rückert J, Ritter C, Schulz TF, and Lührs T (2013). A structural basis for BRD2/4-mediated host chromatin interaction and oligomer assembly of Kaposi sarcoma-associated herpesvirus and murine gammaherpesvirus LANA proteins. PLoS Pathog. 9, e1003640. 10.1371/journal.ppat.1003640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Toth Z, Papp B, Brulois K, Choi YJ, Gao S-J, and Jung JU (2016). LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection. PLoS Pathog. 12, e1005878. 10.1371/journal.ppat.1005878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Cai Q, Banerje S, Cervini A, Lu J, Hislop AD, Dzeng R, and Robertson ES (2013). IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation. PLoS Pathog. 9, e1003751. 10.1371/journal.ppat.1003751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Manzano M, Günther T, Ju H, Nicholas J, Bartom ET, Grundhoff A, and Gottwein E (2020). Kaposi’s Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma. mBio 11, e01457–e01520. 10.1128/mBio.01457-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Kim T-K, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, Harmin DA, Laptewicz M, Barbara-Haley K, Kuersten S, et al. (2010). Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187. 10.1038/nature09033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Park A, Oh S, Jung KL, Choi UY, Lee H-R, Rosenfeld MG, and Jung JU (2020). Global epigenomic analysis of KSHV-infected primary effusion lymphoma identifies functional MYC superenhancers and enhancer RNAs. Proc. Natl. Acad. Sci. USA 117, 21618–21627. 10.1073/pnas.1922216117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Ye X, Zhao Y, and Karijolich J (2019). The landscape of transcription initiation across latent and lytic KSHV genomes. PLoS Pathog. 15, e1007852. 10.1371/journal.ppat.1007852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Brackett K, Mungale A, Lopez-Isidro M, Proctor DA, Najarro G, and Arias C (2021). CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Cells. Viruses 13, 783. 10.3390/v13050783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Wood CD, Veenstra H, Khasnis S, Gunnell A, Webb HM, Shannon-Lowe C, Andrews S, Osborne CS, and West MJ (2016). MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs. Elife 5, e18270. 10.7554/eLife.18270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.McClellan MJ, Wood CD, Ojeniyi O, Cooper TJ, Kanhere A, Arvey A, Webb HM, Palermo RD, Harth-Hertle ML, Kempkes B, et al. (2013). Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming. PLoS Pathog. 9, e1003636. 10.1371/journal.ppat.1003636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Zhou H, Schmidt SCS, Jiang S, Willox B, Bernhardt K, Liang J, Johannsen EC, Kharchenko P, Gewurz BE, Kieff E, and Zhao B (2015). Epstein-Barr virus oncoprotein super-enhancers control B cell growth. Cell Host Microbe 17, 205–216. 10.1016/j.chom.2014.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Liang J, Zhou H, Gerdt C, Tan M, Colson T, Kaye KM, Kieff E, and Zhao B (2016). Epstein-Barr virus super-enhancer eRNAs are essential for MYC oncogene expression and lymphoblast proliferation. Proc. Natl. Acad. Sci. USA 113, 14121–14126. 10.1073/pnas.1616697113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Cohen JI, Wang F, Mannick J, and Kieff E (1989). Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc. Natl. Acad. Sci. USA 86, 9558–9562. 10.1073/pnas.86.23.9558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.Kempkes B, Spitkovsky D, Jansen-Dürr P, Ellwart JW, Kremmer E, Delecluse HJ, Rottenberger C, Bornkamm GW, and Hammerschmidt W (1995). B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2. EMBO J. 14, 88–96. 10.1002/j.1460-2075.1995.tb06978.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80.Mercier A, Arias C, Madrid AS, Holdorf MM, and Ganem D (2014). Site-Specific Association with Host and Viral Chromatin by Kaposi’s Sarcoma-Associated Herpesvirus LANA and Its Reversal during Lytic Reactivation. J. Virol 88, 6762–6777. 10.1128/JVI.00268-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Garber AC, Shu MA, Hu J, and Renne R (2001). DNA Binding and Modulation of Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus. J. Virol 75, 7882–7892. 10.1128/JVI.75.17.7882-7892.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Lotke R, Schneeweiß U, Pietrek M, Günther T, Grundhoff A, Weidner-Glunde M, and Schulz TF (2020). Brd/BET Proteins Influence the Genome-Wide Localization of the Kaposi’s Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus Major Latency Proteins. Front. Microbiol 11, 591778. 10.3389/fmicb.2020.591778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Garber AC, Hu J, and Renne R (2002). Latency-associated Nuclear Antigen (LANA) Cooperatively Binds to Two Sites within the Terminal Repeat, and Both Sites Contribute to the Ability of LANA to Suppress Transcription and to Facilitate DNA Replication. J. Biol. Chem 277, 27401–27411. 10.1074/jbc.M203489200. [DOI] [PubMed] [Google Scholar]
  • 84.Hung KL, Yost KE, Xie L, Shi Q, Helmsauer K, Luebeck J, Schöpflin R, Lange JT, Chamorro González R, Weiser NE, et al. (2021). ecDNA hubs drive cooperative intermolecular oncogene expression. Nature 600, 731–736. 10.1038/s41586-021-04116-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Campbell M, Watanabe T, Nakano K, Davis RR, Lyu Y, Tepper CG, Durbin-Johnson B, Fujimuro M, and Izumiya Y (2018). KSHV episomes reveal dynamic chromatin loop formation with domain-specific gene regulation. Nat. Commun 9, 49. 10.1038/s41467-017-02089-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Sears J, Ujihara M, Wong S, Ott C, Middeldorp J, and Aiyar A (2004). The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes. J. Virol 78, 11487–11505. 10.1128/JVI.78.21.11487-11505.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Abernathy E, Gilbertson S, Alla R, and Glaunsinger B (2015). Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells. Cell Host Microbe 18, 243–253. 10.1016/j.chom.2015.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Hagège H, Klous P, Braem C, Splinter E, Dekker J, Cathala G, De Laat W, and Forné T (2007). Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat. Protoc 2, 1722–1733. 10.1038/nprot.2007.243. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1980926-supplement-1.pdf (2.1MB, pdf)

Data Availability Statement

  • All the CUT&RUN, RNA-seq, ATACme, and HiChIP sequencing data generated in this study have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Original western blot images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
LANA Millipore Clone LN53
GAPDH Proteintech Cat# 60004–1-Ig; RRID: AB_2107436
Phospho-Rpb1 CTD (Ser2) Cell Signaling Technology (CST) Clone E1Z3G; RRID: AB_2798238
Phospho-Rpb1 CTD (Ser5) Cell Signaling Technology (CST) Clone D9N5I; RRID: AB_2798246
Rpb1 Santa Cruz Biotechnology (SCBT) Clone N-20; RRID: AB_632359
H3K27ac Cell Signaling Technology (CST) Clone D5E4; RRID: AB_2904133
H3K27me3 Cell Signaling Technology (CST) Clone C36B11; RRID: AB_2616029
H3K4me1 Cell Signaling Technology (CST) Clone D1A9; RRID: AB_10695148
H3K4me3 Cell Signaling Technology (CST) Clone C42D8; RRID: AB_2616028
BRD2 Cell Signaling Technology (CST) Clone D89B4; RRID: AB_2535181
BRD4 Santa Cruz Cat# ab128874; RRID: AB_11145462
EZH2 Cell Signaling Technology (CST) Clone D2C9; RRID: AB_10694683
IRF4 Cell Signaling Technology (CST) 4964S; RRID: AB_10698467
Chemicals, peptides, and recombinant proteins
Doxycycline Fisher Cat# BP2653–1
Triton X-100 VWR Cat# 0694–1L
Bovine Serum Albumin Rockland Cat# BSA-1000
Tween 20 Fisher Cat# BP337–500
TRIzol Invitrogen Cat# 15596018
Tris Base VWR Cat# 0497–5KG
NaCl Fisher Cat# S271–10
Ribo-lock Thermo Cat# EO0381
M-MLV RT Promega Cat# M170A
PVDF Millipore Cat# IPFL00010
Formaldehyde VWR Cat# M134–500ML
Glycine RPI Cat# G36050–1000
DTT VWR Cat# 0281–5G
SDS VWR Cat# M107–500G
Phenol:chloroform VWR Cat# 0883–400mL
Trypan Blue Corning Cat# 25–900-CI
Dulbecco’s modified Eagle medium (DMEM) Corning Cat# 10–017-CV
RPMI 1640 Medium Thermo Cat# 11875093
Fetal Bovine Serum (FBS) Invitrogen Cat# 26140–079
Penicillin/streptomycin GIBCO Cat# 15140–122
PowerUP SYBR green Applied Biosystems Cat# 100029284
PBS Corning Cat# 21–031-CV
Bromophenol blue Sigma Cat# B0126
Sodium deoxycholate Fisher Cat# BP349
DNase I NEB Cat# M0303S
EDTA RPI Cat# E57020–500
EGTA Sigma Cat# 324626
IGEPAL-630 Sigma Cat# I8896
Phenol:Chloroform, pH8.0 VWR Cat# 0883–100ML
Dynabeads™ MyOne™ Streptavidin C1 Invitrogen Cat# 65002
SureBeads Protein G Magnetic Beads Bio-Rad Cat# 161–4023
cOmplete™, EDTA-free Protease Inhibitor Cocktail Roche Cat# 04693132001
5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) Sigma Cat# D1916
4-Thiouridine (4sU) Sigma Cat# T4509
EZ-Link™ HPDP-Biotin Thermo Cat# A35390
SDS VWR Cat# M107–500G
HindIII NEB Cat# R0104L
T4 DNA Ligase NEB Cat# M0202S
Critical commercial assays
CUT&RUN Assay Kit Cell Signaling Technology (CST) Cat# 86652
truChIP Chromatin Shearing Kit with Formaldehyde Covaris Part# 520154
Arima-HiC + Kit Arima Part# A510008
KAPA Hyper Prep Kit Roche cat#07962363001
DNA Clean and Concentrator-5 Zymo Cat# D4003
DNA Clean and Concentrator-25 Zymo Cat# D4006
Lightning EZ DNA Methylation-Lightning Kit Zymo Cat# D5030
IDT for illumine TruSeq DNA UD indexes IDT Ref# 20021453
NEBNext rRNA Depletion Kit NEB Cat# E6310
NEBNext® Ultra™ II Directional RNA Library Prep NEB Cat# E7760
Deposited data
RNA-seq, CUT&RUN, LANA HiChIP, ATACme This paper GSE231452
RAMPAGE Ye et al.72 GSE129902
GRO-seq Park et al.71 GSE147063
CHD4 CUT&RUN Kumar et al.51 GSE163695
KSHV episome cHiC Campbell85 GSE163695
Original Western blot images https://doi.org/10.17632/dc4fbk8nnm.1 N/A
Experimental models: Cell lines
TREx-BCBL1-RTA Nakamura et al.60 N/A
BCBL1-NTC Brackett et al.73 N/A
BCBL1-LANA-KD Brackett et al.73 N/A
BC-3 ATCC Cat# CRL-3615
Oligonucleotides
See Tables S1 and S2 for PCR oligos for RT-qPCT and 3C-qPCR N/A N/A
Software and algorithms
STAR https://github.com/alexdobin/STAR Version 2.7.3a
featureCounts http://bioinf.wehi.edu.au/featureCounts/ Version 2.0.0
Trimmomatic http://www.usadellab.org/cms/index.php?page=trimmomatic Version 0.39
bowtie2 https://github.com/BenLangmead/bowtie2 Version 2.3.5.1
MACS https://github.com/macs3-project/MACS/wiki/Install-macs2 Version 2.2.7.1
IDR https://github.com/nboley/idr Version 2.0.3
WALT https://github.com/smithlabcode/walt Version 1.0
MethPipe https://github.com/smithlabcode/methpipe Version 5.0.1
HiCUP https://www.bioinformatics.babraham.ac.uk/projects/hicup/ Version 0.8.0
CID http://groups.csail.mit.edu/cgs/gem/cid/ Version 1.0
MICC https://github.com/rakarnik/MICC No release
bedtools https://bedtools.readthedocs.io/en/latest/ Version 2.30.0
samtools http://www.htslib.org/ Version 1.9
deeptools https://deeptools.readthedocs.io/en/latest/ Version 3.3.1
homer http://homer.ucsd.edu/homer/ Version 4.11.1
edgeR https://www.bioconductor.org/packages/release/bioc/html/edgeR.html Version 2.26.5
Open in a new tab

RESOURCES