Biology subjects

Tempera, I.

Publications and source records attributed to Tempera, I..

9 recordsLinked to original sources

bioRxiv2026

NAD Metabolism Drives CTCF-Cohesin Chromatin Regulation in EBV-Transformed B-Cells

Chromatin architecture regulates gene expression, and it is maintained by architectural proteins such as CTCF and the cohesin complex. In Epstein Barr virus infected cells, viral chromatin organization is stabilized through the interaction between PARP1 and CTCF, ensuring maintenance of latent gene expression. EBV B cells exhibit elevated PARP1 activity, which imposes a substantial metabolic burden due to NAD consumption by PARP1 itself. We demonstrate that efficient EBV infection and B-cell transformation rely on the NAD salvage pathway to sustain PARP1 activity and CTCF and cohesin chromatin folding. Pharmacologic inhibition of the rate-limiting enzyme NAMPT with FK866 phenocopies the chromatin architectural disruption induced by PARP1 and changes in NAD levels affects CTCF/cohesin chromatin architectural remodeling and loop extrusion. Moreover, NAMPT inhibition reduces tumor burden in EBV xenograft models. These findings identify a critical metabolic dependency linking NAD homeostasis to CTCF-cohesin mediated chromatin architecture and reveal a therapeutic vulnerability in EBV-associated malignancies.

microbiology↗

bioRxiv2026

EBNA1 INHIBITORS REVEAL CDC7 AND POU2F1 AS DIRECT FUNCTIONAL TARGETS IN EBV EPITHELIAL CANCERS

Epstein-Barr virus (EBV) latent infection is causally linked to several epithelial cancers, including endemic forms of undifferentiated nasopharyngeal carcinoma (NPC) and to a subtype of gastric cancer (GC). EBNA1 is the viral-encoded sequence-specific DNA-binding protein required for episome maintenance but also contributes to host-cell survival through multiple mechanisms including binding to host chromosome. We previously developed small molecule inhibitors of EBNA1 DNA-binding that block host cell cycle progression and growth of EBV+ tumors in vivo. However, the underlying molecular mechanisms of EBNA1 function and inhibition have not been completely elucidated. In this study, we employ VK1727 to inhibit EBNA1 DNA-binding to viral and cellular genomes in three EBV+ epithelial tumors (PDX C15, C666-1 and SNU719). We integrate EBNA1ChIP-seq and transcriptomic RNA-seq analyses to identify the cell cycle dependent kinase CDC7 and a stem cell transcription factor POU2F1 as direct functional targets of EBNA1 in these epithelial cancers. EBNA1 binding to CDC7 promoter and POU2F1 intron promotes RNA Pol II-pS5 to initiate transcription of these two genes. We show that CDC7 inhibitor Simurosertib is epistatic, while Bcl2 inhibitor Venetoclax is synergistic with VK1727 in the inhibition of EBV+ epithelial cancer cell proliferation and survival. Our study reveals new functional gene targets and pathways of VK1727 in EBV+ epithelial cancers that provide new biomarkers and combinatorial strategies to treat EBV-driven cancers. IMPORTANCEEBNA1 is essential for EBV latency and tumorigenesis, but its mechanism of action on host gene expression is not yet known. Small molecule inhibitors of EBNA1 DNA-binding block cell cycle progression and inhibit growth of EBV+ tumors. In this study, we use the EBNA1 small molecule inhibitor VK1727 to identify cellular gene targets that are bound by EBNA1 and deregulated by its pharmacological inhibition in EBV+ epithelial cancer cell lines and an NPC PDX mouse model. We identify cycle dependent kinase CDC7 and the stem cell transcription factor POU2F1 as EBNA1 bound and regulated genes important for EBV epithelial cancer proliferation. These findings not only decipher molecular mechanism how VK1727 blocks cell cycle progression and inhibits cell proliferation but also provide two new cellular gene targets and pathways for therapeutic intervention in EBV+ epithelial cancers.

microbiology↗

bioRxiv2026

Lamin A/C maintains genome topology and regulates transcriptional programs essential for virus-driven B cell activation.

Lamin A/C is a crucial structural component of the nuclear lamina that influences chromatin organization and gene regulation. In this study, we demonstrate that lamin A/C is vital for maintaining higher-order genome organization and transcriptional programs that support EBV-driven B-cell activation. Loss of lamin A/C in a B-lymphoblastoid cell line caused significant three-dimensional reorganization of the genome, evidenced by the loss of long-range chromatin loops, an increase in short-range contacts, and redistribution of H3K9me2- marked heterochromatin. These structural disruptions were linked to widespread changes in gene expression affecting metabolic, signaling, and differentiation pathways. Mechanistically, lamin A/C influences the nuclear positioning and transcription of CTCF-bound loci by preventing their relocation to the periphery and their association with lamin B1. Blocking H3K9me2 deposition mimicked the transcriptional effects of lamin A/C depletion and revealed increased sensitivity to PI3K inhibitors. Overall, our results identify lamin A/C as a key organizer of genome structure and epigenetic regulation in EBV-infected B cells, uncovering a lamin-dependent pathway that connects nuclear architecture, metabolism, and viral disease processes.

microbiology↗

bioRxiv2025

Integrated transcriptomic and methylome analysis reveals retinoic acid pathway activation after decitabine treatment in EBV associated gastric cancer

Epstein-Barr virus associated gastric cancer (EBVaGC) accounts for [~]9-10% of gastric cancers worldwide and is defined by a distinctive molecular profile, including extreme hypermethylation of the DNA. Targeting this aberrant methylation may be a potential therapeutic strategy. EBV+ gastric cancer cell lines (YCCEL1, SNU719) and EBV- lines (AGS, SNU16, MKN74) were treated with a DNA methyl-transferase inhibitor (DNMT), decitabine (DCB), for three days followed by RNA sequencing to identify EBV-specific responses. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) was performed in EBV+ cell-lines and integrated with expression data to identify epigenetically regulated networks. While DCB induced broad transcriptional changes across all lines, EBV+ cells exhibited the strongest transcriptional response, sharing many upregulated genes. Many of these EBV+ specific genes were expressed at lower baseline levels in EBV+ tumors from TCGA. DCB predominantly reduced methylation at highly methylated intergenic CpGs, with a subset of promoters undergoing significant demethylation. Integrated analysis revealed a strong inverse correlation between promoter demethylation and gene expression, implicating multiple cancer-relevant pathways. Upstream regulator analysis and motif enrichment indicated that regions losing methylation were enriched for retinoic acid receptor (RAR) binding motifs, suggesting that DCB-mediated demethylation restores RA pathway accessibility and transcriptional activity. Further, inhibiting RAR signaling reduced DCB induced apoptosis. Although DCB can induce both host gene re-expression and viral lytic gene activation in EBV-positive tumors, its impact on RA signaling in EBVaGC has not been studied. Decitabine promotes extensive epigenetic reprogramming in EBVaGC, with preferential effects in CIMP-positive, EBV-infected cell lines. ImportanceEBV+ gastric cancer contains hypermethylated DNA and despite this distinct molecular phenotype there are currently no EBV-specific treatments available. Using an FDA approved inhibitor to target hypermethylated DNA and multi-omics approach to study the cellular response, we uncovered epigenetically altered transcriptional networks that may be further exploited to improve potential therapy. Among the pathways disrupted, retinoic acid signaling is of particular interest, as retinoid receptors such as RAR and RAR{beta} are frequently hypermethylated and repressed in EBVaGC. Our findings indicate that DNMT inhibition can partially reverse RA receptor silencing, supporting further investigation of DNMTi-RA combination strategies as a novel therapy for EBV+ gastric cancer.

microbiology↗

bioRxiv2025

Histone Variant H2A.Z Cooperates with EBNA1 to Maintain Epstein-Barr Virus Latent Epigenome

Chromatin structure plays a central role in the regulation of Epstein-Barr Virus (EBV) latency. The histone variant H2A.Z.1 has been implicated in chromatin structures associated with initiation of transcription and DNA replication. Here, we investigate the functional role of H2AZ.1 in the regulation of EBV chromatin, gene expression and copy number during latent infection. We found that H2A.Z.1 is highly enriched with EBNA1 binding sites at oriP and Qp, and to a lesser extent with transcriptionally active CTCF binding sites on the EBV genomes in both Mutu I Burkitt lymphoma (BL) and SNU719 EBV-associated gastric carcinoma (EBVaGC) cell lines. RNA-interference depletion of H2A.Z.1 resulted in the reactivation of viral lytic genes (ZTA and EAD) and increases viral DNA copy numbers in both MutuI and SNU719 cells. H2A.Z depletion also led to a decrease in EBNA1 binding to oriP and Qp, on the viral episome as well as on oriP plasmids independently of other viral genes and genomes. H2A.Z.1 depletion also reduced peaks of H3K27ac and H4K20me3 at regulatory elements in the EBV genome. In the cellular genome, H2A.Z.1 colocalized with only a subset of EBNA1 binding sites and H2A.Z.1 depletion altered transcription of genes associated with myc targets and mTORC1 signaling. Taken together, these findings indicate that H2A.Z.1 cooperates with EBNA1 to regulate chromatin structures important for epigenetic programming of the latent episome. ImportanceCellular factors the restrict latent viral reactivation are of fundamental importance. We have found that the cellular histone variant H2A.Z functions in cooperation with the Epstein-Barr Virus (EBV) latency maintenance protein EBNA1 to establish a stable epigenome and restrict lytic cycle reactivation during latency. We show that H2A.Z co-occupies EBNA1 binding sites on the EBV and host genome, and that depletion of H2A.Z leads to robust reactivation of EBV from latency. H2A.Z is important for the function of EBNA1 at the origin of plasmid (oriP) replication and establishing EBV epigenetic marks. H2A.Z binds with EBNA1 at cellular binding sites and controls the expression of cellular genes in the cMyc and mTORC1 pathways that are also implicated in control of EBV latency.

molecular biology↗

bioRxiv2024

The DNA loop release factor WAPL suppresses Epstein-Barr virus latent membrane protein expression to maintain the highly restricted latency I program

Epstein-Barr virus (EBV) uses latency programs to colonize the memory B-cell reservoir, and each program is associated with human malignancies. However, knowledge remains incomplete of epigenetic mechanisms that maintain the highly restricted latency I program, present in memory and Burkitt lymphoma cells, in which EBNA1 is the only EBV-encoded protein expressed. Given increasing appreciation that higher order chromatin architecture is an important determinant of viral and host gene expression, we investigated roles of Wings Apart-Like Protein Homolog (WAPL), a host factor that unloads cohesins to control DNA loop size and that was discovered as an EBNA2-associated protein. WAPL knockout (KO) in Burkitt cells de-repressed LMP1 and LMP2A expression but not other EBV oncogenes to yield a viral program reminiscent of EBV latency II, which is rarely observed in B-cells. WAPL KO also increased LMP1/2A levels in latency III lymphoblastoid cells. WAPL KO altered EBV genome architecture, triggering formation of DNA loops between the LMP promoter region and the EBV origins of lytic replication (oriLyt). Hi-C analysis further demonstrated that WAPL KO reprograms EBV genomic DNA looping. LMP1 and LMP2A de-repression correlated with decreased histone repressive marks at their promoters. We propose that EBV coopts WAPL to negatively regulate latent membrane protein expression to maintain Burkitt latency I. Author SummaryEBV is a highly prevalent herpesvirus etiologically linked to multiple lymphomas, gastric and nasopharyngeal carcinomas, and multiple sclerosis. EBV persists in the human host in B-cells that express a series of latency programs, each of which is observed in a distinct type of human lymphoma. The most restricted form of EBV latency, called latency I, is observed in memory cells and in most Burkitt lymphomas. In this state, EBNA1 is the only EBV-encoded protein expressed to facilitate infected cell immunoevasion. However, epigenetic mechanisms that repress expression of the other eight EBV-encoded latency proteins remain to be fully elucidated. We hypothesized that the host factor WAPL might have a role in restriction of EBV genes, as it is a major regulator of long-range DNA interactions by negatively regulating cohesin proteins that stabilize DNA loops, and WAPL was found in a yeast 2-hybrid screen for EBNA2-interacting host factors. Using CRISPR together with Hi-ChIP and Hi-C DNA architecture analyses, we uncovered WAPL roles in suppressing expression of LMP1 and LMP2A, which mimic signaling by CD40 and B-cell immunoglobulin receptors, respectively. These proteins are expressed together with EBNA1 in the latency II program. We demonstrate that WAPL KO changes EBV genomic architecture, including allowing the formation of DNA loops between the oriLyt enhancers and the LMP promoter regions. Collectively, our study suggests that WAPL reinforces Burkitt latency I by preventing the formation of DNA loops that may instead support the latency II program.

microbiology↗

bioRxiv2023

PARP1 Inhibition Halts EBV+ Lymphoma Progression by Disrupting the EBNA2/MYC Axis

PARP1 has been shown to regulate EBV latency. However, the therapeutic effect of PARP1 inhibitors on EBV+ lymphomagenesis has not yet been explored. Here, we show that PARPi BMN-673 has a potent anti-tumor effect on EBV-driven LCL in a mouse xenograft model. We found that PARP1 inhibition induces a dramatic transcriptional reprogramming of LCLs driven largely by the reduction of the MYC oncogene expression and dysregulation of MYC targets, both in vivo and in vitro. PARP1 inhibition also reduced the expression of viral oncoprotein EBNA2, which we previously demonstrated depends on PARP1 for activation of MYC. Further, we show that PARP1 inhibition blocks the chromatin association of MYC, EBNA2, and tumor suppressor p53. Overall, our study strengthens the central role of PARP1 in EBV malignant transformation and identifies the EBNA2/MYC pathway as a target of PARP1 inhibitors and its utility for the treatment of EBNA2-driven EBV-associated cancers. Significance StatementA promising approach to treating EBV-driven malignancies involves targeting cancer and EBV biology. However, investigating host factors that co-regulate EBV latent gene expression, such as PARP1, has been incomplete. Our study demonstrates that the PARP1 inhibitor BMN-673 effectively reduces EBV-driven tumors and metastasis in an LCL xenograft model. Additionally, we have identified potential dysregulated mechanisms associated with PARP1 inhibition. These findings strengthen the role of PARP1 in EBV+ lymphomas and establish a link between PARP1 and the EBNA2/MYC axis. This has important implications for developing therapeutic approaches to various EBV-associated malignancies.

cancer biology↗

bioRxiv2021

Nuclear Lamina binds the EBV genome during latency and regulates viral gene expression

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus. AUTHOR SUMMARYEpstein-Barr virus (EBV) is a common herpesvirus that establishes a lifelong latent infection in a small fraction of B cells of the infected individuals. In most cases, EBV infection is asymptomatic; however, especially in the context of immune suppression, EBV latent infection is associated with several malignancies. In EBV+ cancer cells, latent viral gene expression plays an essential role in sustaining the cancer phenotype. We and others have established that epigenetic modifications of the viral genome are critical to regulating EBV gene expression during latency. Understanding how the EBV genome is epigenetically regulated during latent infection may help identify new specific therapeutic targets for treating EBV+ malignancies. The nuclear lamina is involved in regulating the composition and structure of the cellular chromatin. In the present study, we determined that the nuclear lamina binds the EBV genome during latency, influencing viral gene expression. Depleting one component of the nuclear lamina, lamin A/C, increased the expression of latent EBV genes associated with cellular proliferation, indicating that the binding of the nuclear lamina with the viral genome is essential to control viral gene expression in infected cells. Our data show for the first time that the nuclear lamina may be involved in the cellular response against EBV infection by restricting viral gene expression.

microbiology↗

bioRxiv2019

EBV latent membrane protein 1 induces lipogenesis and confers sensitivity to inhibition of fatty acid synthase in B cells.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkins and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitts lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1 and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B-cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1 and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could potentially be used in the treatment of LMP1+ EBV associated malignancies by targeting a LMP1-specific dependency on lipogenesis. ImportanceDespite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of viral-associated malignancies. Non-cancerous cells preferentially obtain fatty acids from dietary sources whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1 and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis could be an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

microbiology↗