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Complex Interactions between Cohesin and CTCF in Regulation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription.

ABSTRACT: CTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long-range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with both proteins previously reported to act as restrictive factors for lytic cycle transcription and virion production. In this study, we examined the interdependence of CTCF and cohesin binding to the KSHV genome. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that cohesin binding to the KSHV genome is highly CTCF dependent, whereas CTCF binding does not require cohesin. Furthermore, depletion of CTCF leads to the almost complete dissociation of cohesin from sites at which they colocalize. Thus, previous studies that examined the effects of CTCF depletion actually represent the concomitant depletion of both CTCF and cohesin components. Analysis of the effects of single and combined depletion indicates that CTCF primarily activates KSHV lytic transcription, whereas cohesin has primarily inhibitory effects. Furthermore, CTCF or cohesin depletion was found to have regulatory effects on cellular gene expression relevant for the control of viral infection, with both proteins potentially facilitating the expression of multiple genes important in the innate immune response to viruses. Thus, CTCF and cohesin have both positive and negative effects on KSHV lytic replication as well as effects on the host cell that enhance antiviral defenses.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to Kaposi's sarcoma and several lymphoproliferative diseases. KSHV, like other herpesviruses, intermittently reactivates from latency and enters a lytic cycle in which numerous lytic mRNAs and proteins are produced, culminating in infectious virion production. These lytic proteins may also contribute to tumorigenesis. Reactivation from latency is controlled by processes that restrict or activate the transcription of KSHV lytic genes. KSHV gene expression is modulated by binding of the host cell proteins CTCF and cohesin complex to the KSHV genome. These proteins bind to and modulate the conformation of chromatin, thereby regulating transcription. We have analyzed the interdependence of binding of CTCF and cohesin and demonstrate that while CTCF is required for cohesin binding to KSHV, they have very distinct effects, with cohesin primarily restricting KSHV lytic transcription. Furthermore, we show that cohesin and CTCF also exert effects on the host cell that promote antiviral defenses.

SUBMITTER: Li D

PROVIDER: S-EPMC6955261 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Complex Interactions between Cohesin and CTCF in Regulation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription.

Li Dajiang D Mosbruger Tim T Verma Dinesh D Swaminathan Sankar S

Journal of virology 20200106 2

CTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long-range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with ...[more]

PMID: 31666380

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as gene-specific activators of some promoters, but differ in acting as global negative regulators of transcription.

Project description:The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.

Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle-viral latency and the productive lytic cycle-and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.ImportanceKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, in which they escape immune detection by restricting viral gene expression. Importantly, however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B [ARID3B]) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle-dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.

Dataset Information

Complex Interactions between Cohesin and CTCF in Regulation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription.

Publications

Complex Interactions between Cohesin and CTCF in Regulation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription.

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