The association between REST reduction and the development of neuroendocrine prostate cancer (NEPC), a novel drug-resistant and lethal variant of castration-resistant prostate cancer (CRPC), is well established. To better understand the mechanisms underlying this process, we aimed to identify REST-repressed long noncoding RNAs (lncRNAs) that promote neuroendocrine differentiation (NED), thus facilitating targeted therapy-induced resistance. In this study, we used data from REST knockdown RNA sequencing combined with siRNA screening to determine that LINC01801 was upregulated and played a crucial role in NED in prostate cancer (PCa). Using The Cancer Genome Atlas (TCGA) prostate adenocarcinoma database and CRPC samples collected in our laboratory, we demonstrated that LINC01801 expression is upregulated in NEPC. Functional experiments revealed that overexpression of LINC01801 had a slight stimulatory effect on the NED of LNCaP cells, while downregulation of LINC01801 significantly inhibited the induction of NED. Mechanistically, LINC01801 is transcriptionally repressed by REST, and transcriptomic analysis revealed that LINC01801 preferentially affects the autophagy pathway. LINC01801 was found to function as a competing endogenous RNA (ceRNA) to regulate the expression of autophagy-related genes by sponging hsa-miR-6889-3p in prostate cancer cells. In conclusion, our data expand the current knowledge of REST-induced NED and highlight the contribution of the REST-LINC01801-hsa-miR-6889-3p axis to autophagic induction, which may provide promising avenues for therapeutic opportunities.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.

Abstract Background Lineage plasticity is recognized as a treatment-induced resistance mechanism in prostate cancer (PCa) and contributes to the development of neuroendocrine prostate cancer (NEPC), a lethal variant of castration-resistant prostate cancer (CRPC), that is increasing in the era of second-generation anti-hormonal therapy. At present, there are no effective treatments for NEPC. Conclusions and perspectives Following our long-standing interest in studying RE1-silencing transcription factor (REST), a pivotal repressor of neuroendocrine differentiation (NED), we conducted a siRNA screening targeting 147 REST-repressing long non-coding RNAs (lncRNAs) and identified prostate cancer transcript-neuroendocrine 1 (PCAT-NE1) as a novel lncRNA that induces NED through activating autophagy signaling, crucial for NED of prostate adenocarcinoma cell. Analyses of clinical data and samples indicate that PCAT-NE1 is elevated in NEPC. Through gain- and loss-of-function experiments, we find that PCAT-NE1 promotes NED. Mechanistically, PCAT-NE1 acts as a competing endogenous RNA (ceRNA) that sponge hsa-miR-6889-3p, leading to the up-regulation of autophagy-related gene VPS13A and autophagy activation. These findings provide new insight into lncRNA-mediated mechanism for autophagy activation in NEPC and suggest PCAT-NE1 as a potential diagnostic biomarker and therapeutic target for NEPC.

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model with 2kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by CTCF and SMC1, and the KSHV transactivator was recruited to these sites during reactivation. Triggering KSHV gene expression decreased pre-wired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help coordinate viral gene transcription. Importance The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 base pair resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral lifecycle.

Studies on “HIT&RUN” effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses. Importance Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects – those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

In previous studies, we have shown that expression of a viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we identify viral lncRNA binding proteins, and show that an underlying molecular mechanism regulating the KSHV latency-lytic replication switch is via a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Proximity biotin labeling, single cell transcriptomics, and siRNA screening along with complementation studies identified that CHD4's enzymatic activity silences viral gene expression by preventing transcription factory formation. Furthermore, Capture Hi-C, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and proteomics approaches together identify KSHV episome docking sites on host chromosomes and colocalization with a CHD4 protein complex, ChAHP, at epigenetically active genomic regions. PAN RNA binds and competes with CHD4 DNA binding in vitro, and KSHV episomes detached from these host genomic loci sites when reactivation is triggered. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible genomic looping, and is therefore important for the ability of KSHV to establish and maintain latency in a "poised" state at selected host genomic loci.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding , and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

KDM4A is a histone lysine demethylase that has been described as an oncogene in various types of cancer. The importance of KDM4A-mediated epigenetic regulation in tumorigenesis is just emerging. Here, by using Kaposi's sarcoma associated herpesvirus (KSHV) as a screening model, we identified 6 oncogenic virus-induced long non-coding RNAs (lncRNAs) with the potential to open chromatin. RNA immunoprecipitation revealed KSHV-induced KDM4A-associated transcript (KIKAT)/LINC01061 as a binding partner of KDM4A. Integrated ChIP-seq and RNA-seq analysis showed that the KIKAT/LINC01061 interaction may mediate relocalization of KDM4A from the transcription start site (TSS) of the AMOT promoter region and transactivation of AMOT, an angiostatin binding protein that regulates endothelial cell migration. Knockdown of AMOT diminished the migration ability of uninfected SLK and iSLK-BAC16 cells in response to KIKAT/LINC01061 overexpression. Thus, we conclude that KIKAT/LINC01061 triggered shifting of KDM4A as a potential epigenetic mechanism regulating gene transactivation. Dysregulation of KIKAT/LINC01061 expression may represent a novel pathological mechanism contributing to KDM4A oncogenicity.

Neuroendocrine prostate cancer (NEPC) is a drug-resistant lethal variant of castration-resistant prostate cancer (CRPC) that is becoming increasingly common following the application of second-generation anti-hormonal therapy. However, the underlying mechanisms of neuroendocrine differentiation (NED) of prostate cancer (PCa) remain unclear. Long non-coding RNAs (lncRNAs) have recently been implicated in promoting lineage plasticity, a biological process that contributes to the acquisition of neuroendocrine (NE) characteristics. Here, we identified a novel lncRNA LINC01801 that is overexpressed in NEPC and which contributes to NED of PCa. We designated this lncRNA prostate cancer associated transcript-neuroendocrine 1 (PCAT-NE1). PCAT-NE1 acts as a competing endogenous RNA (ceRNA) that sponge hsa-miR-6889-3p, leading to the up-regulation of autophagy-related gene VPS13A and autophagy activation. Forced expression of miR-6889-3p and knockdown of VPS13A both inhibit NED induced by PCAT-NE1 overexpression. This study demonstrates that a novel PCa-associated lncRNA PCAT-NE1 promotes NED of PCa by decreasing the inhibitory effect of miR-6889-3p on autophagy signaling. Therefore, PCAT-NE1-miR-6889-3p-VPS13A axis may provide a new prognostic biomarker and therapeutic strategy for treatment-induced NEPC.

Hi-C (high throughput chromosome conformation capture) is a powerful tool designed to explore the genome-wide collection of chromosomal contacts within a cell population. However, the highly complex nature of Hi-C libraries often precludes the robust identification of interactions between individual restriction fragments with high resolution and statistical significance. This challenge led to the incorporation of sequence capture technology into Hi-C protocols, giving rise to a Capture Hi-C methodology. In this chapter, we review the general principles of the technique and provide a view emanating from our experience employing this approach to study the dynamics of episomal viral DNA contacts during specific stages of the viral life cycle.

Kaposi's sarcoma-associated herpesvirus (KSHV), also designated human herpesvirus 8 (HHV-8), has been linked to Kaposi's sarcoma, as well as to primary effusion lymphoma (PEL), and a subset of multicentric Castleman's disease. KSHV genomes are maintained as episomes within infected cells and the virus exhibits a biphasic life cycle consisting of a life-long latent phase during which only a few viral genes are expressed and no viral progeny are produced and a transient lytic reactivation phase, in which a full repertoire of ~ 80 lytic genes are activated in a temporally regulated manner culminating in the release of new virions. Lytic replication is initiated by a single viral protein, K-Rta (ORF50), which activates more than 80 viral genes from multiple resident viral episomes (i.e., viral chromosomes). One of the major targets of K-Rta is a long non-coding nuclear RNA, PAN RNA (polyadenylated nuclear RNA), a lncRNA that accumulates to exceedingly high levels in the nucleus during viral reactivation. K-Rta directly binds to the PAN RNA promoter and robustly activates PAN RNA expression. Although PAN RNA has been known for over 20 years, its role in viral replication is still incompletely understood. In this perspective, we will briefly review the current understanding of PAN RNA and then describe our current working model of this RNA. The model is based on our observations concerning events that occur during KSHV lytic reactivation including (i) a marked accumulation of RNA Pol II at the PAN promoter, (ii) genomic looping emanating from the PAN locus, (iii) interaction of a second viral lytic protein (ORF57) with K-Rta, PAN RNA and RNA Pol II, (iv) the essential requirement for PAN RNA expression in cis for optimal transcriptional execution needed for the entire lytic program, and (v) ORF57 recruitment of RNA Pol II to the PAN genomic locus. Together our results generate a model in which the PAN locus serves as a hub for sequestration/trapping of the cellular transcriptional machinery proximal to viral episomes. Sequestration at the PAN locus facilitates high levels of viral transcription throughout the viral genome during lytic replication. ORF57 acts as a transcription-dependent transactivator at the PAN locus by binding to both Rta and PAN to locally trap RNA Pol II. The resulting accumulation of high levels of nuclear PAN RNA created by this process is an inducible enhancer-derived (eRNA) by-product that litters the infected cell nucleus.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that infects humans and exhibits a biphasic life cycle consisting of latent and lytic phases. Following entry into host cells, the KSHV genome undergoes circularization and chromatinization into an extrachromosomal episome ultimately leading to the establishment of latency. The KSHV episome is organized into distinct chromatin domains marked by variations in repressive or activating epigenetic modifications, including DNA methylation, histone methylation, and histone acetylation. Thus, the development of KSHV latency is believed to be governed by epigenetic regulation. In the past decade, interrogation of the KSHV epitome by genome-wide approaches has revealed a complex epigenetic mark landscape across KSHV genome and has uncovered the important regulatory roles of epigenetic modifications in governing the development of KSHV latency. Here, we highlight many of the findings regarding the role of DNA methylation, histone modification, post-translational modification (PTM) of chromatin remodeling proteins, the contribution of long non-coding RNAs (lncRNAs) in regulating KSHV latency development, and the role of higher-order episomal chromatin architecture in the maintenance of latency and the latent-to-lytic switch.

In mammals, microRNAs can be actively secreted from cells to blood. miR-29b-3p has been shown to play a pivotal role in muscle atrophy, but its role in intercellular communication is largely unknown. Here, we showed that miR-29b-3p was upregulated in normal and premature aging mouse muscle and plasma. miR-29b-3p was also upregulated in the blood of aging individuals, and circulating levels of miR-29b-3p were negatively correlated with relative appendicular skeletal muscle. Consistently, miR-29b-3p was observed in exosomes isolated from long-term differentiated atrophic C2C12 cells. When C2C12-derived miR-29b-3p-containing exosomes were uptaken by neuronal SH-SY5Y cells, increased miR-29b-3p levels in recipient cells were observed. Moreover, miR-29b-3p overexpression led to downregulation of neuronal-related genes and inhibition of neuronal differentiation. Interestingly, we identified HIF1α-AS2 as a novel c-FOS targeting lncRNA that is induced by miR-29b-3p through down-modulation of c-FOS and is required for miR-29b-3p-mediated neuronal differentiation inhibition. Our results suggest that atrophy-associated circulating miR-29b-3p may mediate distal communication between muscle cells and neurons.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus that infects humans and exhibits a biphasic life cycle consisting of latent and lytic phases. Following entry into host cells, the KSHV genome undergoes circularization and chromatinization into an extrachromosomal episome ultimately leading to the establishment of latency. The KSHV episome is organized into distinct chromatin domains marked by variations in repressive or activating epigenetic modifications, including DNA methylation, histone methylation, and histone acetylation. Thus, the development of KSHV latency is believed to be governed by epigenetic regulation. In the past decade, interrogation of the KSHV epitome by genome-wide approaches has revealed a complex epigenetic mark landscape across KSHV genome and has uncovered the important regulatory roles of epigenetic modifications in governing the development of KSHV latency. Here, we highlight many of the findings regarding the role of DNA methylation, histone modification, post-translational modification (PTM) of chromatin remodeling proteins, the contribution of long non-coding RNAs (lncRNAs) in regulating KSHV latency development, and the role of higher-order episomal chromatin architecture in the maintenance of latency and the latent-to-lytic switch.

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

Non-invasive far infrared radiation (FIR) has been observed to improve the health of patients with coronary artery disease (CAD). Endothelial colony forming cells (ECFCs) contribute to vascular repair and CAD. The goal of this study was to uncover the role of FIR in ECFCs function and to reveal potential biomarkers for indication of FIR therapy in CAD patients. FIR significantly enhanced in vitro migration (transwell assay) and tube formation (tube length) capacities in a subpopulation of CAD ECFCs. Clinical parameters associated with the responsiveness of ECFCs to FIR include smoking and gender. ECFCs from CAD patients that smoke did not respond to FIR in most cases. In contrast, ECFCs from females showed a higher responsiveness to FIR than ECFCs from males. To decipher the molecular mechanisms by which FIR modulates ECFCs functions, regardless of sex, RNA sequencing analysis was performed in both genders of FIR-responsive and FIR-non/unresponsive ECFCs. Gene Ontology (GO) analysis of FIR up-regulated genes indicated that the pathways enriched in FIR-responsive ECFCs were involved in cell viability, angiogenesis and transcription. Small RNA sequencing illustrated 18 and 14 miRNAs that are up- and down-regulated, respectively, in FIR-responsive CAD ECFCs in both genders. Among the top 5 up- and down-regulated miRNAs, down-regulation of miR-548aq-3p in CAD ECFCs after FIR treatment was observed in FIR-responsive CAD ECFCs by RT-qPCR. Down-regulation of miR-548aq-3p was correlated with the tube formation activity of CAD ECFCs enhanced by FIR. After establishment of the down-regulation of miR-548aq-3p by FIR in CAD ECFCs, we demonstrated through overexpression and knockdown experiments that miR-548aq-3p contributes to the inhibition of the tube formation of ECFCs. This study suggests the down-regulation of miR-548aq-3p by FIR may contribute to the improvement of ECFCs function, and represents a novel biomarker for therapeutic usage of FIR in CAD patients.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency virus (HIV)-infected AIDS patients has been ascribed to an interaction between HIV type 1 (HIV-1) and KSHV, focusing on secretory proteins. The HIV-1 secreted protein HIV Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long noncoding RNA (lncRNA) that interacts with HIV Tat. We found that LINC00313 overexpression inhibits cell migration, invasion, and tube formation, and this suppressive effect was relieved by HIV Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components, and this interaction was disrupted by HIV Tat, suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and that HIV Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313, which is upregulated during KSHV reactivation, interacts with HIV Tat to promote endothelial cell motility. These results demonstrate that an lncRNA serves as a novel connector in HIV-KSHV interactions. IMPORTANCE KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

Long non-coding RNAs (lncRNAs) are emerging as novel diagnostic markers of prostate cancer (PCa) and new determinants of castration-resistant PCa (CRPC), an aggressive and metastatic form of PCa. In addition to androgen receptor (AR) signaling, neuroendocrine differentiation (NED) is associated with CRPC. Recent reports demonstrate that the downregulation of repressor element-1 silencing transcription factor (REST) protein is a key step in NED of PCa cells. Here, we report HOTAIR as a novel REST-repressed lncRNA that is upregulated in NED PCa cells and in CRPC. HOTAIR overexpression is sufficient to induce, whereas knockdown of HOTAIR suppressed NED of PCa cells. Gene ontology (GO) analysis of differentially expressed genes under HOTAIR overexpression and in CRPC versus benign prostatic hyperplasia (BPH) suggests that HOTAIR may participate in PCa progression. Taken together, our results provide the first evidence of lncRNA HOTAIR as a driver for NED of PCa cells.

The three-dimensional structure of chromatin organized by genomic loops facilitates RNA polymerase II access to distal promoters. The Kaposi's sarcoma-associated herpesvirus (KSHV) lytic transcriptional program is initiated by a single viral transactivator, K-Rta. Here we report the KSHV genomic structure and its relationship with K-Rta recruitment sites using Capture Hi-C analyses. High-resolution 3D viral genomic maps identify a number of direct physical, long-range, and dynamic genomic interactions. Mutant KSHV chromosomes harboring point mutations in the K-Rta responsive elements (RE) significantly attenuate not only the directly proximate downstream gene, but also distal gene expression in a domain-specific manner. Genomic loops increase in the presence of K-Rta, while abrogation of K-Rta binding impairs the formation of inducible genomic loops, decreases the expression of genes networked through the looping, and diminishes KSHV replication. Our study demonstrates that genomic architectural dynamics plays an essential role in herpesvirus gene expression.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.