Chie Izumiya

KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation

Localized chromatin modifications of histone tails play an important role in regulating gene transcription, and aberration of these processes leads to carcinogenesis. Methylated histone lysine residues, a key player in chromatin remodeling, are demethylated by the JmjC class of enzymes. Here we show that JMJD5 (now renamed KDM8), a JmjC family member, demethylates H3K36me2 and is required for cell cycle progression. Chromatin immunoprecipitation assays applied to human genome tiling arrays in conjunction with RNA microarray revealed that KDM8 occupies the coding region of cyclin A1 and directly regulates transcription. Mechanistic analyses showed that KDM8 functioned as a transcriptional activator by inhibiting HDAC recruitment via demethylation of H3K36me2, an epigenetic repressive mark. Tumor array experiments revealed KDM8 is overexpressed in several types of cancer. In addition, loss-of-function studies in MCF7 cells leads to cell cycle arrest. These studies identified KDM8 as an important cell cycle regulator.

Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Is a Coregulator of K-Rta: Physical Association and Promoter-Dependent Transcriptional Repression

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposis sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

Ku Is a Novel Transcriptional Recycling Coactivator of the Androgen Receptor in Prostate Cancer Cells

The androgen receptor (AR) dynamically assembles and disassembles multicomponent receptor complexes in order to respond rapidly and reversibly to fluctuations in androgen levels. We are interested in identifying the basal factors that compose the AR aporeceptor and holoreceptor complexes and impact the transcriptional process. Using tandem mass spectroscopy analysis, we identified the trimeric DNA-dependent protein kinase (DNA-PK) complex as the major AR-interacting proteins. AR directly interacts with both Ku70 and Ku80 in vivo and in vitro, as shown by co-immunoprecipitation, glutathione S-transferase pull-down, and Sf9 cell/baculovirus expression. The interaction was localized to the androgen receptor ligand binding domain and is independent of DNA interactions. Ku interacts with AR in the cytoplasm and nucleus regardless of the presence or absence of androgen. Ku acts as a coactivator of AR activity in a luciferase reporter assay employing both Ku-defective cells and Ku small interfering RNA knock-down in a prostate cancer cell line. DNA-PK catalytic subunit (DNA-PKcs) also acts as a coactivator of androgen receptor activity in a luciferase reporter assay employing DNA-PKcs defective cells. AR nuclear translocation is not affected in Ku defective cells, implying Ku functionality may be mainly nuclear. Chromatin immunoprecipitation experiments demonstrated that both Ku70 and Ku80 interact with the prostate-specific antigen promoter in an androgen-dependant manner. Finally, in vitro transcription assays demonstrated Ku involvement in transcriptional recycling with androgen dependent promoters.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

ABSTRACT The oncogenic herpesvirus, Kaposis sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.

Cell cycle regulation by Kaposi's sarcoma-associated herpesvirus K-bZIP: Direct interaction with cyclin-CDK2 and induction of G1 growth arrest

ABSTRACT In order to cope with hostile host environments, many viruses have developed strategies to perturb the cellular machinery to suit their replication needs. Some herpesvirus genes protect cells from undergoing apoptosis to prolong the lives of infected cells, while others, such as Epstein-Barr virus Zta, slow down the G1/S transition phase to allow ample opportunity for transcription and translation of viral genes before the onset of cellular genomic replication. In this study, we investigated whether Kaposis sarcoma-associated herpesvirus (KSHV) K-bZIP, a homologue of the Epstein-Barr virus transcription factor BZLF1 (Zta), plays a role in cell cycle regulation. Here we show that K-bZIP physically associates with cyclin-CDK2 and downmodulates its kinase activity. The association can be detected in the natural environment of KSHV-infected cells without artificial overexpression of either component. With purified protein, it can be shown that the interaction between K-bZIP and cyclin-CDK2 is direct and that K-bZIP alone is sufficient to inhibit CDK2 activity. The interacting domain of K-bZIP has been mapped to the basic region. The result of these associations is a prolonged G1 phase, accompanied by the induction of p21 and p27 in a naturally infected B-cell line. Thus, in addition to the previously described transcription and genome replication functions, a new role of K-bZIP in KSHV replication is identified in this report.

NF-κB Serves as a Cellular Sensor of Kaposi's Sarcoma-Associated Herpesvirus Latency and Negatively Regulates K-Rta by Antagonizing the RBP-Jκ Coactivator

ABSTRACT Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposis sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-κB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-κB expression was induced. Many K-Rta-responsive promoters modulated by NF-κB contain the sequence of the RBP-Jκ binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-κB. Gel shift assays demonstrated that NF-κB inhibits the binding of RBP-Jκ and forms a complex with RBP-Jκ. Our results support a model in which a balance between K-Rta/RBP-Jκ and NF-κB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jκ and NF-κB on viral promoters controls viral gene expression mediated by K-Rta.

A Lytic Viral Long Noncoding RNA Modulates the Function of a Latent Protein

ABSTRACT Latent Kaposis sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.

A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus

Kaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposis sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters were repressed by K-bZIP. The occupancy of K-Rta and K-bZIP across KSHV promoters was analyzed by chromatin immunoprecipitation with a viral promoter-chip in BCBL-1 cells. In addition, acetylation of local histones was examined to determine accessibility of promoters during latency and reactivation. Finally, 10 promoters were selected to study the dynamics of transcription factor recruitment. This study provides a comprehensive overview of the responsiveness of KSHV promoters to K-Rta and K-bZIP, and describes key chromatin changes during viral reactivation.

Protein Arginine Methyltransferase 1-directed Methylation of Kaposi Sarcoma-associated Herpesvirus Latency-associated Nuclear Antigen

Background: Post-translational modifications generate functional heterogeneity of viral regulatory factors. Results: Viral chromatin association by Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is modulated by protein arginine methyltransferase 1 (PRMT1)-directed methylation. Conclusion: Methylation of KSHV LANA antagonizes viral reactivation. Significance: Protein methylation contributes to the functional properties of viral regulatory proteins, including KSHV LANA. The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.

Kaposi's Sarcoma-Associated Herpesvirus K-Rta Exhibits SUMO-Targeting Ubiquitin Ligase (STUbL) Like Activity and Is Essential for Viral Reactivation

The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposis sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rtas ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.