Progress 04/01/15 to 03/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Presentations: Mr. Yifei Lio (Graduate Student, TAMU) 2017 TAMU Student Research Week (March 29th, 2017) Title: Marek's disease virus encoded US3 protein interacts and stabilizes Meq oncoprotein Author: Yifei Liao, Kanika Bajwa, Owais A. Khan, Mohammad AI-Mahmood, Yoshihiro Izumiya, Blanca Lupiani and Sanjay M. Reddy 23rd Annual COM-GSO Research Symposium (April 20th, 2018) Title: Role of Marek's disease virus (MDV) US3 protein kinase in phosphorylation of MDV Meq and cellular CREB protein Author: Yifei Liao, Kanika Bajwa, Owais A. Khan, Mohammad AI-Mahmood, Yoshihiro Izumiya, Blanca Lupiani and Sanjay M. Reddy (Abstract booklet: Poster #29, p36) Dr. Yoshihiro Izumiya FAANG-Plant and Animal Genome XXVI Conference Title: Herpesvirus Reactivation as a Model to Study Spatiotemporal Gene Regulation (invited speaker and poster presentation) Training: Ms. Di Jin (Visiting graduate student in Izumiya's lab) received bioinformatics training by attending 4 days bioinformatics workshop at UC Davis. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
MDV Meq decreases histone acetylation globally Marek's disease virus (MDV) causes T-cell lymohomas in chickens that lead to significant economic impact in poultry industry world-wide. Responsible viral gene (Meq), which is essential to induce T-cell lymphoma has been identified. Meq is a basic leucine zipper (bZIP) protein. Through the leucine zipper region, Meq forms homodimer and heterodimer with othe bZIP proteins such as c-Jun, JunB and Fos. The Meq homodimers and heterodimers bind to specific DNA sequence called Meq-responsive elements I and II (MERE I and MERE II). The Meq/c-Jun heterodimers bind with high affinity to AP-l like MERE I site to upregulate transcription of various gene promoters including meq promoter, while Meq homodimers bind to MERE II sites and repress the transcription of bi-directional promoter between pp38 and pp14. We examined if Meq regulates histone modifications to alter gene expression. We found that overexpression of Meq in CEF decreased H3K9Ac in transfected cells. Co-immunoprecipitation studies with MDV naturally infected cells showed that Meq intreacts with histone deacetylase 1 and 2, and co-migrate with strong cellular repressor CtBP1 (C-terminal binding protein). These studies indicated that Meq may recruit histone deacetylase to the chromatin by overexpresion and decrease histone acetylation. MDV latency associate transcripts (LAT) and Meq During course of our studies, we accidentally found that MDV LAT, a viral long non-coding RNA, expresses significantly higher copies in MDV infected T-cells. The viral latent transcript expresses about 100 to 1,000 times more than other viral genes. We further developed technique that are used to visualize MDV LAT and chicken telomere DNA sequence same time in situ. The study visualized MDV transcript in naturally infected T-cells first time and we found that LAT signals were significantly overlapped with host telomere sequence. We also found that DAPI signal was clearly depleted at the nuclear region where the LAT were expressed. We speculated that higher gene transcription at host telomere genomic region may disrupt host chromatin structures as we saw as depletion of DAPI and induce chromosome instability; this mechanism may contribute to the potent MDV mediated tumorigenesis. It is important to mention that there are other herpesvirus (HHV-6, 7, EHV2) encodes telomere like sequence in their genomes and integrate into host telomere sequence region. Furthermore, our ChIP-seq studies showed that Meq and chicken cJun binding sites were enriched at LAT promoter region, indicating a function of the latent viral transcription factor (Meq) may be to maintain LAT transcription in latently infected cells. These studies indicated that epigenetic alteration induced by MDV infection is caused by Meq though activation of LAT from host telomere genomic region. Drastic chromatin instability induced by integration of active MDV genome, which is maintained by Meq, might be the molecular mechanism of MDV mediated tumorigenesis. To further elucidate the function of LAT in MDV life cycle and tumorigenesis, we have generated recombinant MDV, which harbors strong poly(A) signals at immediate after LAT transcription initiation site. We are planning to infect the recombinant MDV into chicken and examine viral replication and tumorigenesis. Genomic looping formation between viral genome and host genome in naturally infected cells. We recently reported viral genomic looping formation in cis and its significance in viral gene expression (Campbell et al., 2018 Nature Communications). We extended the studies to examine interactions between viral and host chromosome. First, we performed chromosome conformation capture analyses and examined genomic loop formation within MDV genomes. The results showed that MDV LAT region forms genomic loops with other viral genomic region at much higher frequencies. Interestingly, MDV LAT region was found to be associated with Meq promoter region in cis, indicating LAT promoter activation is linked to MDV Meq protein expression and vice versa. We are also performing capture Hi-C studies to generate comprehensive MDV genomic interaction map as well as identifying interaction sites on host genomic region. We speculate genomic loops between MDV LAT region and host chromosome at host telomere will have significant impact on the host gene expression at interacting sites, because telomere region has been shown to enrich specific histone modifying enzymes to maintain chromosome stability. We have been working hard to prepare Capture Hi-C samples; however, we have been failing to obtain high quality Hi-C ligation product in order to prepare for the sequencing libraries. We recently adapted new protocol from a company and we will continue to repeat the experiments. Revealing these novel epigenetic gene regulations though viral genome (not viral protein) may identify new oncogenic mechanisms; MDV with chicken infection model will be the best viral oncogenesis model to study such possibility. Specific Progress doe listed objects: Biochemical and genetic studies listed in major goals have been completed. Interaction of specific cJun/Meq complex in the chicken genome in vivo was changed a little as we focus on more on global effects of MDV genome integration at chicken telomere region. Based on epigenetic histone modification at specific genomic regions in different chicken T-cell lines, we feel such histone modification changes at specific genomic sites would be a "by product" of the induced chromatin instabilities. We decided to focus more on larger changes by the insertion of active MDV genome in the host telomere region and effects of Meq protein in LAT expression from the host telomere regions. We feel that studying genomic instablity by the MDV genome integration and expression of LAT will be different project. We will submit new grant application to further pursue the research direction.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Yuanzhi Lyu, Kazushi Nakano, Ryan R. Davis, Clifford G. Tepper, Mel Campbell, Yoshihiro Izumiya. Zic2 Is Essential for Maintenance of Latency and is a Target of Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation. Journal of Virology 91 (21) e00980-17
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mel Campbell, Tadashi Watanabe, Kazushi Nakano, Ryan R. Davis, Yuanzhi Lyu, Clifford G. Tepper, Blythe Durbin-Johnson, Masahiro Fujimuro, Yoshihiro Izumiya. KSHV episomes reveal dynamic chromatin loop formation with domain-specific gene regulation. Nature Communications 9 (1): 49
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Christopher P. Chen, Frank Chuang, Yoshihiro Izumiya. Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy. Journal of Visual Experiments 131: e56832
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Chie Izumiya, Di Jin, Kazushi Nakano, Sanjay Reddy, Yoshihiro Izumiya. Regulation of Marek's disease virus Reactivation by association with CtBP1. Journal of Virology
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Progress 04/01/16 to 03/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Presentations: Dr. Aijun Sun(Postdoctoral research fellow) Oral presentation Title:Marek's disease virus encoded LORF9 but not LORF10 is involved in Pathogenesis 11th International Symposium on Marek's Disease and Avian Herpesviruses, Tours, France Dr. Guoqing Zhuang (Postdoctoral research fellow) Oral presentation Title:Marek's disease virus cluster 3 microRNAs regulates early cytolytic infection leading to reduced pathogenesis 11th International Symposium on Marek's Disease and Avian Herpesviruses, Tours, France Ms. Kanika Bajwa (graduate student) Oral presentation Title: Marek's disease virus encoded, Meq-vIL8 splice variant is not essential for in vitro replication 11th International Symposium on Marek's Disease and Avian Herpesviruses, Tours, France Dr. Yuanzhi Lyu (Postdoctoral fellow) Oral presentation and poster presentation Title: ZIC2 is degraded by KSHV K-Rta through the ubiquitin-proteasome pathway and is required for the maintenance of KSHV latency 19th International Workshop on KSHV and Related Agent, Los Angeles, California. Dr. Kazushi Nakano (visiting assistant professor) Poster presentation Title: KSHV Replication in Oral Epithelial Cells; RNA-Sequence Analyses During KSHV Lytic Replication 19th International Workshop on KSHV and Related Agent, Los Angeles, California. Yoshihiro Izumiya 1. Epigenetic Alternation via Marek's disease virus infection, San Diego, CA Epigenetic USDA, principle investigator meeting. (Poster presentation) 2. KSHV Chromatin Looping Facilitates Effective Gene Expression; Gene Cluster Activation via Direct K-Rta Binding at a Viral Chromatin Hub (Oral and poster presentation),19th International Workshop on KSHV and Related Agent, Los Angeles, California. 3. PEP005 in combination with JQ1 as an effective therapeutic approach for KSHV-associated primary effusion lymphoma through inhibition of IL-6 production and induction of oncolysis by viral reactivation (Oral and poster presentation),19th International Workshop on KSHV and Related Agent, Los Angeles, California. Seminars: Title: 4D Viral Gene Regulation, Yoshihiro Izumiya, March 15, 2016 (UC Davis), Davis California Training: Drs. Kazushi Nakano and Yuanzhi Lyu Receiving bioinformatics training by attending 4 days bioinformatics workshop at UC Davis. Ms. Di Jin joined our laboratory from Nanjing agricultural university from October 2015 for one year. She is studying molecular biology of MDV gene regulation in my laboratory. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are expecting to receive larger amount of MDV tumor samples from different chickens to perform multiple next generation seqeuncing studies. Our "lock-down" probes will be used to enrich MDV genomic sequence for the seqeuncing analyses. If we could not obtain sufficient amount of MDV tumor samples, we will instead perform RNA sequencing studies. With RNA-seqeuncing studies with our lock-down probes, we will generate next generation of transcriptional map, which provides very detail of multiple splicing variants as well as novel fusion transcripts of MDV genes that are expressed in MDV infected tissue samples. Such studies can not be done without enrichment of MDV transcripts and we will take advantage of our resources generated by the grant. Notheless, our primary focus is to obtain sufficient amount of tissue samples by infecting MDV and isolate multiple tumor samples from different birdsand T-cells at different time points after infection.
Impacts
What was accomplished under these goals?
(1) Research Proress on MDV studies Meq/CtBP and Meq/cJun recruitment sites on MDV and chicken genomes in MDV-infected Chicken T-cells. Marek's disease is a highly contagious lymphoproliferative disease of chickens, which caused significant economic impact in poultry industry world-wide. The disease is caused by a herpesvirus, Marek's disease virus (MDV). MDV encodes a basic leucine zipper (bZIP) protein, Meq (MDV EcoQ), which is the major oncoprotein of MDV. Like other oncoproteins, Meq participates in the shut-off of tumor suppressor pathway. Indeed, Meq is found to physically interact with tumor suppressors CtBP and p53. Meq is known to provide a host cell survival function, but the mechanism is not known. We demonstrate that Meq binds directly to the promoter regulatory regions and up- /down-regulates the transcription of host and viral genes that are important for the initiation of tumorigenesis of MDV-infected cells. In this project period, we performed ChIP-Seq analyses of Meq, CtBP, and cJun in chicken T-cell lines. Data from ChIP-Seq with MDV-infected chicken T cell lines were aligned to the Gullus_gullus-5.0 chicken genome by using the Bowtie2 algorithm. Peak calling was performed by using the MACS2 algorithm with combined IgG as a reference set. All peaks with false discovery rates (FDRs) of 1% for 2 cell lines were overlapped to create a list of unique binding sites. ChIP-Seq analyses allowed successful peak-called in MDV infected chicken T-cells. We identified six major recruitment sites of the MEQ, CtBP, and c-Jun complex. One genomic region was bound primarily by Meq/CtBP complex and the others were mixtures of three proteins. In addition, we identified number of Meq/CtBP and Meq/cJun recruitment sites on the chicken genomes. Although we have tried to perform same set of experiments with MDV infected tumor samplesthat were isolated from MDV infected chickens;however the amount genomic DNA precipitated from chicken tumor samples were too low to process forsequence analyses.With viral oncoprotein binding in our hands, we are performing unbiased chromosome conformation capture studies with Hi-C analyses on MDV infected-chicken T cells to examine relationship between Meq bindings and genomic looping formations. We are examining ifMeq induces genomic structure changes to deregulate host cell gene expression. Detail classification of MDV genes and establishment of MDV PCR arrays To study effects of Meq recruitment on MDV genome in genome-wide, we have prepared MDV PCR array, in which we designed primer pair for every MDV open reading flame. MDV infected T-cells were reactivated by combination of TPA and sodium butylate, and total RNA was prepared. Reactivated samples were then used to verify the MDV primer pairs. With the MDV PCR array, we can examine entire MDV gene expression in a single plate conveniently; this should facilitate our studies. Importantly, we found that a few MDV gene expression was significantly downregulated during reactivation and also found MDV latent associated transcript (LAT) is dynamically regulated during reactivation. Marek's disease virus encoded, Meq-vIL8, RLORF4-vIL8 and RLORF5-vIL8 splice variants are not essential for in vitro replication The MDV genome encodes for an oncogene, meq, and a chemo-attractant of chicken peripheral blood mononuclear cells, vIL8. Chickens infected with a recombinant virus lacking the oncogene meq do not develop tumors, while chickens infected with a mutant virus lacking the vIL8 gene show impaired disease progression and or development. However, it is unclear whether these phenotypes are due to loss of meq and vIL8, individually, or because of splice variants that fuse vIL8 to certain upstream open reading frames including Meq, RLORF4 and RLORF5. To specifically examine the role of these splice variants in MDV pathogenesis, recombinants viruses unable to express splice variants Meq-vIL8 or RLORF4-vIL8 and RLORF5-vIL8 were generated. In vitro growth kinetics showed that these splice variants are not essential for virus replication. Absence of expression of these splice transcripts was confirmed by RT-PCR. Future studies will examine the role of these splice variants in pathogenesis. (2) Specific progresses for listed objectives. (1) Finished. (2) We are still waiting for the approval of our animal protocol to reapet experiments with a larger scale. (3&4) Meq/CtBP and MEq/cJun binding sites were identified and prepared liste of targets genes. Verification of such binding sites with MDV infected chicken T-cell samples has not been performed yet. We are waiting for the appval for the in vivo experiments. (5) Fisnished
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Christopher Chen, Yuanzhi Lyu, Frank Chuang, Kazushi Nakano, Chie Izumiya, Di Jin, Mel Campbell, Yoshihiro Izumiya . Kaposis Sarcoma-associated Herpesvirus Hijacks RNA Polymerase II to Create a Viral Transcriptional Factory . Journal of Virology 91: e02491-16
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Sanjay M. Reddy, Yoshihiro Izumiya, Blanca Lupiani. Marek's disease vaccines: Current status, and strategies for improvement and development of vector vaccines. Veterinary Microbiology.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Yuanzhi Lyu, Kazushi Nakano, Ryan R. Davis, Clifford G. Tepper, Mel Campbell, Yoshihiro Izumiya. Zic2 Is Essential for Polycomb-Mediated Maintenance of KSHV Latency and a Target of K-Rta. PLoS Pathogens.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Brian Wakeman, Yoshihiro Izumiya, Samuel Speck. Identification of Novel Kaposis Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential. Journal of Virology, 91(1): 1-23.
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Progress 04/01/15 to 03/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctral fellow (Aijun Sun) CVM graduate student and postdoctoral research symposium, Jan 29, 2015 Title: Double Deletion of Marek¹s Disease Virus as a Potention Vaccine Candidate. Special award: Second Place Poster Presentation, January 29, 2015 Graduate student (Kanika Bajawa) Student research week, Texas A&M University, March 26, 2015 Bajwa K, Wu Y, Sun A, Lupiani Blanca, Sanjay M. Reddy. Title: Marek's Disease virus encoded UL55 gene is not essential for pathogenesis Special Award: Second Place Poster Award, March 26, 2015 Award: John Paul Delaplane Award, April 1, 2016 Training: Kazushi Nakano (Research fellow) Receiving next generation serquencing training through attending 3 days bioinformatics courses at UC Davis. Seminars: Title: Studying epigenetic gene regulation with herpesvirus infection as a unique model system Yoshihiro Izumiya April 23, 2015 (University of Caifornia, Davis), Sacramento, California, USA Title: Chromatin hub formation and gene expression; new mechanism of deregulation of cellular gene expression by KSHV infection Yoshihro Izumiya May 15, 2015 (University of Osaka), Osaka, Japan How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are expecting that new recombinant MDV will increase the efficacy of immuno-precipitation to have enough amount of DNA for ChIP-seq analyses. We are also planning to increase number of chickens for infection studies to have enough starting materials. Finally, we designed "lock-down" probes to enrich the MDV genome after generation of sequence samples. This approach was used for our KSHV research in my laboratory. We could achieve 600-fold enrichment of the KSHV genome by the approach. This method is similar to exome-sequencing, and we adapted the approach by custom designing the probe sets. We expect that the probe sets allow us to have analytical amounts of sequence reads to cover entire MDV genome and also perform several unique studies with tumor samples. In case we have some difficulties to obtain tumor samples from chickens, we will use chicken T-cell lines that are transformed by MDV infection. We have collected four different chicken T-cell lines that are infected with two different types of recombinant MDV. We will also use the cell lines to perform ChIP-seq analyses and compare these results with those from actual tumor samples. We are actively recruiting graduate students to perform the experiments. For the biochemical studies, we will perform the interaction assays with transient transfection by using DF-1 cells in next project year.
Impacts
What was accomplished under these goals?
Accumulating evidence has suggested that epigenetic alterations are as important as genetic mutations in cancer development. It is proposed that tumors arise through "malignant reprogramming" driven by a combination of both genetic and epigenetic changes. This is underscored by the facts that 1) epigenetic modifications (e.g., histone acetylation and methylation, DNA cytosine methylation) are pivotal regulators of transcription and chromosome dynamics, 2) aberrant epigenetic activities and signatures are indicators of aggressive cancer, and 3) since de-regulated expression of a number of epigenetic proteins is known to be a potent driver of tumor progression and now serve as diagnostic predictive biomarkers. Recent studies also highlighted that microRNA expression plays an important role in tumor development and progression through gene regulation in an epigenetic manner. Marek's disease costs the U.S. poultry industry millions of dollars annually. Marek's disease virus (MDV) is one of the most potent of cancerous viruses, which induces a highly contagious T-cell lymphoma (Marek's disease) in its natural host within a month following infection. MDV infection is also an attractive infectious animal model for herpesvirus-related cancer research. MDV encodes a defined oncoprotein, Meq. We had identified two chicken proteins, a strong gene silencer (CtBP) and a transcriptional factor (JunD), that form a protein complex with Meq in naturally infected chicken T-cells. Accordingly, our focus is to dissect the protein complex and define the function of this complex in the context of MDV infection and T-cell transformation. This will be done by identification of protein recruitment sites of each cellular and viral factor in the MDV and host genomes. Association of these sites with local histone modification and gene expression will be examined. (1) MDV viral microRNA attenuates viral gene expression to regulate viral replication. It has been know that MDV encodes cluster of viral microRNAs in the genome. Similarly, human oncogenic herpesviruses also encode several viral microRNAs in their genomes; however, the lack of a suitable animal model has hampered functional analyses of viral microRNAs in vivo. Because microRNA plays significant role in epigenetic gene regulation and the cluster of the MDV microRNAs is regulated by the Meq complex through AP-1 binding sites, we examined the role of MDV microRNA cluster mdv1-miR-M8-M10 in chickens, its natural host. Our results demonstrated that deletion of MDV-miR-M8-M10 did not affect viral replication in tissue culture but did significantly accelerate early cytolytic replication in vivo by elevating early gene expression. Viral latency establishment and maintenance, and thus tumorigenesis, were not affected. Interestingly, deletion of the viral microRNA resulted in significantly higher viral reactivation from latently infected lymphocytes. In addition, the deletion of mdv1-miR-M8-M10 resulted in more severe atrophy of lymphoid organs, leading to shortened mean death time, and impaired IL8 cytokine production in the infected hosts. We confirmed these results by generating an mdv1-miR-M8-M10 revertant virus with a restored wild type phenotype. To the best of our knowledge, our study is the first to reveal that mdv1-miR-M8-M10 plays a role in modulating virus cytolytic replication and reactivation, two features highly relevant to MDV pathogenesis. (2) Specific progresses for listed objectives. 1. Preparation of recombinant MDV virus, which contains multiple tags in front of Meq. Previously, we prepared the recombinant virus with both Flag and HA tags at the Meq N-terminal region. The construct had extra translational initiation sites immediately after the Flag tag initiation site. Accordingly, we have modified the construct by deleting a methionine after the tag sequence to avoid internal translation initiation. By deleting the second initiation site, we expect to increase expression of Flag-HA tag MEQ expression in chicken tumors. We finished preparing the construct and generated new recombinant MDV. 2. Isolation of MEQ protein complex from tumors in chicken. This experiment will be done by infecting recombinant MDV, which expresses Flag-HA-MEQ in infected chickens. We just finished making recombinant MDV and we will infect the recombinant MDV in chickens in the next project year. 3&4. Identify MEQ-CtBP complex/MEQ-JunD binding sites on MDV and chicken genome. We performed ChIP analyses to identify Meq binding sites on the genomes in tumors once. However, we could not precipitate sufficient amounts of DNA by chromatin immunoprecipitations for robust analysis. In order to obtain enough starting material for chromatin immunoprecipitation, we decided to increase number of chickens for infection studies. These experiments are slightly delayed due to renovation of SPF-facilities at Texas A&M University. We will start once the renovation is complete. 5. Dissecting MEQ-CtBP and MEQ-JunD complexes with biochemical approaches. Recombinant Meq and CtBP were expressed with recombinant baculoviruses and purified from infected insect cells. We will prepare recombinant chicken JunD protein and perform in vitro interaction analyses to map the interaction domain. 6. Generation of mutant MDV and evaluation of oncogenicity in chicken. We will do the experiment in year 3.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Acceleration of Highly Oncogenic Viral Pathogenesis by Infectious Phase Regulation via a Virus-Encoded MicroRNA Cluster
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
KSHV Chromatin Looping Facilitates Effective KSHV Gene Expression; Gene Cluster Activation via Direct K-Rta Binding at a Viral Chromatin Hub
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