Bridging Place-Based Astrobiology Education with Genomics, Including Descriptions of Three Novel Bacterial Species Isolated from Mars Analog Sites of Cultural Relevance.

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T).

KSHV ORF59 and PAN RNA Recruit Histone Demethylases to the Viral Chromatin during Lytic Reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes multiple malignancies in immunocompromised individuals. KSHV primarily establishes a lifelong latency in infected humans during which only a subset of viral genes is expressed while most of the viral genome remains transcriptionally silent with condensed chromatin. However, during the lytic phase, the viral genome undergoes dramatic changes in chromatin landscape leading to a transcriptionally active state with the expression of most of the viral genes and production of progeny virions. Multiple cellular and viral factors influence the epigenetic gene regulation and transitioning of virus from latency to the lytic state. We have earlier shown that KSHV ORF59, viral processivity factor, binds to a protein arginine methyl transferase 5 (PRMT5) to alter the histone arginine methylation during reactivation. Additionally, ORF59 has been shown to interact with most abundantly expressed KSHV long noncoding polyadenylated nuclear RNA (PAN RNA), which associates with the viral epigenome during reactivation. Interestingly, PAN RNA interacts with UTX and JMJD3, cellular H3K27me3 demethylases, and removes the repressive marks on the chromatin. In this study, we report that the recruitment of histone demethylases to the viral chromatin is facilitated by the expression of ORF59 protein and PAN RNA. Using biochemical and localization assays including co-immunoprecipitation and immunofluorescence, we demonstate ORF59 localizes with UTX and JMJD3. Our results confirm that PAN RNA enhances the interaction of ORF59 with the chromatin modifying enzymes UTX and JMJD3.

Kaposi's Sarcoma-Associated Herpesvirus Deregulates Host Cellular Replication during Lytic Reactivation by Disrupting the MCM Complex through ORF59.

Minichromosome maintenance proteins (MCMs) play an important role in DNA replication by binding to the origins as helicase and recruiting polymerases for DNA synthesis. During the S phase, MCM complex is loaded to limit DNA replication once per cell cycle. We identified MCMs as ORF59 binding partners in our protein pulldown assays, which led us to hypothesize that this interaction influences DNA replication. ORF59's interactions with MCMs were confirmed in both endogenous and overexpression systems, which showed its association with MCM3, MCM4, MCM5, and MCM6. Interestingly, MCM6 interacted with both the N- and C-terminal domains of ORF59, and its depletion in BCBL-1 and BC3 cells led to an increase in viral genome copies, viral late gene transcripts, and virion production compared to the control cells following reactivation. MCMs perform their function by loading onto the replication competent DNA, and one means of regulating chromatin loading/unloading, in addition to enzymatic activity of the MCM complex, is by posttranslational modifications, including phosphorylation of these factors. Interestingly, a hypophosphorylated form of MCM3, which is associated with reduced loading onto the chromatin, was detected during lytic reactivation and correlated with its inability to associate with histones in reactivated cells. Additionally, chromatin immunoprecipitation showed lower levels of MCM3 and MCM4 association at cellular origins of replication and decreased levels of cellular DNA synthesis in cells undergoing reactivation. Taken together, these findings suggest a mechanism in which KSHV ORF59 disrupts the assembly and functions of MCM complex to stall cellular DNA replication and promote viral replication.IMPORTANCE KSHV is the causative agent of various lethal malignancies affecting immunocompromised individuals. Both lytic and latent phases of the viral life cycle contribute to the progression of these cancers. A better understanding of how viral proteins disrupt functions of a normal healthy cell to cause oncogenesis is warranted. One crucial lytic protein produced early during lytic reactivation is the multifunctional ORF59. In this report, we elucidated an important role of ORF59 in manipulating the cellular environment conducive for viral DNA replication by deregulating the normal functions of the host MCM proteins. ORF59 binds to specific MCMs and sequesters them away from replication origins in order to sabotage cellular DNA replication. Blocking cellular DNA replication ensures that cellular resources are utilized for transcription and replication of viral DNA.

Quantifying Symmetrically Methylated H4R3 on the Kaposi's Sarcoma-associated Herpesvirus (KSHV) Genome by ChIP-Seq.

Post-translational modifications to histone tails contribute to the three-dimensional structure of chromatin and play an important role in detegrmining the relative expression of nearby genes. One such modification is symmetric di-methylation of arginine residues, which may exhibit different effects on gene expression including blocking the binding of transcriptional activators, or recruiting repressive effector molecules. Recent ChIP-Seq studies have demonstrated the importance of cross-talk between different histone modifications in gene regulation. Thus, to acquire a comprehensive understanding of the combined efforts of these epigenetic marks, ChIP-Seq must be utilized for identifying specific enrichment on the chromatin. Tumorigenic herpesvirus KSHV, employs epigenetic mechanisms for gene regulation, and by evaluating relative abundance of multiple histone modifications in a thorough, unbiased way, using ChIP-Seq, we can get a superior insight concerning the complex mechanisms of viral replication and pathogenesis.