The Role of Epstein-Barr Virus in Modulating Key Tumor Suppressor Genes in Associated Malignancies: Epigenetics, Transcriptional, and Post-Translational Modifications

Epstein-Barr virus (EBV) is ubiquitous and carried by approximately 90% of the world’s adult population. Several mechanisms and pathways have been proposed as to how EBV facilitates the pathogenesis and progression of malignancies, such as Hodgkin’s lymphoma, Burkitt’s lymphoma, nasopharyngeal carcinoma, and gastric cancers, the majority of which have been linked to viral proteins that are expressed upon infection including latent membrane proteins (LMPs) and Epstein-Barr virus nuclear antigens (EBNAs). EBV expresses microRNAs that facilitate the progression of some cancers. Mostly, EBV induces epigenetic silencing of tumor suppressor genes, degradation of tumor suppressor mRNA transcripts, post-translational modification, and inactivation of tumor suppressor proteins. This review summarizes the mechanisms by which EBV modulates different tumor suppressors at the molecular and cellular levels in associated cancers. Briefly, EBV gene products upregulate DNA methylases to induce epigenetic silencing of tumor suppressor genes via hypermethylation. MicroRNAs expressed by EBV are also involved in the direct targeting of tumor suppressor genes for degradation, and other EBV gene products directly bind to tumor suppressor proteins to inactivate them. All these processes result in downregulation and impaired function of tumor suppressors, ultimately promoting malignances.

Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded β-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

Combination of Genomic and Transcriptomic Approaches Highlights Vascular and Circadian Clock Components in Multiple Sclerosis

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER–lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

Shenkang Injection and Its Three Anthraquinones Ameliorates Renal Fibrosis by Simultaneous Targeting IƙB/NF-ƙB and Keap1/Nrf2 Signaling Pathways

Oxidative stress and inflammation are important and critical mediators in the development and progression of chronic kidney disease (CKD) and its complications. Shenkang injection (SKI) has been widely used to treat patients with CKD. Although the anti-oxidative and anti-inflammatory activity was involved in SKI against CKD, its bioactive components and underlying mechanism remain enigmatic. A rat model of adenine-induced chronic renal failure (CRF) is associated with, and largely driven by, oxidative stress and inflammation. Hence, we identified the anti-oxidative and anti-inflammatory components of SKI and further revealed their underlying mechanism in the adenine-induced CRF rats. Compared with control rats, the levels of creatinine, urea, uric acid, total cholesterol, triglyceride, and low-density lipoprotein cholesterol in serum were significantly increased in the adenine-induced CRF rats. However, treatment with SKI and its three anthraquinones including chrysophanol, emodin, and rhein could reverse these aberrant changes. They could significantly inhibit pro-fibrotic protein expressions including collagen I, α-SMA, fibronectin, and vimentin in the kidney tissues of the adenine-induced CRF rats. Of note, SKI and rhein showed the stronger inhibitory effect on these pro-fibrotic protein expressions than chrysophanol and emodin. Furthermore, they could improve dysregulation of IƙB/NF-ƙB and Keap1/Nrf2 signaling pathways. Chrysophanol and emodin showed the stronger inhibitory effect on the NF-κB p65 protein expression than SKI and rhein. Rhein showed the strongest inhibitory effect on p65 downstream target gene products including NAD(P)H oxidase subunits (p47phox, p67phox, and gp91phox) and COX-2, MCP-1, iNOS, and 12-LO in the kidney tissues. However, SKI and rhein showed the stronger inhibitory effect on the significantly downregulated anti-inflammatory and anti-oxidative protein expression nuclear Nrf2 and its target gene products including HO-1, catalase, GCLC, and NQO1 in the Keap1/Nrf2 signaling pathway than chrysophanol and emodin. This study first demonstrated that SKI and its major components protected against renal fibrosis by inhibiting oxidative stress and inflammation via simultaneous targeting IƙB/NF-ƙB and Keap1/Nrf2 signaling pathways, which illuminated the potential molecular mechanism of anti-oxidative and anti-inflammatory effects of SKI.

Ebf Activates Expression of a Cholinergic Locus in a Multipolar Motor Ganglion Interneuron Subtype in Ciona

Conserved transcription factors termed “terminal selectors” regulate neuronal sub-type specification and differentiation through combinatorial transcriptional regulation of terminal differentiation genes. The unique combinations of terminal differentiation gene products in turn contribute to the functional identities of each neuron. One well-characterized terminal selector is COE (Collier/Olf/Ebf), which has been shown to activate cholinergic gene batteries in C. elegans motor neurons. However, its functions in other metazoans, particularly chordates, is less clear. Here we show that the sole COE ortholog in the non-vertebrate chordate Ciona robusta, Ebf, controls the expression of the cholinergic locus VAChT/ChAT in a single dorsal interneuron of the larval Motor Ganglion, which is presumed to be homologous to the vertebrate spinal cord. We propose that, while the function of Ebf as a regulator of cholinergic neuron identity conserved across bilaterians, its exact role may have diverged in different cholinergic neuron subtypes (e.g., interneurons vs. motor neurons) in chordate-specific motor circuits.

Isolation and Characterization of Levoglucosan Metabolizing Bacteria

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented diverse genera of Microbacterium, Paenibacillus , Shinella , and Klebsiella . Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069 T and Shinella sumterensis MEC087 T , while the remaining isolates were closely related to either Microbacterium lacusdiani or Klebsiella pneumoniae . The genetic sequence of LGDH, lgdA , was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in E. coli . Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. Importance Levoglucosan is the most prevalent soluble carbohydrate remaining after high temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae . A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species which degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria which metabolize levoglucosan.

Real-Time Monitoring of Key Gene Products Involved in Rice Photoperiodic Flowering

Flowering is an important biological process through which plants determine the timing of reproduction. In rice, florigen mRNA is induced more strongly when the day length is shorter than the critical day length through recognition of 30-min differences in the photoperiod. Grain number, plant height, and heading date 7 (Ghd7), which encodes a CCT-domain protein unique to monocots, has been identified as a key floral repressor in rice, and Heading date 1 (Hd1), a rice ortholog of the Arabidopsis floral activator CONSTANS (CO), is another key floral regulator gene. The Hd1 gene product has been shown to interact with the Ghd7 gene product to form a strong floral repressor complex under long-day conditions. However, the mRNA dynamics of these genes cannot explain the day-length responses of their downstream genes. Thus, a real-time monitoring system of these key gene products is needed to elucidate the molecular mechanisms underlying accurate photoperiod recognition in rice. Here, we developed a monitoring system using luciferase (LUC) fusion protein lines derived from the Ghd7-LUC and Hd1-LUC genes. We successfully obtained a functionally complemented gene-targeted line for Ghd7-LUC. Using this system, we found that the Ghd7-LUC protein begins to accumulate rapidly after dawn and reaches its peak more rapidly under a short-day condition than under a long-day condition. Our system provides a powerful tool for revealing the accurate time-keeping regulation system incorporating these key gene products involved in rice photoperiodic flowering.