KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Effects of Chronic Arginase Inhibition with Norvaline on Tau Pathology and Brain Glucose Metabolism in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is an insidious neurodegenerative disorder representing a serious continuously escalating medico-social problem. The AD-associated progressive dementia is followed by gradual formation of amyloid plaques and neurofibrillary tangles in the brain. Though, converging evidence indicates apparent metabolic dysfunctions as key AD characteristic. In particular, late-onset AD possesses a clear metabolic signature. Considerable brain insulin signaling impairment and a decline in glucose metabolism are common AD attributes. Thus, positron emission tomography (PET) with glucose tracers is a reliable non-invasive tool for early AD diagnosis and treatment efficacy monitoring. Various approaches and agents have been trialed to modulate insulin signaling. Accumulating data point to arginase inhibition as a promising direction to treat AD via diverse molecular mechanisms involving, inter alia, the insulin pathway. Here, we use a transgenic AD mouse model, demonstrating age-dependent brain insulin signaling abnormalities, reduced brain insulin receptor levels, and substantial energy metabolism alterations, to evaluate the effects of arginase inhibition with Norvaline on glucose metabolism. We utilize fluorodeoxyglucose whole-body micro-PET to reveal a significant treatment-associated increase in glucose uptake by the brain tissue in-vivo. Additionally, we apply advanced molecular biology and bioinformatics methods to explore the mechanisms underlying the effects of Norvaline on glucose metabolism. We demonstrate that treatment-associated improvement in glucose utilization is followed by significantly elevated levels of insulin receptor and glucose transporter-3 expression in the mice hippocampi. Additionally, Norvaline diminishes the rate of Tau protein phosphorylation. Our results suggest that Norvaline interferes with AD pathogenesis. These findings open new avenues for clinical evaluation and innovative drug development.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing.

OBJECTIVES: Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. METHODS: To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. RESULTS: Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. CONCLUSIONS: Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Arginase Inhibition Supports Survival and Differentiation of Neuronal Precursors in Adult Alzheimer's Disease Mice.

Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer's disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administered an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and applied an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidenced a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline showed no significant effect upon the progenitor cell number and constitution. We demonstrated that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identified a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.

Norvaline Restores the BBB Integrity in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the leading cause of dementia. The disease progression is associated with the build-up of amyloid plaques and neurofibrillary tangles in the brain. However, besides the well-defined lesions, the AD-related pathology includes neuroinflammation, compromised energy metabolism, and chronic oxidative stress. Likewise, the blood-brain barrier (BBB) dysfunction is suggested to be a cause and AD consequence. Accordingly, therapeutic targeting of the compromised BBB is a promising disease-modifying approach. We utilized a homozygous triple-transgenic mouse model of AD (3×Tg-AD) to assess the effects of L-norvaline on BBB integrity. We scrutinized the perivascular astrocytes and macrophages by measuring the immunopositive profiles in relation to the presence of ß-amyloid and compare the results with those found in wild-type animals. Typically, 3×Tg-AD mice display astroglia cytoskeletal atrophy, associated with the deposition of ß-amyloid in the endothelia, and declining nitric oxide synthase (NOS) levels. L-norvaline escalated NOS levels, then reduced rates of BBB permeability, amyloid angiopathy, microgliosis, and astrodegeneration, which suggests AD treatment agent efficacy. Moreover, results undergird the roles of astrodegeneration and microgliosis in AD-associated BBB dysfunction and progressive cognitive impairment. L-norvaline self-evidently interferes with AD pathogenesis and presents a potent remedy for angiopathies and neurodegenerative disorders intervention.

L-Norvaline, a new therapeutic agent against Alzheimer's disease.

Growing evidence highlights the role of arginase activity in the manifestation of Alzheimer's disease (AD). Upregulation of arginase was shown to contribute to neurodegeneration. Regulation of arginase activity appears to be a promising approach for interfering with the pathogenesis of AD. Therefore, the enzyme represents a novel therapeutic target. In this study, we administered an arginase inhibitor, L-norvaline (250 mg/L), for 2.5 months to a triple-transgenic model (3×Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes. Then, the neuroprotective effects of L-norvaline were evaluated using immunohistochemistry, proteomics, and quantitative polymerase chain reaction assays. Finally, we identified the biological pathways activated by the treatment. Remarkably, L-norvaline treatment reverses the cognitive decline in AD mice. The treatment is neuroprotective as indicated by reduced beta-amyloidosis, alleviated microgliosis, and reduced tumor necrosis factor transcription levels. Moreover, elevated levels of neuroplasticity related postsynaptic density protein 95 were detected in the hippocampi of mice treated with L-norvaline. Furthermore, we disclosed several biological pathways, which were involved in cell survival and neuroplasticity and were activated by the treatment. Through these modes of action, L-norvaline has the potential to improve the symptoms of AD and even interferes with its pathogenesis. As such, L-norvaline is a promising neuroprotective molecule that might be tailored for the treatment of a range of neurodegenerative disorders. The study was approved by the Bar-Ilan University Animal Care and Use Committee (approval No. 82-10-2017) on October 1, 2017.

The effects of AtRad52 over-expression on homologous recombination in Arabidopsis.

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.

Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta.

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase(®) reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.

Ectopic expression of human Cdk2 and its yeast homolog, Ime2, is deleterious to Saccharomyces cerevisiae.

Entry into and precise progression through the cell cycle depends on the sequential expression and activation of cyclin dependent kinases (CDK). In accord, CDK dysregulation is a hallmark of many cancers. The function of Cdk2 is still an enigma as in vitro studies revealed that it is required for S phase-entry, whereas in vivo studies showed that Cdk2 is not an essential gene. Moreover, unlike other Cdks, or its cyclin E regulator, Cdk2-overexpressing tumors were reported only in one type of tumor. In this report we used budding yeast as a tool to explore Cdk2 function. We showed that hCdk2 promoted S phase in cells carrying a temperature-sensitive mutation in yCDK1, albeit, only when expressed at low or moderate levels. Overexpression of hCdk2 resulted in a defect in the G1 to S transition and a reduction in viability. The same phenotypes were observed in cells overexpressing its yeast functional homolog, Ime2, which is a meiosis-specific CDK-like kinase. A genetic interaction with the DNA damage checkpoint was demonstrated by showing an increased toxicity of hCdk2 and Ime2 in RAD53-deleted cells, and delayed Rad53 activation in response to MMS treatment in cells overexpressing hCdk2 or Ime2.

A switch from a gradient to a threshold mode in the regulation of a transcriptional cascade promotes robust execution of meiosis in budding yeast.

Tight regulation of developmental pathways is of critical importance to all organisms, and is achieved by a transcriptional cascade ensuring the coordinated expression of sets of genes. We aimed to explore whether a strong signal is required to enter and complete a developmental pathway, by using meiosis in budding yeast as a model. We demonstrate that meiosis in budding yeast is insensitive to drastic changes in the levels of its consecutive positive regulators (Ime1, Ime2, and Ndt80). Entry into DNA replication is not correlated with the time of transcription of the early genes that regulate this event. Entry into nuclear division is directly regulated by the time of transcription of the middle genes, as premature transcription of their activator NDT80, leads to a premature entry into the first meiotic division, and loss of coordination between DNA replication and nuclear division. We demonstrate that Cdk1/Cln3 functions as a negative regulator of Ime2, and that ectopic expression of Cln3 delays entry into nuclear division as well as NDT80 transcription. Because Ime2 functions as a positive regulator for premeiotic DNA replication and NDT80 transcription, as well as a negative regulator of Cdk/Cln, we suggest that a double negative feedback loop between Ime2 and Cdk1/Cln3 promotes a bistable switch from the cell cycle to meiosis. Moreover, our results suggest a regulatory mode switch that ensures robust meiosis as the transcription of the early meiosis-specific genes responds in a graded mode to Ime1 levels, whereas that of the middle and late genes as well as initiation of DNA replication, are regulated in a threshold mode.

Faithful modeling of transient expression and its application to elucidating negative feedback regulation.

Modeling and analysis of genetic regulatory networks is essential both for better understanding their dynamic behavior and for elucidating and refining open issues. We hereby present a discrete computational model that effectively describes the transient and sequential expression of a network of genes in a representative developmental pathway. Our model system is a transcriptional cascade that includes positive and negative feedback loops directing the initiation and progression through meiosis in budding yeast. The computational model allows qualitative analysis of the transcription of early meiosis-specific genes, specifically, Ime2 and their master activator, Ime1. The simulations demonstrate a robust transcriptional behavior with respect to the initial levels of Ime1 and Ime2. The computational results were verified experimentally by deleting various genes and by changing initial conditions. The model has a strong predictive aspect, and it provides insights into how to distinguish among and reason about alternative hypotheses concerning the mode by which negative regulation through Ime1 and Ime2 is accomplished. Some predictions were validated experimentally, for instance, showing that the decline in the transcription of IME1 depends on Rpd3, which is recruited by Ime1 to its promoter. Finally, this general model promotes the analysis of systems that are devoid of consistent quantitative data, as is often the case, and it can be easily adapted to other developmental pathways.