Transgene Silencing Research Articles

A Molecular Analysis of the Aminopeptidase P-Related Domain of PID-5 from Caenorhabditis elegans.

A novel protein, PID-5, has been shown to be a requirement for germline immortality and has recently been implicated in RNA-induced epigenetic silencing in the Caenorhabditis elegans embryo. Importantly, it has been shown to contain both an eTudor and aminopeptidase P-related domain. However, the silencing mechanism has not yet been fully characterised. In this study, bioinformatic tools were used to compare pre-existing aminopeptidase P molecular structures to the AlphaFold2-predicted aminopeptidase P-related domain of PID-5 (PID-5 APP-RD). Structural homology, metal composition, inhibitor-bonding interactions, and the potential for dimerisation were critically assessed through computational techniques, including structural superimposition and protein-ligand docking. Results from this research suggest that the metallopeptidase-like domain shares high structural homology with known aminopeptidase P enzymes and possesses the canonical 'pita-bread fold'. However, the absence of conserved metal-coordinating residues indicates that only a single Zn2+ may be bound at the active site. The PID-5 APP-RD may form transient interactions with a known aminopeptidase P inhibitor and may therefore recognise substrates in a comparable way to the known structures. However, loss of key catalytic residues suggests the domain will be inactive. Further evidence suggests that heterodimerisation with C. elegans aminopeptidase P is feasible and therefore PID-5 is predicted to regulate proteolytic cleavage in the silencing pathway. PID-5 may interact with PID-2 to bring aminopeptidase P activity to the Z-granule, where it could influence WAGO-4 activity to ensure the balanced production of 22G-RNA signals for transgenerational silencing. Targeted experiments into APPs implicated in malaria and cancer are required in order to build upon the biological and therapeutic significance of this research.

“Who’d Have Thought?”: Unravelling Ancestors’ Hidden Histories and Their Impact on Dharug Ngurra Presences, Places and People

As a means of opening the lid on transgenerational silencing—which was a survival strategy for thousands of Indigenous families against intended cultural genocide—while balancing the place of auto/biography in that journey, this paper focuses on the impact of Ancestors’ hidden histories and how the discovery of those histories drives complex identifications when woven with Presences, places, and people on Dharug Ngurra/Country. Using my own family’s recently uncovered early colonial Ancestral storying, histories that involve Dharug traditional custodian, African slave, and Anglo characters, some as First Fleet arrivals, the paper considers the place of auto/biography as a form of agency that brings past into presence, and which, in turn, opens opportunities to heal, decolonise, and transform Dharug and, more broadly, Indigenous communities, their knowledges, practices, and ontologies. When this activation involves most of the metropolis known as Sydney, Australia, we recognise its transformative potential to change non-Indigenous people’s perspectives. When we recognise auto/biography as a form of ‘truth-telling’, it allows a space to re-story relationality, both human and other-than-human, and restores Indigenous presence into Ngurra for biodiverse justice in a climate-changing world. Addressing these matters through poetic multimedia allows a place of safety between the pain and the healing.

In planta dynamics, transport biases, and endogenous functions of mobile siRNAs in Arabidopsis.

In RNA interference (RNAi), small interfering RNAs (siRNAs) produced from double-stranded RNA guide ARGONAUTE (AGO) proteins to silence sequence-complementary RNA/DNA. RNAi can propagate locally and systemically in plants, but despite recent advances in our understanding of the underlying mechanisms, basic questions remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata (PDs), yet how its dynamics in planta compares with that of established symplastic diffusion markers remains unknown. Also is why select siRNA species, or size classes thereof, are apparently recovered in RNAi recipient tissues, yet only under some experimental settings. Shootward movement of endogenous RNAi in micro-grafted Arabidopsis is also yet to be achieved, while potential endogenous functions of mobile RNAi remain scarcely documented. Here, we show (i) that temporal, localized PD occlusion in source leaves' companion cells (CCs) suffices to abrogate all systemic manifestations of CC-activated mobile transgene silencing, including in sink leaves; (ii) that the presence or absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA length selectivity observed upon vascular movement; (iii) that stress enhancement allows endo-siRNAs of a single inverted repeat (IR) locus to translocate against the shoot-to-root phloem flow; and (iv) that mobile endo-siRNAs generated from this locus have the potential to regulate hundreds of transcripts. Our results close important knowledge gaps, rationalize previously noted inconsistencies between mobile RNAi settings, and provide a framework for mobile endo-siRNA research.

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

Synergistic action of the Arabidopsis spliceosome components PRP39a and SmD1b in promoting posttranscriptional transgene silencing.

Besides regulating splicing, the conserved spliceosome component SmD1 (Small nuclear ribonucleoprotein D1)b promotes posttranscriptional silencing of sense transgenes (S-PTGS [post-transcriptional genesilencing]). Here, we show that the conserved spliceosome component PRP39 (Pre-mRNA-processing factor 39)a also plays a role in S-PTGS in Arabidopsis thaliana. However, PRP39a and SmD1b actions appear distinct in both splicing and S-PTGS. Indeed, RNAseq-based analysis of expression level and alternative splicing in prp39a and smd1b mutants identified different sets of deregulated transcripts and noncoding RNAs. Moreover, double mutant analyses involving prp39a or smd1b and RNA quality control (RQC) mutants revealed distinct genetic interactions for SmD1b and PRP39a with nuclear RQC machineries, suggesting nonredundant roles in the RQC/PTGS interplay. Supporting this hypothesis, a prp39a smd1b double mutant exhibited enhanced suppression of S-PTGS compared to the single mutants. Because the prp39a and smd1b mutants (i) showed no major changes in the expression of PTGS or RQC components or in small RNA production and (ii) do not alter PTGS triggered by inverted-repeat transgenes directly producing dsRNA (IR-PTGS), PRP39a, and SmD1b appear to synergistically promote a step specific to S-PTGS. We propose that, independently from their specific roles in splicing, PRP39a and SmD1b limit 3'-to-5' and/or 5'-to-3' degradation of transgene-derived aberrant RNAs in the nucleus, thus favoring the export of aberrant RNAs to the cytoplasm where their conversion into double-stranded RNA (dsRNA) initiates S-PTGS.

Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering

AbstractCell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9–mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering.

Advances in epigenetic regulation of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3ug of genomic DNA, 3hours of hands-on sample preparation time, and 3days of sequencing time to locate transgenes in a host genome.

Building a pipeline to identify and engineer constitutive and repressible promoters.

To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII.

Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

ET-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes.

Post-transcriptional gene silencing using double-stranded RNA has revolutionized the field of functional genetics, allowing fast and easy disruption of gene function in various organisms. In Drosophila, many transgenic RNAi lines have been generated in large-scale efforts, including the Drosophila Transgenic RNAi Project (TRiP), to facilitate in vivo knockdown of virtually any Drosophila gene with spatial and temporal resolution. The available transgenic RNAi lines represent a fundamental resource for the fly community, providing an unprecedented opportunity to address a vast range of biological questions relevant to basic and biomedical research fields. However, caution should be applied regarding the efficiency and specificity of the RNAi approach. Here, we demonstrate that pVALIUM10-based RNAi lines, representing ∼13% of the total TRiP collection (1,808 of 13,410 pVALIUM TRiP-based RNAi lines), cause unintended off-target silencing of transgenes expressed from Gateway destination vectors. The silencing is mediated by targeting attB1 and attB2 sequences generated via site-specific recombination and included in the transcribed mRNA. Deleting these attB sites from the Gateway expression vector prevents silencing and restores expected transgene expression.

The Caenorhabditis elegans ARIP-4 DNA helicase couples mitochondrial surveillance to immune, detoxification, and antiviral pathways

Surveillance of Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4, is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4, animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.

A family of C. elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing.

Germline Argonautes direct transcriptome surveillance within perinuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in and promote the formation of nuage. Previous studies have implicated GLH proteins in inherited silencing, but direct roles in small-RNA production, Argonaute binding, or mRNA targeting have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute target mRNAs, and promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates direct binding to the Argonaute WAGO-1, which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.

Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing.

An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5' or 3' processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.

Advances in Crop Breeding Through Precision Genome Editing.

The global climate change and unfavourable abiotic and biotic factors are limiting agricultural productivity and therefore intensifying the challenges for crop scientists to meet the rising demand for global food supply. The introduction of applied genetics to agriculture through plant breeding facilitated the development of hybrid varieties with improved crop productivity. However, the development of new varieties with the existing gene pools poses a challenge for crop breeders. Genetic engineering holds the potential to broaden genetic diversity by the introduction of new genes into crops. But the random insertion of foreign DNA into the plant’s nuclear genome often leads to transgene silencing. Recent advances in the field of plant breeding include the development of a new breeding technique called genome editing. Genome editing technologies have emerged as powerful tools to precisely modify the crop genomes at specific sites in the genome, which has been the longstanding goal of plant breeders. The precise modification of the target genome, the absence of foreign DNA in the genome-edited plants, and the faster and cheaper method of genome modification are the remarkable features of the genome-editing technology that have resulted in its widespread application in crop breeding in less than a decade. This review focuses on the advances in crop breeding through precision genome editing. This review includes: an overview of the different breeding approaches for crop improvement; genome editing tools and their mechanism of action and application of the most widely used genome editing technology, CRISPR/Cas9, for crop improvement especially for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield and quality improvement, reduction of anti-nutrients, and improved shelf life; and an update on the regulatory approval of the genome-edited crops. This review also throws a light on development of high-yielding climate-resilient crops through precision genome editing.

Characterization of demethylating DNA glycosylase ROS1 from Nicotiana tabacum L.

One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of 5-methylcytosine (mC), which is further recognized by regulatory proteins. In mammals, methylation mainly occurs in CG dinucleotides, while in plants it targets CG, CHG, and CHH sequences (H is any base but G). Correct maintenance of the DNA methylation status is based on the balance of methylation, passive demethylation, and active demethylation. While in mammals active demethylation is based on targeted regulated damage to mC in DNA followed by the action of repair enzymes, demethylation in plants is performed by specialized DNA glycosylases that hydrolyze the N-glycosidic bond of mC nucleotides. The genome of the model plant Arabidopsis thaliana encodes four paralogous proteins, two of which, DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1), possess 5-methylcytosine-DNA glycosylase activity and are necessary for the regulation of development, response to infections and abiotic stress and silencing of transgenes and mobile elements. Homologues of DME and ROS1 are present in all plant groups; however, outside A. thaliana, they are poorly studied. Here we report the properties of a recombinant fragment of the ROS1 protein from Nicotiana tabacum (NtROS1), which contains all main structural domains required for catalytic activity. Using homologous modeling, we have constructed a structural model of NtROS1, which revealed folding characteristic of DNA glycosylases of the helix– hairpin–helix structural superfamily. The recombinant NtROS1 protein was able to remove mC bases from DNA, and the enzyme activity was barely affected by the methylation status of CG dinucleotides in the opposite strand. The enzyme removed 5-hydroxymethylcytosine (hmC) from DNA with a lower eff iciency, showing minimal activity in the presence of mC in the opposite strand. Expression of the NtROS1 gene in cultured human cells resulted in a global decrease in the level of genomic DNA methylation. In general, it can be said that the NtROS1 protein and other homologues of DME and ROS1 represent a promising scaffold for engineering enzymes to analyze the status of epigenetic methylation and to control gene activity.

Functional Characterization of Genes Coding for Novel β-D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Centaurium erythraea Rafn.

Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with β-D glucose linked at C1 position. Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes β-glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two β-D-glucosidases (named CeBGlu1 and CeBGlu2) from centaury (Centaurium erythraea Rafn; fam. Gentianaceae), belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using in vitro assays with recombinant proteins, following their heterologous expression in E. coli and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid C. erythraea plants. Transient overexpression of CeBGlu1 and CeBGlu2 in C. erythraea leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI − MS2 profiling of leaves of diploid and tetraploid C. erythraea genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when CeBGlu1 and CeBGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that in planta metabolic engineering adopting transient overexpression of CeBGlu1 and CeBGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of C. erythraea.