Transcriptional Gene Silencing Research Articles

H3K27me3 timely dictates uterine epithelial transcriptome remodeling and thus transformation essential for normal embryo implantation.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation.

Euchromatin histone-lysine N-methyltransferase 2 regulates the expression of potassium-sodium-activated channel subfamily T member 1 in primary sensory neurons and contributes to remifentanil-induced pain sensitivity

Intraoperative remifentanil administration has been linked to increased postoperative pain sensitivity. Recent studies have identified the involvement of euchromatic histone-lysine N-methyltransferase 2 (Ehmt2/G9a) in neuropathic pain associated with the transcriptional silencing of many potassium ion channel genes. This study investigates whether G9a regulates the potassium sodium-activated channel subfamily T member 1 (Slo2.2) in remifentanil-induced post-incisional hyperalgesia (RIH) in rodents. We performed remifentanil infusion (1 μg·kg-1·min-1 for 60 min) followed by plantar incision to induce RIH in rodents. Our results showed that RIH was accompanied by increased G9a and H3K9me2 production and decreased Slo2.2 expression 48 h postoperatively. Deletion of G9a rescued Slo2.2 expression in DRG and reduced RIH intensity. Slo2.2 overexpression also reversed this hyperalgesia phenotype. G9a overexpression decreased Slo2.2-mediated leak current and increased excitability in the small-diameter DRG neurons and laminal II small-diameter neurons in the spinal dorsal horn, which was implicated in peripheral and central sensitization. These results suggest that G9a contributes to the development of RIH by epigenetically silencing Slo2.2 in DRG neurons, leading to decreased central sensitization in the spinal cord. The findings may have implications for the development of novel therapeutic targets for the treatment of postoperative pain.

The clade-specific target recognition mechanisms of plant RISCs.

Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent silencing of target genes. The model plant Arabidopsis thaliana encodes 10 different AGOs, categorized into three distinct clades based on amino acid sequence similarity. While clade 1 and 2 RISCs are known for their roles in post-transcriptional gene silencing, and clade 3 RISCs are associated with transcriptional gene silencing in the nucleus, the specific mechanisms of how RISCs from each clade recognize their targets remain unclear. In this study, I conducted quantitative binding analyses between RISCs and target nucleic acids with mismatches at various positions, unveiling distinct target binding characteristics unique to each clade. Clade 1 and 2 RISCs require base pairing not only in the seed region but also in the 3' supplementary region for stable target RNA binding, with clade 1 exhibiting a higher stringency. Conversely, clade 3 RISCs tolerate dinucleotide mismatches beyond the seed region. Strikingly, they bind to DNA targets with an affinity equal to or surpassing that of RNA, like prokaryotic AGO complexes. These insights challenge existing views on plant RNA silencing and open avenues for exploring new functions of eukaryotic AGOs.

Abstract LB201: ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery

Abstract RNA sequencing has improved our understanding of cellular heterogeneity by providing in-depth transcriptome information at the single cell level. However, this “dataset infodemic” did not translate into an abundance of new validated targets due to the difficulties in performing gene-to-function studies. The main goal of the ENIGMAC™ discovery platform is to support gene-to-function studies and to deliver new validated macrophage targets. ENIGMAC™ combines custom, disease relevant assays and unique genome editing methods supported by multi-omics bioinformatics analyses yielding selected gene sets for interrogation. We use a human Induced Pluripotent Stem Cell (iPSC) line that yields macrophages phenotypically and functionally similar to MDM. By employing this iPSC system, we can produce millions of macrophages per week, which allows multiple high throughput assays to support target validation and drug discovery. Furthermore, we developed several proprietary technologies allowing fast and reliable gene editing of macrophages, including gene Knock In (KI), Knock Out (KO) and Knock Down (KD) with high efficiency both at iPSC and macrophage level while maintaining expression/silencing during macrophage differentiation. Here we report preliminary results of the CRISPRi screen, aiming to identify novel regulators of INFg-mediated PD-L1 upregulation on macrophages, a surrogate readout for a suppressive phenotype. To this aim, we developed a proprietary human iPSC line that constitutively expresses dCAS9-KRAB protein, allowing for a highly gene specific guide RNA (sgRNA)-mediated method of silencing of gene transcription. Notably, our proprietary anti-silencing system allows to maintains dCAS9-KRAB expression throughout the entire macrophage differentiation process. During the screen, 100 million iPSC-derived microphages were transduced with a 6500 gRNAs library targeting 1300 genes selected from disease-relevant macrophage datasets. Macrophages were subsequently stimulated with INFg and sorted for loss of PD-L1 expression using flow cytometry. Sequencing of the PD-L1 low macrophage population successfully identified PD-L1 regulators such as IFNGR1, IFNGR2, JAK1, STAT1 and IRF1 among other hits. The novel hits are being now individually validated, and selected ones will be progressed to drug discovery. Importantly, this macrophage CRISPRi screen provides technical proof of concept for the ENIGMAC™ platform to perform screens at scale and to identify novel hits. In conclusion, the ENIGMAC™ platform represents a unique tool for gene to function studies using human macrophages. Furthermore, it is disease agnostic and can be integrated with a variety of disease-specific conditions and phenotypic readouts. Citation Format: Thomas W. Monteiro Crozier, Martha Lopez-Yrigoyen, Helena Engman, Moritz Haneklaus, Samuel Witham, Krzystof B. Wicher, Steven Myatt, Luca Cassetta, Carola Ries. ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB201.

The Role of H3K27me3-Mediated Th17 Differentiation in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a common chronic progressive inflammatory autoimmune disease. T helper 17 (Th17) cells are the major effector cells mediating AS inflammation. Histone 3 Lys 27 trimethylation (H3K27me3) is an inhibitory histone modification that silences gene transcription and plays an important role in Th17 differentiation. The objective of this study was to investigate the expression of H3K27me3 in patients with AS and to explore its epigenetic regulation mechanism of Th17 differentiation during AS inflammation. We collected serum samples from 45 patients with AS at various stages and 10 healthy controls to measure their Interleukin-17 (IL-17) levels using ELISA. A quantitative polymerase chain reaction was used to quantify the mRNA levels of RORc and the signaling molecules of the JAK2/STAT3 pathway, JMJD3, and EZH2. Additionally, Western blot analysis was performed to quantify the protein levels of H3K27me3, RORγt, JAK2, STAT3, JMJD3, and EZH2 in cell protein extracts. The results showed that H3K27me3 expression in peripheral blood mononuclear cells (PBMCs) was significantly lower in patients with active AS compared to both the normal control groups and those with stable AS. Moreover, a significant negative correlation was observed between H3K27me3 expression and the characteristic transcription factor of Th17 differentiation, RORγt. We also discovered that patients with active AS exhibited significantly higher levels of JMJD3, an inhibitor of H3K27 demethylase, compared to the normal control group and patients with stable AS, while the expression of H3K27 methyltransferase (EZH2) was significantly lower. These findings suggest that H3K27me3 may be a dynamic and important epigenetic modification in AS inflammation, and JMJD3/EZH2 regulates the methylation level of H3K27me3, which may be one of the key regulatory factors in the pathogenesis of AS. These findings contribute to our understanding of the role of epigenetics in AS and may have implications for the development of novel therapeutic strategies for AS.

RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise.

Insect pests rank among the major limiting factors in agricultural production worldwide. In addition to direct effect on crops, some phytophagous insects are efficient vectors for plant disease transmission. Large amounts of conventional insecticides are required to secure food production worldwide, with a high impact on the economy and environment, particularly when beneficial insects are also affected by chemicals that frequently lack the desired specificity. RNA interference (RNAi) is a natural mechanism gene expression regulation and protection against exogenous and endogenous genetic elements present in most eukaryotes, including insects. Molecules of double-stranded RNA (dsRNA) or highly structured RNA are the substrates of cellular enzymes to produce several types of small RNAs (sRNAs), which play a crucial role in targeting sequences for transcriptional or post-transcriptional gene silencing. The relatively simple rules that underlie RNAi regulation, mainly based in Watson-Crick complementarity, have facilitated biotechnological applications based on these cellular mechanisms. This includes the promise of using engineered dsRNA molecules, either endogenously produced in crop plants or exogenously synthesized and applied onto crops, as a new generation of highly specific, sustainable, and environmentally friendly insecticides. Fueled on this expectation, this article reviews current knowledge about the RNAi pathways in insects, and some other applied questions such as production and delivery of recombinant RNA, which are critical to establish RNAi as a reliable technology for insect control in crop plants.

The P2 nucleic acid binding protein of Sugarcane bacilliform virus is a viral pathogenic factor.

Saccharum spp. is the primary source of sugar and plays a significant role in global renewable bioenergy. Sugarcane bacilliform virus (SCBV) is one of the most important viruses infecting sugarcane, causing severe yield losses and quality degradation. It is of great significance to reveal the pathogenesis of SCBV and resistance breeding. However, little is known about the viral virulence factors or RNA silencing suppressors and the molecular mechanism of pathogenesis. To systematically investigate the functions of the unknown protein P2 encoded by SCBV ORF2. Phylogenetic analysis was implemented to infer the evolutionary relationship between the P2 of SCBV and other badnaviruses. The precise subcellular localization of P2 was verified in the transient infiltrated Nicotiana benthamiana epidermal mesophyll cells and protoplasts using the Laser scanning confocal microscope (LSCM). The post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) RNA silencing suppressor activity of P2 was analyzed, respectively. Furthermore, restriction digestion and RT-qPCR assays were conducted to verify the probable mechanism of P2 on repressing DNA methylation. To explore the pathogenicity of P2, a potato virus X-based viral vector was used to heterologously express SCBV P2 and the consequent H2O2 accumulation was detected by the 3,3'-diaminobenzidine (DAB) staining method. Phylogenetic analysis shows that SCBV has no obvious sequence similarity and low genetic relatedness to Badnavirus and Tungrovirus representatives. LSCM studies show that P2 is localized in both the cytoplasm and nucleus. Moreover, P2 is shown to be a suppressor of PTGS and TGS, which can not only repress ssRNA-induced gene silencing but also disrupt the host RNA-directed DNA methylation (RdDM) pathway. In addition, P2 can trigger an oxidative burst and cause typical hypersensitive-like response (HLR) necrosis in systemic leaves of N. benthamiana when expressed by PVX. Overall, our results laid a foundation for deciphering the molecular mechanism of SCBV pathogenesis and made progress for resistance breeding.

Structural features of T-DNA that induce transcriptional gene silencing during agroinfiltration.

Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefaciens cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.

Two H3K36 methyltransferases differentially associate with transcriptional activity and enrichment of facultative heterochromatin in rice blast fungus

Di- and tri-methylation of lysine 36 on histone H3 (H3K36me2/3) is catalysed by histone methyltransferase Set2, which plays an essential role in transcriptional regulation. Although there is a single H3K36 methyltransferase in yeast and higher eukaryotes, two H3K36 methyltransferases, Ash1 and Set2, were present in many filamentous fungi. However, their roles in H3K36 methylation and transcriptional regulation remained unclear. Combined with methods of RNA-seq and ChIP-seq, we revealed that both Ash1 and Set2 are redundantly required for the full H3K36me2/3 activity in Magnaporthe oryzae, which causes the devastating worldwide rice blast disease. Ash1 and Set2 distinguish genomic H3K36me2/3-marked regions and are differentially associated with repressed and activated transcription, respectively. Furthermore, Ash1-catalysed H3K36me2 was co-localized with H3K27me3 at the chromatin, and Ash1 was required for the enrichment and transcriptional silencing of H3K27me3-occupied genes. With the different roles of Ash1 and Set2, in H3K36me2/3 enrichment and transcriptional regulation on the stress-responsive genes, they differentially respond to various stresses in M. oryzae. Overall, we reveal a novel mechanism by which two H3K36 methyltransferases catalyze H3K36me2/3 that differentially associate with transcriptional activities and contribute to enrichment of facultative heterochromatin in eukaryotes.

Loss of cohesin regulator PDS5A reveals repressive role of Polycomb loops

Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) are conserved epigenetic regulators that promote transcriptional gene silencing. PRC1 and PRC2 converge on shared targets, catalyzing repressive histone modifications. Additionally, a subset of PRC1/PRC2 targets engage in long-range interactions whose functions in gene silencing are poorly understood. Using a CRISPR screen in mouse embryonic stem cells, we found that the cohesin regulator PDS5A links transcriptional silencing by Polycomb and 3D genome organization. PDS5A deletion impairs cohesin unloading and results in derepression of a subset of endogenous PRC1/PRC2 target genes. Importantly, derepression is not linked to loss of Polycomb chromatin domains. Instead, PDS5A removal causes aberrant cohesin activity leading to ectopic insulation sites, which disrupt the formation of ultra-long Polycomb loops. We show that these loops are important for robust silencing at a subset of PRC1/PRC2 target genes and that maintenance of cohesin-dependent genome architecture is critical for Polycomb regulation.

Abstract A010: Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line

Abstract Regulatory T cells play an important role to modulate the balance between immunotolerance and immunosurveillance. These cells have the property of inhibiting effector lymphocytes and may antagonize antitumor immunity. Regulatory T cells are originated in the thymus, or even converted from peripheral lymphocytes by factors produced in the tumor microenvironment. Clinical data suggests that regulatory T cell infiltration correlates with poor prognosis in the treatment of solid tumors. The FOXP3 transcription factor is considered a master key to control the phenotype of regulatory T cells. It was previously shown, that the ectopic expression of FOXP3 can induce an immunosuppressive phenotype in lymphocytes. In contrast, it has been observed that mutations in the FOXP3 gene can cause impaired immunosuppressive activity mediated by regulatory T cells, such as the autoimmunity syndrome known as IPEX. In this sense, the FOXP3 transcription factor may be an interesting target, looking for inactivation of the immunosuppressive phenotype of regulatory T cells, to potentiate antitumor response. In this work, we present an assay development to investigate the potential of transcriptional interference RNA candidates to silence FOXP3 expression. In contrast to post-transcriptional gene silencing method, which targets messenger RNA and depends on a constant supply or efficient turn-over of the inhibitory molecule, the transcriptional gene silencing targets the cellular DNA genome, inducing epigenetic changes that may control the target gene transcription. We employed the human MCF-7 tumor cell line, which has endogenous and constitutive expression of FOXP3 as a target model to test transcriptional gene silencing candidates. This cell line is transduced with lentiviral vectors harboring interfering RNA sequences driven to the FOXP3 promoter region. The RNA interference candidates are then evaluated for their ability to induce DNA methylation, by bisulfite method, and transcriptional gene silencing of FOXP3, by qPCR. We performed the screening of candidates for FOXP3 transcriptional silencing, in comparison to a post-transcriptional interference RNA control. The lentiviral transduced cells are easily expanded and allow a temporal analysis of target gene expression. In this model, we observed the possibility of finding transcriptional interference RNA candidates that exhibited high efficiency and stability, that may be used for research purposes, or even for the investigation of new therapeutic possibilities in immuno-oncology. Citation Format: Carolinne T Fogagnolo, Daniela S Mizobuti, Marcio C Bajgelman. Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A010.

Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways.

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

Small RNAs: Promising Molecules to Tackle Climate Change Impacts in Coffee Production.

Over the centuries, human society has evolved based on the ability to select and use more adapted species for food supply, which means making plant species tastier and more productive in particular environmental conditions. However, nowadays, this scenario is highly threatened by climate change, especially by the changes in temperature and greenhouse gasses that directly affect photosynthesis, which highlights the need for strategic studies aiming at crop breeding and guaranteeing food security. This is especially worrying for crops with complex phenology, genomes with low variability, and the ones that support a large production chain, such as Coffea sp. L. In this context, recent advances shed some light on the genome function and transcriptional control, revealing small RNAs (sRNAs) that are responsible for environmental cues and could provide variability through gene expression regulation. Basically, sRNAs are responsive to environmental changes and act on the transcriptional and post-transcriptional gene silencing pathways that regulate gene expression and, consequently, biological processes. Here, we first discuss the predicted impact of climate changes on coffee plants and coffee chain production and then the role of sRNAs in response to environmental changes, especially temperature, in different species, together with their potential as tools for genetic improvement. Very few studies in coffee explored the relationship between sRNAs and environmental cues; thus, this review contributes to understanding coffee development in the face of climate change and towards new strategies of crop breeding.

Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

H4K20me3 is important for Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin in a fungal pathogen.

Facultative heterochromatin controls development and differentiation in many eukaryotes. In metazoans, plants, and many filamentous fungi, facultative heterochromatin is characterized by transcriptional repression and enrichment with nucleosomes that are trimethylated at histone H3 lysine 27 (H3K27me3). While loss of H3K27me3 results in derepression of transcriptional gene silencing in many species, additional up- and downstream layers of regulation are necessary to mediate control of transcription in chromosome regions enriched with H3K27me3. Here, we investigated the effects of one histone mark on histone H4, namely H4K20me3, in the fungus Zymoseptoria tritici, a globally important pathogen of wheat. Deletion of kmt5, the gene encoding the sole methyltransferase responsible for H4K20 methylation, resulted in global derepression of transcription, especially in regions of facultative heterochromatin. Derepression in the absence of H4K20me3 not only affected known genes but also a large number of novel, previously undetected transcripts generated from regions of facultative heterochromatin on accessory chromosomes. Transcriptional activation in kmt5 deletion strains was accompanied by a complete loss of Ash1-mediated H3K36me3 and chromatin reorganization affecting H3K27me3 and H3K4me2 distribution in regions of facultative heterochromatin. Strains with H4K20L, M or Q mutations in the single histone H4 gene of Z. tritici recapitulated these chromatin changes, suggesting that H4K20me3 is important for Ash1-mediated H3K36me3. The ∆kmt5 mutants we obtained were more sensitive to genotoxic stressors than wild type and both, ∆kmt5 and ∆ash1, showed greatly increased rates of accessory chromosome loss. Taken together, our results provide insights into an unsuspected mechanism involved in the assembly and maintenance of facultative heterochromatin.

Canonical transcriptional gene silencing may contribute to long-term heat response and recovery through MOM1.

Plant canonical transcriptional gene silencing (TGS) is involved in epigenetic mechanisms that mediate genomic imprinting and the suppression of transposable elements (TEs). It has been recognised that long-term heat disrupts epigenetic silencing, with the ensuing activation of TEs. However, the physiological involvement of the TGS machinery under prolonged high temperatures has not yet been established. Here, we performed non-lethal extended periodic heat stress and recovery treatments on Arabidopsis thaliana lines mutated on key TGS factors, analysing transcriptomic changes of coding-protein genes and TEs. Plants bearing MET1, DRM2 and CMT3, and MOM1 mutated alleles showed novel transcriptional properties compatible with functionalities concerning the induction/repression of partially shared or private heat-triggered transcriptome networks. Certain observations supported the idea that some responses are based on thermal de-silencing. TEs transcriptional activation uncovered the interaction with specific epigenetic layers, which may play dedicated suppressing roles under determinate physiological conditions such as heat. Furthermore, physiological experimentation suggested that MOM1 is required to resume growth after stress. Our data thus provide initial evidence that at least one canonical TGS factor may contribute to plant acclimation and recovery from non-lethal long-term heat despite the stress-induced epigenetic disturbance.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.

In vitro antioxidant, antibacterial, cytotoxic, and epigenetic screening of crude extract and fractions of the marine sponge Neopetrosia exigua from Mauritius waters

The marine sponge Neopetrosia exigua is known as a goldmine of novel compounds, yet its pharmacological activities remain poorly characterised. Herein, this study investigates the bioactivities of N. exigua collected from Mauritius waters. The crude extract (dichloromethane: methanol), hexane, ethyl acetate and aqueous fractions obtained from N. exigua were subjected to in vitro antioxidant assays. Their antibacterial activities were evaluated using the broth microdilution method to determine the minimum inhibitory concentration (MIC). The cytotoxic and epigenetic activities were further screened using the MTT assay and a cell-based image system that measures de-repression of a silenced Green Fluorescent Protein (GFP) reporter gene, respectively. Higher antioxidant activity was recorded for the ethyl acetate fraction as demonstrated by its significant ferric reducing antioxidant power, radical scavenging, and metal chelating activities relative to control (p < 0.05). The best antibacterial profile was presented by the ethyl acetate fraction against Cutibacterium acnes (MIC: 0.039 mg/ml), Streptococcus mutans (MIC: 0.078 mg/ml) and Mycobacterium smegmatis (MIC: 0.313 mg/ml). Similarly, the fraction displayed significant cytotoxicity against the human liposarcoma SW872 cells with IC50 value of 44.34 ± 2.64 µg/ml and GFP re-activation capacity of 43.79 ± 3.19% (p < 0.05). This work conveys interesting data on the antioxidant, antimicrobial, and anticancer properties of N. exigua. In particular, this study indicates the promising potential of N. exigua as a reservoir of epigenetically active agents that can modulate transcription of silenced genes involved in carcinogenesis. Hence, further investigations to isolate the active constituents is actively warranted.

A Study of DNA Methylation of Bladder Cancer Biomarkers in the Urine of Patients with Neurogenic Lower Urinary Tract Dysfunction.

Background: Bladder cancer (BCa) in patients suffering from neurogenic lower urinary tract dysfunction (NLUTD) is a significant concern due to its advanced stage at diagnosis and high mortality rate. Currently, there is a scarcity of specific guidelines for BCa screening in these patients. The development of urine biomarkers for BCa seems to be an attractive non-invasive method of screening or risk stratification in this patient population. DNA methylation is an epigenetic modification, resulting in the transcriptional silencing of tumor suppression genes, that is frequently detected in the urine of BCa patients. Objectives: We aimed to investigate DNA hypermethylation in five gene promoters, previously associated with BCa, in the urine of NLUTD patients, and in comparison with healthy controls. Design, setting and participants: This was a prospective case-control study that recruited neurourology outpatients from a public teaching hospital who had suffered from NLUTD for at least 5 years. They all underwent cystoscopy combined with biopsy for BCa screening following written informed consent. DNA was extracted and DNA methylation was assessed for the RASSF1, RARβ, DAPK, TERT and APC gene promoters via quantitative methylation-specific PCR in urine specimens from the patients and controls. Results: Forty-one patients of mixed NLUTD etiology and 35 controls were enrolled. DNA was detected in 36 patients' urine specimens and in those of 22 controls. In the urine specimens, DNA was hypermethylated in at least one of five gene promoters in 17/36 patients and in 3/22 controls (47.22% vs. 13.64%, respectively, p = 0.009). RASSF1 was hypermethylated in 10/17 (58.82%) specimens with detected methylation, APC in 7/17 (41.18%), DAPK in 4/17 (23.53%), RAR-β2 in 3/17 (17.56%) and TERT in none. According to a multivariate logistic regression analysis, NLUTD and male gender were significantly associated with hypermethylation (OR = 7.43, p = 0.007 and OR = 4.21; p = 0.04, respectively). In the tissue specimens, histology revealed TaLG BCa in two patients and urothelial squamous metaplasia in five patients. Chronic bladder inflammation was present in 35/41 bladder biopsies. Conclusions: DNA hypermethylation in a panel of five BCa-associated genes in the urine was significantly more frequent in NLUTD patients than in the controls. Our results warrant further evaluation in longitudinal studies assessing the clinical implications and possible associations between DNA hypermethylation, chronic inflammation and BCa in the NLUTD population.

DNA Methylation in Alcohol Use Disorder.

Excessive drinking damages the central nervous system of individuals and can even cause alcohol use disorder (AUD). AUD is regulated by both genetic and environmental factors. Genes determine susceptibility to alcohol, and the dysregulation of epigenome drives the abnormal transcription program and promotes the occurrence and development of AUD. DNA methylation is one of the earliest and most widely studied epigenetic mechanisms that can be inherited stably. In ontogeny, DNA methylation pattern is a dynamic process, showing differences and characteristics at different stages. DNA dysmethylation is prevalent in human cancer and alcohol-related psychiatric disorders, resulting in local hypermethylation and transcriptional silencing of related genes. Here, we summarize recent findings on the roles and regulatory mechanisms of DNA methylation, the development of methyltransferase inhibitors, methylation alteration during alcohol exposure at different stages of life, and possible therapeutic options for targeting methylation in human and animal studies.