Genomic Silencing Research Articles

The TOP-2/condensin II axis silences transcription during germline specification in C. elegans.

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 protein's ability to globally silence mRNA transcription in germline precursor cells, also known as the P lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of TOP-2/condensin II activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P lineage. We also present evidence that while both PIE-1 and TOP-2/condensin II are required for genome silencing in the P lineage, PIE-1 can silence transcription independently of TOP-2/condensin II when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active. Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

Aberrant DNA methylation associated with the development of metabolic dysfunction-associated fatty liver disease

The literature review deals with DNA methylation, a key epigenetic mechanism that controls the activity of gene transcription, plays a decisive role in the formation of genomic imprinting, gene silencing, X-chromosome inactivation, RNA splicing, DNA repair, cell differentiation and cell reprogramming, and also determines the occurrence and development of liver steatotic lesions and metabolic disorders. Methylation of DNA cytosine dinucleotide (CpG) can be represented in two types: de novo CpG methylation, which is carried out by 5mC DNA writers — DNA-(cytosine-5)-methyltransferase (DNMT) 3a and 3b, and suppor­ting DNA methylation, which is performed by DNMT1 during DNA replication. It has been found that the maintenance DNA methylation allows the preservation of the methylation pattern characteristic of progenitor cells in the cells of the new generation, and the DNA methylation of the gene body is associated with its increased expression. Active demethylation of 5mC is carried out by TET dioxygenases, including three enzymatic representatives: TET1, TET2 and TET3. It has been demonstrated that aberrant methylation of DNA nucleotides is directly related to the activity of lipid synthesis, the degree of oxidative stress, the development of liver steatosis, low-grade inflammation, insulin resistance, and the progression of liver fibrosis. The authors presented in detail the functions and features of DNA methyltransferases, erasers, and readers of 5mC sites; possible violations of the balance of activity of writers and erasers of 5mC DNA; DNA methylation landscape and patterns; clinical significance of DNA methylation signatures in metabolic dysfunction-associated fatty liver disease. Global hypomethylation of genome, at least 55 genes, is observed in patients with metabolic dysfunction-associated fatty liver disease. The authors emphasize that the use of DNA methylation signatures is a promising direction for early diagnosis and prognosis of the course of metabolic dysfunction-associated fatty liver disease, while the study of molecular components of DNA methylation mechanisms involved in the regulation of gene expression, the dependence of their activity on exposure to the exposome will allow to persona­lize and improve recommendations for lifestyle and diet modification in patients with metabolic dysfunction-associated fatty liver disease.

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this ‘privileged’ repertoire. Our experiments identify early transcription as a potential ‘epigenetic’ contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Breeding strategies for late blight resistance in potato crop: recent developments.

Late blight (LB) is a serious disease that affects potato crop and is caused by Phytophthora infestans. Fungicides are commonly used to manage this disease, but this practice has led to the development of resistant strains and it also poses serious environmental and health risks. Therefore, breeding for resistance development can be the most effective strategies to control late blight. Various Solanum species have been utilized as a source of resistance genes to combat late blight disease. Several potential resistance genes and quantitative resistance loci (QRLs) have been identified and mapped through the application of molecular techniques. Furthermore, molecular markers closely linked to resistance genes or QRLs have been utilized to hasten the breeding process. However, the use of single-gene resistance can lead to the breakdown of resistance within a short period. To address this, breeding programs are now being focused on development of durable and broad-spectrum resistant cultivars by combining multiple resistant genes and QRLs using advanced molecular breeding tools such as marker-assisted selection (MAS) and cis-genic approaches. In addition to the strategies mentioned earlier, somatic hybridization has been utilized for the development and characterization of interspecific somatic hybrids. To further broaden the scope of late blight resistance breeding, approaches such as genomic selection, RNAi silencing, and various genome editing techniques can be employed. This study provides an overview of recent advances in various breeding strategies and their applications in improving the late blight resistance breeding program.

Characterization of factors that underlie transcriptional silencing in C. elegans oocytes.

While it has been appreciated for decades that prophase-arrested oocytes are transcriptionally silenced on a global level, the molecular pathways that promote silencing have remained elusive. Previous work in C. elegans has shown that both topoisomerase II (TOP-2) and condensin II collaborate with the H3K9me heterochromatin pathway to silence gene expression in the germline during L1 starvation, and that the PIE-1 protein silences the genome in the P-lineage of early embryos. Here, we show that all three of these silencing systems, TOP-2/condensin II, H3K9me, and PIE-1, are required for transcriptional repression in oocytes. We find that H3K9me3 marks increase dramatically on chromatin during silencing, and that silencing is under cell cycle control. We also find that PIE-1 localizes to the nucleolus just prior to silencing, and that nucleolar dissolution during silencing is dependent on TOP-2/condensin II. Our data identify both the molecular components and the trigger for genome silencing in oocytes and establish a link between PIE-1 nucleolar residency and its ability to repress transcription.

Review: Recent Applications of Gene Editing in Fish Species and Aquatic Medicine.

Gene editing and gene silencing techniques have the potential to revolutionize our knowledge of biology and diseases of fish and other aquatic animals. By using such techniques, it is feasible to change the phenotype and modify cells, tissues and organs of animals in order to cure abnormalities and dysfunctions in the organisms. Gene editing is currently experimental in wide fields of aquaculture, including growth, controlled reproduction, sterility and disease resistance. Zink finger nucleases, TALENs and CRISPR/Cas9 targeted cleavage of the DNA induce favorable changes to site-specific locations. Moreover, gene silencing can be used to inhibit the translation of RNA, namely, to regulate gene expression. This methodology is widely used by researchers to investigate genes involved in different disorders. It is a promising tool in biotechnology and in medicine for investigating gene function and diseases. The production of food fish has increased markedly, making fish and seafood globally more popular. Consequently, the incidence of associated problems and disease outbreaks has also increased. A greater investment in new technologies is therefore needed to overcome such problems in this industry. To put it concisely, the modification of genomic DNA and gene silencing can comprehensively influence aquatic animal medicine in the future. On the ethical side, these precise genetic modifications make it more complicated to recognize genetically modified organisms in nature and can cause several side effects through created mutations. The aim of this review is to summarize the current state of applications of gene modifications and genome editing in fish medicine.

Effects of Defective Unloading and Recycling of PCNA Revealed by the Analysis of ELG1 Mutants.

Timely and complete replication of the genome is essential for life. The PCNA ring plays an essential role in DNA replication and repair by contributing to the processivity of DNA polymerases and by recruiting proteins that act in DNA replication-associated processes. The ELG1 gene encodes a protein that works, together with the Rfc2-5 subunits (shared by the replication factor C complex), to unload PCNA from chromatin. While ELG1 is not essential for life, deletion of the gene has strong consequences for the stability of the genome, and elg1 mutants exhibit sensitivity to DNA damaging agents, defects in genomic silencing, high mutation rates, and other striking phenotypes. Here, we sought to understand whether all the roles attributed to Elg1 in genome stability maintenance are due to its effects on PCNA unloading, or whether they are due to additional functions of the protein. By using a battery of mutants that affect PCNA accumulation at various degrees, we show that all the phenotypes measured correlate with the amount of PCNA left at the chromatin. Our results thus demonstrate the importance of Elg1 and of PCNA unloading in promoting proper chromatin structure and in maintaining a stable genome.

Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.

ABSTRACTCandida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis.IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.

Unveiling the nature's fruit basket to computationally identify Citrus sinensis csi-mir169–3p as a probable plant miRNA against Reference and Omicron SARS-CoV-2 genome

The fundamental role of microRNAs (miRNAs) has long been associated with regulation of gene expression during transcription and post transcription of mRNA's 3′UTR by the RNA interference mechanism. Also, the process of how miRNAs tend to induce mRNA degradation has been predominantly studied in many infectious diseases. In this article, we would like to discuss the interaction of dietary plant miRNAs derived from fresh fruits against the viral genome of the causative agent of COVID-19, specifically targeting the 3′UTR of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) genome. Expanding the analysis, we have also identified plant miRNAs that interact against the Omicron (B.1.1.529) variant of SARS-CoV-2 across 37 countries/territories throughout the world. This cross-species virus-plant interaction led us to identify the alignment of dietary plant miRNAs found in fruits like Citrus sinensis (Orange), Prunus persica (Peaches), Vitis vinifera (Grapes) and Malus domestica (Apple) onto the viral genomes. In particular, the interaction of C. sinensis miRNA - csi-miR169–3p and SARS-CoV-2 is noteworthy, as the targeted 3′UTR region “CTGCCT” is found conserved amongst all curated 772 Omicron variants across the globe. Hence this site “CTGCCT” and miRNA csi-miR169–3p may become promising therapeutic candidates to induce viral genome silencing. Thereby, this study reveals the mechanistic way of how fruits tend to enact a fight against viruses like SARS-CoV-2 and aid in maintaining a strong immune system of an individual.

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Local Immune Control of Latent Herpes Simplex Virus Type 1 in Ganglia of Mice and Man.

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen. HSV-1 genomes persist in trigeminal ganglia neuronal nuclei as chromatinized episomes, while epithelial cells are typically killed by lytic infection. Fluctuations in anti-viral responses, broadly defined, may underlay periodic reactivations. The ganglionic immune response to HSV-1 infection includes cell-intrinsic responses in neurons, innate sensing by several cell types, and the infiltration and persistence of antigen-specific T-cells. The mechanisms specifying the contrasting fates of HSV-1 in neurons and epithelial cells may include differential genome silencing and chromatinization, dictated by variation in access of immune modulating viral tegument proteins to the cell body, and protection of neurons by autophagy. Innate responses have the capacity of recruiting additional immune cells and paracrine activity on parenchymal cells, for example via chemokines and type I interferons. In both mice and humans, HSV-1-specific CD8 and CD4 T-cells are recruited to ganglia, with mechanistic studies suggesting active roles in immune surveillance and control of reactivation. In this review we focus mainly on HSV-1 and the TG, comparing and contrasting where possible observational, interventional, and in vitro studies between humans and animal hosts.

Genetic analysis implicates a molecular chaperone complex in regulating epigenetic silencing of methylated genomic regions

DNA cytosine methylation confers stable epigenetic silencing in plants and many animals. However, the mechanisms underlying DNA methylation-mediated genomic silencing are not fully understood. We conducted a forward genetic screen for cellular factors required for the silencing of a heavily methylated p35S:NPTII transgene in the Arabidopsis thaliana rdm1-1 mutant background, which led to the identification of a Hsp20 family protein, RDS1 (rdm1-1 suppressor 1). Loss-of-function mutations in RDS1 released the silencing of the p35S::NPTII transgene in rdm1-1 mutant plants, without changing the DNA methylation state of the transgene. Protein interaction analyses suggest that RDS1 exists in a protein complex consisting of the methyl-DNA binding domain proteins MBD5 and MBD6, two other Hsp20 family proteins, RDS2 and IDM3, a Hsp40/DNAJ family protein, and a Hsp70 family protein. Like rds1 mutations, mutations in RDS2, MBD5, or MBD6 release the silencing of the transgene in the rdm1 mutant background. Our results suggest that Hsp20, Hsp40, and Hsp70 proteins may form a complex that is recruited to some genomic regions with DNA methylation by methyl-DNA binding proteins to regulate the state of silencing of these regions.

Autophagy-Mediated Clearance of Free Genomic DNA in the Cytoplasm Protects the Growth and Survival of Cancer Cells.

The cGAS (GMP-AMP synthase)-mediated senescence-associated secretory phenotype (SASP) and DNA-induced autophagy (DNA autophagy) have been extensively investigated in recent years. However, cGAS-mediated autophagy has not been elucidated in cancer cells. The described investigation revealed that active DNA autophagy but not SASP activity could be detected in the BT-549 breast cancer cell line with high micronucleus (MN) formation. DNA autophagy was identified as selective autophagy of free genomic DNA in the cytoplasm but not nucleophagy. The process of DNA autophagy in the cytosol could be initiate by cGAS and usually cooperates with SQSTM1-mediated autophagy of ubiquitinated histones. Cytoplasmic DNA, together with nuclear proteins such as histones, could be derived from DNA replication-induced nuclear damage and MN collapse. The inhibition of autophagy through chemical inhibitors as well as the genomic silencing of cGAS or SQSTM1 could suppress the growth and survival of cancer cells, and induced DNA damage could increase the sensitivity to these inhibitors. Furthermore, expanded observations of several other kinds of human cancer cells indicated that high relative DNA autophagy or enhancement of DNA damage could also increase or sensitize these cells to inhibition of DNA autophagy.

Germ granule dysfunction is a hallmark and mirror of Piwi mutant sterility

In several species, Piwi/piRNA genome silencing defects cause immediate sterility that correlates with transposon expression and transposon-induced genomic instability. In C. elegans, mutations in the Piwi-related gene (prg-1) and other piRNA deficient mutants cause a transgenerational decline in fertility over a period of several generations. Here we show that the sterility of late generation piRNA mutants correlates poorly with increases in DNA damage signaling. Instead, sterile individuals consistently exhibit altered perinuclear germ granules. We show that disruption of germ granules does not activate transposon expression but induces multiple phenotypes found in sterile prg-1 pathway mutants. Furthermore, loss of the germ granule component pgl-1 enhances prg-1 mutant infertility. Environmental restoration of germ granule function for sterile pgl-1 mutants restores their fertility. We propose that Piwi mutant sterility is a reproductive arrest phenotype that is characterized by perturbed germ granule structure and is phenocopied by germ granule dysfunction, independent of genomic instability.

Inhibition of Critical DNA Dioxygenase Activity in IDH1/2 Mutant Myeloid Neoplasms

Neomorphic mutations in IDH1/2 producing R-2-Hydroxyglutrate (R-2HG), are common in myeloid malignancies and in various solid cancers. A diffuse hypermethylated status is the biological consequence of the R-2HG-mediated inhibition of several α-ketoglutarate (αKG)-dependent enzymes including DNA dioxygenases TET1, TET2 and TET3.1,2 Specifically, the inhibition of TET2, either induced by the interaction with R-2HG or by direct genomic silencing (as in case of TET2 loss of function mutations) is responsible for the block of the DNA cytosine demethylation pathway, inducing changes in expression patterns, (e.g. decreasing expression of tumor suppressor genes) and impairing execution of differentiation programs. Analysis of genomic data from a Cleveland Clinic (CCF) cohort of AML/MDS patients combined in a meta-analytic fashion with BeatAML3 and Tumor Cancer Genome Atlas (TCGA) cohorts (1119 profiled patients) showed that IDH1/2 mutations are mutually exclusive (only 3% [N=4/106] of AML IDH1/2 mutated cases had TET2 mutations, expected to be at a frequency of 18% [N=110/585] in IDH1/2 wild type cases, p=.000125). In this scenario we suggest that the loss of TET2 activity due to mutations prevents the expansion of IDH1/2 mutant myeloid neoplasms (MNs) because of phenotypic redundancies inducing synthetic lethality. With this premise we stipulated that a critical level of DNA dioxygenase activity exists and thus cells with low TET2 activity will not tolerate further inhibition by R-2HG. Here we propose to apply pharmacologic inhibition of TET2 to produce an additive effect on DNA dioxygenases to investigate whether this will result in a synthetic lethality of IDH1/2 mutant cells. Specifically we hypothesize that TET-dioxygenase inhibition may be implemented as a possible therapeutic strategy in neomorphic IDH1/2 mutant MNs. To explore this hypothesis we conducted a series of in vitro experiments in different isogenic cell lines expressing either mutant or wild type IDH1 or IDH2, that were simultaneously mutant, wild type (WT) or knock down (KD) for TET2 (TF1-IDH2R140Q, K562-IDH1R132C both WT for TET2 gene, and K18-IDH1R132CTET2KD and SIGM5-IDH1R132C TET2MT, both with a doxycycline inducible promoter for mutant IDH1). First we found that the doxycycline induction of ectopic IDH1R132C expression led to R-2HG increase (~10,000-fold over the baseline) and induced cell death in TET2-deficient cells (experiments conducted in SIGM5-IDH1R132C cells showing 70% of decrease in cell growth after five days of IDH induction with doxycycline), confirming the cytotoxic effect of cellular R-2HG. We then tested in IDH1/2MT cells sensitivity towards TETi76, a specific TET inhibitor designed on R-2HG scaffold (with more than 200 fold potency compared to R-2HG in cell-free assays of 5-hydroxy-methyl cytosine [5hMC] production).4 This compound showed particular selectivity towards inhibition of DNA dioxygenases when a set of 23 other dioxygenase inhibitors were screened. Most importantly, consistent with our hypothesis, TETi76 preferentially inhibited the proliferation of IDH1/2MT cells either following doxycycline-induction both in TET2WTand TET2 deficient models (K562 TET2WT, K18 TET2kD, SIGM-5 TET2MT cell lines), or in models not carrying the inducible promoter (TF1 TETWT) (Growth inhibition: 20-25% in IDHWT vs 70-80% in IDHMT cell lines after 72h of co-culture with TETi76 treatment for concentrations ranging between 1 and 5 µM. P-value range: 0.04-0.001 in pairwise comparisons with untreated controls). Overall, our findings are consistent with the idea that neomorphic IDH1/2MT phenocopies loss of function TET2MT, through R-2HG, down-modulating pathways fundamental for cell homeostasis, division and differentiation. If a residual TET-activity is needed for the function of IDH1/2MT cells, the complete block of the residual activity appears to inevitably disrupt this phenotype impairing cell growth and proliferation. This is also in agreement with the paucity of TET3 and TET1 mutation in the context of TET2MT carriers. In summary, results shown here represent an important proof of concept that the increased inhibition of DNA dioxygenase activity, instead of being more leukemogenic, can be synthetically lethal. Our observations may have implications with regard to the therapy of IDH1/2 mutated neoplasms including AML and MDS Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Rac1 Promotes Cell Motility by Controlling Cell Mechanics in Human Glioblastoma.

The failure of existing therapies in treating human glioblastoma (GBM) mostly is due to the ability of GBM to infiltrate into healthy regions of the brain; however, the relationship between cell motility and cell mechanics is not well understood. Here, we used atomic force microscopy (AFM), live-cell imaging, and biochemical tools to study the connection between motility and mechanics in human GBM cells. It was found thatRac1 inactivation by genomic silencing and inhibition with EHT 1864 reduced cell motility, inhibited cell ruffles, and disrupted the dynamics of cytoskeleton organization and cell adhesion. These changes were correlated with abnormal localization of myosin IIa and a rapid suppression of the phosphorylation of Erk1/2. At the same time, AFM measurements of the GBM cells revealed a significant increase in cell elasticity and viscosity following Rac1 inhibition. These results indicate that mechanical properties profoundly affect cell motility and may play an important role in the infiltration of GBM. It is conceivable that the mechanical characters might be used as markers for further surgical and therapeutical interventions.

Telomeric small RNAs in the genus Caenorhabditis.

Telomeric DNA is composed of simple tandem repeat sequences and has a G-rich strand that runs 5′ to 3′ toward the chromosome terminus. Small RNAs with homology to telomeres have been observed in several organisms and could originate from telomeres or from interstitial telomere sequences (ITSs), which are composites of degenerate and perfect telomere repeat sequences found on chromosome arms. We identified Caenorhabditis elegans small RNAs composed of the Caenorhabditis telomere sequence (TTAGGC)n with up to three mismatches, which might interact with telomeres. We rigorously defined ITSs for genomes of C. elegans and for two closely related nematodes, Caenorhabditis briggsae and Caenorhabditis remanei. Most telomeric small RNAs with mismatches originated from ITSs, which were depleted from mRNAs but were enriched in introns whose genes often displayed hallmarks of genomic silencing. C. elegans small RNAs composed of perfect telomere repeats were very rare but their levels increased by several orders of magnitude in C. briggsae and C. remanei. Major small RNA species in C. elegans begin with a 5′ guanine nucleotide, which was strongly depleted from perfect telomeric small RNAs of all three Caenorhabditis species. Perfect G-rich or C-rich telomeric small RNAs commonly began with 5′ UAGGCU and 5′ UUAGGC or 5′ CUAAGC, respectively. In contrast, telomeric small RNAs with mismatches had a mixture of all four 5′ nucleotides. We suggest that perfect telomeric small RNAs have a mechanism of biogenesis that is distinct from known classes of small RNAs and that a dramatic change in their regulation occurred during recent Caenorhabditis evolution.