Silencer Element Research Articles

A Synonymous Exonic Splice Silencer Variant in IRF6 as a Novel and Cryptic Cause of Non-Syndromic Cleft Lip and Palate.

Missense, nonsense, splice site and regulatory region variants in interferon regulatory factor 6 (IRF6) have been shown to contribute to both syndromic and non-syndromic forms of cleft lip and/or palate (CL/P). We report the diagnostic evaluation of a complex multigeneration family of Honduran ancestry with a pedigree structure consistent with autosomal-dominant inheritance with both incomplete penetrance and variable expressivity. The proband’s grandmother bore children with two partners and CL/P segregates on both sides of each lineage. Through whole-exome sequencing of five members of the family, we identified a single shared synonymous variant, located in the middle of exon 7 of IRF6 (p.Ser307Ser; g.209963979 G>A; c.921C>T). The variant was shown to segregate in the seven affected individuals and through three unaffected obligate carriers, spanning both sides of this pedigree. This variant is very rare, only being found in three (all of Latino ancestry) of 251,352 alleles in the gnomAD database. While the variant did not create a splice acceptor/donor site, in silico analysis predicted it to impact an exonic splice silencer element and the binding of major splice regulatory factors. In vitro splice assays supported this by revealing multiple abnormal splicing events, estimated to impact >60% of allelic transcripts. Sequencing of the alternate splice products demonstrated the unmasking of a cryptic splice site six nucleotides 5′ of the variant, as well as variable utilization of cryptic splice sites in intron 6. The ectopic expression of different splice regulatory proteins altered the proportion of abnormal splicing events seen in the splice assay, although the alteration was dependent on the splice factor. Importantly, each alternatively spliced mRNA is predicted to result in a frame shift and prematurely truncated IRF6 protein. This is the first study to identify a synonymous variant as a likely cause of NS-CL/P and highlights the care that should be taken by laboratories when considering and interpreting variants.

Nanoscale Properties of the Methylation in GpC Dinucleotide Systems

AbstractThe incorporation of a methyl group to DNA nucleotides (methylation) is a well known phenomenon in epigenetic regulation. Methylation can result in gene silencing. Here we investigate the electronic properties of cytosine methylation in different DNA systems using first‐principles density functional theory calculations. Specifically, four systems are investigated consisting of two stacked base pairs, including the deoxyribose‐phosphate backbone. These four systems are the unmethylated GpC‐CpG, the single methylated GpC‐mCpG, the double symmetrical methylated GpCm‐mCpG, and the quadruple symmetrical GpCmm‐mmCpG. Single base‐pair cases are also considered with and without the deoxyribose‐phosphate backbone (with: pG‐pC, pG‐pCm, and pG‐pCmm; without: G−C, G−Cm, and G‐Cmm). These structures are relaxed using the conjugate gradient method. Nanoscale properties, such as the electronic density of states, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) wave functions, charge Mulliken population, hydrogen bond energy, binding energy, and dipole moments between unmethylated and methylated relaxed structures are obtained and compared. These results demonstrate that the deoxyribose‐phosphate backbone plays a crucial role in the spatial distribution of HOMO and LUMO wave functions in the stacked systems modulating reactivity and stability. Cytosine methylation on the GpC‐CpG system results in a stabilization effect. Dipole moment modification in the studied systems could favor specific protein‐DNA interactions due to the methyl group non‐polarity, producing hydrophobic DNA regions. These findings suggest specific rules of interaction between methylated regions and gene silencing elements at the nanoscale level.

Candidate silencer elements for the human and mouse genomes

The study of gene regulation is dominated by a focus on the control of gene activation or increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have identified enhancers, however, genome-wide identification of silencers by computational or experimental approaches are lacking. Here, we first define uncharacterized cis-regulatory elements likely containing silencers and find that 41.5% of ~7500 tested elements show silencer activity using massively parallel reporter assay (MPRA). We trained a support vector machine classifier based on MPRA data to predict candidate silencers in over 100 human and mouse cell or tissue types. The predicted candidate silencers exhibit characteristics expected of silencers. Leveraging promoter-capture HiC data, we find that over 50% of silencers are interacting with gene promoters having very low to no expression. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.

Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development.

SummaryIn metazoan development, lineage specific gene expression is modulated by the delicate balance between transcription activation and repression. Despite much of our knowledge in the enhancer-centered transcription activation, silencers and their roles in normal development are poorly understood. Here, we performed chromatin interaction analyses of Polycomb repressive complex 2 (PRC2), a key regulator inducing transcriptional gene silencing, to uncover silencers, their molecular identity and associated chromatin connectivity. Systematic analysis of the cis-regulatory silencer elements reveals their chromatin features and gene targeting specificity. Deletion of these PRC2-bound silencers in mice results in transcriptional derepression of their interacting genes and pleiotropic developmental phenotypes, including embryonic lethality. While functioning as PRC2-bound silencers in pluripotent cells, they can transition into active tissue-specific enhancers during development, suggesting their regulatory versatility. Our study characterizes the molecular nature of silencers, their associated chromatin architectures, and offers the exciting possibility of targeted re-activation of epigenetically silenced genes.

Decapentaplegic signaling regulates Wingless ligand production and target activation during Drosophila wing development.

The Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways are essential for animal development. However, how these two signals are integrated in distinct tissues is not fully understood. Here, we describe a novel mode of Dpp-Wg crosstalk during Drosophila wing development. We show that the canonical Dpp signaling is required for Wg target gene activation. In addition, Dpp signaling inhibits the transcription of wg through the schnurri (shn) repressor complex. A Dpp-responsive shn/pMad/Med silencer element (SSE) is identified in the genomic loci of the wg gene. ChIP analysis suggests that shn interacts with this element invivo. Our findings support a model in which Dpp signaling plays a dual role in transcriptional regulation of both the wg gene and downstream targets.

Escape From X-Chromosome Inactivation: An Evolutionary Perspective.

Sex chromosomes originate as a pair of homologus autosomes that then follow a general pattern of divergence. This is evident in mammalian sex chromosomes, which have undergone stepwise recombination suppression events that left footprints of evolutionary strata on the X chromosome. The loss of genes on the Y chromosome led to Ohno’s hypothesis of dosage equivalence between XY males and XX females, which is achieved through X-chromosome inactivation (XCI). This process transcriptionally silences all but one X chromosome in each female cell, although 15–30% of human X-linked genes still escape inactivation. There are multiple evolutionary pathways that may lead to a gene escaping XCI, including remaining Y chromosome homology, or female advantage to escape. The conservation of some escape genes across multiple species and the ability of the mouse inactive X to recapitulate human escape status both suggest that escape from XCI is controlled by conserved processes. Evolutionary pressures to minimize dosage imbalances have led to the accumulation of genetic elements that favor either silencing or escape; lack of dosage sensitivity might also allow for the escape of flanking genes near another escapee, if a boundary element is not present between them. Delineation of the elements involved in escape is progressing, but mechanistic understanding of how they interact to allow escape from XCI is still lacking. Although increasingly well-studied in humans and mice, non-trivial challenges to studying escape have impeded progress in other species. Mouse models that can dissect the role of the sex chromosomes distinct from sex of the organism reveal an important contribution for escape genes to multiple diseases. In humans, with their elevated number of escape genes, the phenotypic consequences of sex chromosome aneuplodies and sexual dimorphism in disease both highlight the importance of escape genes.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defense system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied by enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE, PlantCare, and PlantPAN. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% identity) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin.

In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.

The FlpTRAP system for purification of specific, endogenous chromatin regions

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

Abstract 2756: A high-resolution G-quadruplex structure involved in cmyc oncogene regulation

Abstract Gene expression needs strict regulation in human cells to prevent tumorigenesis. Among other control mechanisms, guanine-rich sequences which are clustered in promoter regions of oncogenes can form four stranded nucleic acid structures, called G-quadruplexes (G4), and regulate their transcription. The cmyc protein is the most important cellular regulator for proliferation and is often overexpressed in cancer cells. The DNA G-quadruplex formed in the cmyc promoter is a transcriptional silencer element and stabilization of this G4 by small molecules suppresses cmyc transcription, making it a promising target for cancer therapy. High-resolution structures of the highly diverse G4s provide the opportunity to understand their molecular interactions with transcription factors and to design small molecules that specifically bind a particular G4 conformer. Characteristically, multiple G4s can be formed in gene promoter regions because their G-rich sequence generally contains more than the minimum of four G-stretches. While all reported cmyc G4s share the same core with parallel oriented strands, their overall shape is individualized by distinct combinations of varying flanking and loop motifs. In this study, we present a new high-resolution NMR structure of a G4 found to form in the cmyc promoter region. The determined cmyc G4 has a 1:6:1 loop-size arrangement and its conformation is largely defined by its long central loop of 6 nt. We show that this G4 structure is the preferred target of nucleolin protein in comparison to other G4s formed in the cmyc promoter, demonstrating how the polymorphism of a G-rich sequence can modulate the interaction with transcription factors and other regulators. Our NMR structure shows that, despite the length of the 6-nt loop motif and the associated increased flexibility, the folding converges well due to a base pairing between the central loop and the 5’-flanking. Thus, the long loop is pulled towards the 5’-end while analogous loop-flanking interactions are prevented at the opposite 3’-site. In conclusion, the presented new addition to the ensemble of possible cmyc promoter G4 conformations extends the folding landscape for this promoter region, in particular due to the long central loop. This may shed light on how proteins such as nucleolin can target specific G4 and shift the equilibrium of folding to fine-tune gene expression. Finally, this new structure is another promising target for cancer drug development. Citation Format: Jonathan Dickerhoff, Luying Chen, Guanhui Wu, Buket Onel, Clement Lin, Danzhou Yang. A high-resolution G-quadruplex structure involved in cmyc oncogene regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2756.

Partial isolation of distal promoter sequence of the NPR1 gene from local chilli Pepper [Capsicum annuum L.] genotype berangkai

Understanding of NPR1gene expression is a crucial aspect in improvement of chilli pepper resistant. The NPR1 gene is known to confer broad spectrum resistance toward many phytopathogens by activating SARmechanism. For that reason, characterization of the NPR1-gene promotor is necessary. The distal promoter of the NPR1-gene in general is characterized by the presence of enhancer, silencer, and cis-acting elements, located 5,950 bp from the ATG starting point. Therefore this study is focused to the isolateion of the distal promoter sequence of the NPR1 gene from a local Capsicum annul L. Genotype Berangkai. Specific primer pairs designatedas PD_CbNPR1-F1 [FR] and PD_CbNPR1-F2 [FR] were designed to amplify the whole targeted segments. However, only 1,873 bp in total [31.5 %] of sequences could successfully be elucidated from the first round, covering 970 and 993 bp from its upstream and downstream segments respectively. Hhomology analysis using BLAST tool successfully confirmed 99 and 100 % homology with its reference sequence derived from cv. Zunla-1. These result indicated that the NPR1 distal promoter sequence is successfully isolated from chilli pepper [Capsicum annuum L] genotype Berangkai. However, ffurther analysis using primer walking strategy should be undertaken in order to identify the entire sequence of its promoter region.

Optimized reactive silencers composed of closely-spaced elongated side-branch resonators.

This paper reports a theoretical study of the sound propagation in a rectangular waveguide loaded by closely-spaced elongated side-branch resonators forming a simple low-frequency broadband reactive silencer. Semi-analytical calculations account for the evanescent modes both in the main waveguide and side-branch resonators and for the viscothermal losses in the silencer elements. Reasonable accuracy is maintained in the evaluation of transmission, reflection, and absorption coefficients, while the calculation time is reduced by a few hundred times in comparison with the finite element method. Therefore, the proposed method is particularly suitable for optimization procedure. The lengths of the individual equally spaced side-branch resonators are optimized by a heuristic evolutionary algorithm that maximizes the minimum transmission loss (TL) over a pre-defined frequency range. Numerical results indicate that the minimum TL of the optimized silencers is reduced due to the destructive effect of the evanescent coupling from the resonators of the nearest side-branches. In the opposite, the TL increases linearly with the number of the side-branch resonators.

Investigating the exon 6 sequence changes of interleukin 7 receptor A (IL7RA) gene in patients with relapsing-remitting multiple sclerosis.

Interleukin 7 receptor alpha (IL7RA) gene that encodes a subunit of IL7 receptor has been reported to be associated with different immunologic disease. Multiple Sclerosis (MS) patients have shown an aberrant blood level of soluble form of IL7R protein. The genomic changes in the sequence of this gene have been suggested to be correlated with its altered splicing specially, variants in the exon 6 of the gene have been reported to influence the maintenance or skipping of this exon and control the soluble or insoluble form of the final product. In order to evaluate this changes in the IL7RA gene and to determine a possible correlation between these changes and the MS susceptibility the whole sequence of the exon 6 and 7 and their flanking sequences were analyzed. In this regard, we investigate the sequence changes of the exon 6 and 7 of the IL7RA gene in 75 relapsing-remitting MS patients and compare the results with 75 healthy control using sequence analyzing. The results of the sequence analysis were used in two aspects. The allelic and genotypic estimated frequencies of a reported risk variant rs6897932 in patients and controls in our population confirmed its association with the disease (P= 0.009, OR = 6.273, for TT genotype). Also, we report a possible hazardous cutoff for changes in a potential exon splicing silencer element (ESS (nt. 20-24)) and its correlation with rs6897932 to confer the risk of developing MS. In conclusion our results confirm the association between IL7RA exon 6 sequence changes and increased susceptibility for multiple sclerosis.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defence system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied with enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% homology) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

Improving the acoustic efficiency of laminated dissipative noise silencers for boiler gas-air paths

The operation of thermal power facilities is associated with the negative impact on the environment and humans of such factors as air, water, soil pollution, electromagnetic radiation, and noise. This article is devoted to improving the noise reduction efficiency of gas and air paths of boilers of thermal power plants. In world practice, to reduce noise in gas-air ducts, dissipative noise silencers are used. The authors propose a new design of sound attenuation elements for dissipative plate-type silencers. The article defines the acoustic and aerodynamic characteristics of noise silencers with new sound attenuation elements. We compare the characteristics of silencers made of new silencing elements and the characteristics of silencers consisting of plates of common design with flat surfaces of the side walls, and demonstrate an increase in acoustic efficiency and a decrease in pressure loss when using the proposed new silencing elements in noise silencers. Recommendations on the use of dissipative plate-type silencers with developed sound attenuation elements for gas-air paths of boilers are given.

SU88 - AN OPRD1 VARIANT PREDICTS CONTINUED OPIOID USE IN AFRICAN-AMERICANS UNDERGOING BUPRENORPHINE TREATMENT

Background Opioid Use Disorder (OUD) is a significant public health issue in the United States and globally. FDA-approved treatment for OUD includes Opioid Substitution Therapy (OST) with medications such as methadone and buprenorphine. While OST has been shown to be effective, a meta-analysis of outcome data from OST studies indicated that a significant percentage of opioid dependent patients treated with OST do not sustain abstinence or reduce their illicit opioid use. Understanding the factors that predict reductions in illicit opioid use during treatment would allow clinicians to make more informed therapeutic decisions, selecting medications that are most likely to benefit individual patients. Methods The START trial was a 24-week open-label randomized trial of methadone and buprenorphine for the treatment of OUD. Weekly urine drug screens were performed. Treatment efficacy was defined by the proportion of opioid positive urine tests. A Generalized Estimating Equation (GEE) was used to determine if variants in the OPRD1 gene were associated with treatment efficacy in African-Americans (AA; n=77). A replication population (n=75) was collected from AA patients who had previously participated in OST trials in Baltimore. Urine drug screens were performed three times per week. For a meta-analysis, weeks 21–24 were analyzed since they represent the last month of data available for both data sets. Responders were defined as patients who a) were retained in treatment until at least week 21 and b) were opioid positive for less than 50% of their urine drug screens in weeks 21–24. For in vitro analysis, 15 bp regions surrounding the C or T allele of rs678849 were cloned into luciferase vectors and transfected into neuroblastoma cells. Luciferase activity was measured by dual-luciferase reporter assay system. Results In the START trial, rs678849 was associated with the efficacy of methadone (p=0.001) and buprenorphine (p=0.008) in treating OUD in AA patients. The pharmacogenetic effect on buprenorphine (p=0.01), but not methadone (p=0.23), was replicated in the independent AA population from Baltimore. In a meta-analysis of weeks 21–24 of both data sets, patients with the C/C genotype had a significantly higher proportion of opioid positive urine drug screens (59%) than T allele carriers (26%, p Discussion These data suggest that a variant in the OPRD1 gene has a large pharmacogenetic effect on buprenorphine efficacy in the AA population and that the variant may have effects on expression. We hypothesize that the C allele of rs678849 functions as a silencer element, binding transcriptional repressors and downregulating OPRD1 expression. We also hypothesize that the T allele of the variant is an enhancer element and upregulates OPRD1 expression through interactions with a unique group of transcription factors. Further in vitro studies will be needed to dissect the complete mechanism and a prospective clinical trial that confirms this pharmacogenetic effect will be necessary before rs678849 can be used to guide treatment decisions.

MS-11, a mimetic of the mSin3-binding helix in NRSF, ameliorates social interaction deficits in a prenatal valproic acid-induced autism mouse model

Growing evidence suggests pivotal roles for epigenetic mechanisms in both animal models of and individuals with autism spectrum disorders (ASD). Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencing elements in neuronal genes and recruits co-repressors, such as mSin3, to epigenetically inhibit neuronal gene expression. Because dysregulation of NRSF is related to ASD, here we examined the effects of mS-11, a chemically optimized mimetic of the mSin3-binding helix in NRSF, on the behavioral and morphological abnormalities found in a mouse model of valproic acid (VPA)-induced ASD. Chronic treatment with mS-11 improved prenatal VPA-induced deficits in social interaction. Additionally, we found that NRSF mRNA expression was greater in the somatosensory cortex of VPA-exposed mice than of controls. Agreeing with these behavioral findings, mice that were prenatally exposed to VPA showed lower dendritic spine density in the somatosensory cortex, which was reversed by chronic treatment with mS-11. These findings suggest that mS-11 has the potential for improving ASD-related symptoms through inhibition of mSin3-NRSF binding.

Major G-Quadruplex Form of HIV-1 LTR Reveals a (3 + 1) Folding Topology Containing a Stem-Loop.

Nucleic acids can form noncanonical four-stranded structures called G-quadruplexes. G-quadruplex-forming sequences are found in several genomes including human and viruses. Previous studies showed that the G-rich sequence located in the U3 promoter region of the HIV-1 long terminal repeat (LTR) folds into a set of dynamically interchangeable G-quadruplex structures. G-quadruplexes formed in the LTR could act as silencer elements to regulate viral transcription. Stabilization of LTR G-quadruplexes by G-quadruplex-specific ligands resulted in decreased viral production, suggesting the possibility of targeting viral G-quadruplex structures for antiviral purposes. Among all the G-quadruplexes formed in the LTR sequence, LTR-III was shown to be the major G-quadruplex conformation in vitro. Here we report the NMR structure of LTR-III in K+ solution, revealing the formation of a unique quadruplex–duplex hybrid consisting of a three-layer (3 + 1) G-quadruplex scaffold, a 12-nt diagonal loop containing a conserved duplex-stem, a 3-nt lateral loop, a 1-nt propeller loop, and a V-shaped loop. Our structure showed several distinct features including a quadruplex–duplex junction, representing an attractive motif for drug targeting. The structure solved in this study may be used as a promising target to selectively impair the viral cycle.

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

Abstract 4798: Targeting hTERT for treatment of glioblastoma (GBM)

Abstract Approximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis. Citation Format: Saumya Reddy Bollam, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Targeting hTERT for treatment of glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4798.