Transcript Clusters Research Articles

Impact of protein kinase CK2 downregulation and inhibition on oncomir clusters 17 ~ 92 and 106b ~ 25 in prostate, breast, and head and neck cancers

BackgroundProtein kinase CK2 is a ubiquitous and highly conserved protein Ser/Thr kinase with diverse cell functions. CK2 is upregulated in various cancers and affects numerous aspects of their underlying pathobiology. The important role of microRNAs (miRNAs) referred to as oncomirs is also recognized in various cancers. Elevation of both CK2 and altered miRNA expression in cancers raised the question whether there was a connection between CK2 function and oncomirs in cancer.MethodsPCR array analysis was used to examine the effects of CK2 siRNA-mediated downregulation on miRNA levels in C4-2 prostate cancer cells. We employed prostate cancer, breast cancer, and head and neck squamous cell carcinoma (HNSCC) cells as well as a prostate cancer xenograft orthotopic tumor model to examine the effects of CK2 siRNA-mediated downregulation or chemical inhibition on oncomir cluster miR-17 ~ 92 and miR-106b ~ 25 constituent miRNAs by quantitative reverse-transcriptase stem-loop PCR. Pri-miRNAs were measured in cancer cell lines by quantitative reverse-transcriptase PCR. Protein levels were assessed by western blot. PC3-LN4 prostate cancer orthotopic xenograft tumors and blood were collected from nude mice following repeated treatments with tenfibgen ligand nanocapsules containing RNAi-CK2 or RNAi-Control cargoes.ResultsPCR array analysis demonstrated effect on a subset of miRNAs following CK2 downregulation; we focused our investigation on CK2 regulation of miR-17 ~ 92 and 106b ~ 25 oncomir clusters. Chemical inhibition or molecular downregulation of CK2 greatly reduced expression of miR-17 ~ 92 and 106b ~ 25 in prostate, breast and head and neck cancer cells in vitro. CK2α and CK2α´ protein levels were significantly correlated with many of the miR-17 ~ 92 and some of the miR-106b ~ 25 constituent members in prostate cancer cells. Decreased pri-miRNA levels for the miR-17 ~ 92 gene cluster transcript were observed for 5 of 6 cancer cell lines tested following CK2 downregulation. Nanocapsule-mediated delivery of RNAi-CK2 reduced CK2 protein expression in orthotopic prostate xenograft tumors and decreased intra-tumoral and serum levels of the oncomirs.ConclusionsTargeting CK2 for the development of new cancer therapies is under active investigation in many laboratories and pharmaceutical companies. Our data suggest a new role for CK2 in cell signaling and survival in multiple cancer types through maintenance of miR-17 ~ 92 and 106b ~ 25 biogenesis.

Prediction and functional interpretation of inter-chromosomal genome architecture from DNA sequence with TwinC.

Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal (cis) folding and less characterized inter-chromosomal (trans) interfaces. Current predictive models of 3D genome folding can effectively infer pairwise cis-chromatin interactions from the primary DNA sequence but generally ignore trans contacts. There is an unmet need for robust models of trans-genome organization that provide insights into their underlying principles and functional relevance. We present TwinC, an interpretable convolutional neural network model that reliably predicts trans contacts measurable through genome-wide chromatin conformation capture (Hi-C). TwinC uses a paired sequence design from replicate Hi-C experiments to learn single base pair relevance in trans interactions across two stretches of DNA. The method achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from Hi-C experiments in heart tissue. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding and G-quadruplexes in forming trans contacts. In summary, TwinC models and interprets trans genome architecture, shedding light on this poorly understood aspect of gene regulation.

MRNA Nuclear Clustering Leads to a Difference in Mutant Huntingtin mRNA and Protein Silencing by siRNAs In Vivo.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein invivo and are considered a viable therapeutic strategy. However, the extent to which they silence huntingtin mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a divalent scaffold and delivered to two mouse models of HD. In both models, divalent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type HTT protein was observed in both models. Subsequent fluorescent in situ hybridization analysis shows that divalent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

#1121 Identification of kidney transcripts associated with prognosis in ANCA-associated glomerulonephritis

Abstract Background and Aims Kidney involvement in ANCA-associated vasculitis (AAV-GN) predicts poor patient and kidney survival. Deciphering the transcriptomic landscape in AAV-GN may provide insights into pathogenic mechanisms or identify biomarkers for refining diagnosis and/or prognosis, which would help stratify risk and tailor therapeutic management. We aimed to investigate the potential prognostic value of kidney transcripts associated with kidney survival. Method This study included adult patients with AAV-GN from the French Maine-Anjou Registry. Immune gene transcript analysis was performed on RNA extracted from 97 AAV-GN kidney biopsies using NanoString technology. Transcripts of interest were selected, and their prognostic performance was assessed with respect to current histological-based classifications. Following the identification of a possible role for clusterin (CLU), the relationship between serum CLU and prognosis was assessed. Results Among the 750 evaluated transcripts, we identified a 4-gene signature (XRCC6, PRKCD, TEK, and CLU) that was strongly associated with kidney survival (Fig. 1A). This signature predicted kidney survival better than histological-based classifications (global C-Index 0.87 vs. 0.65 for Berden classification or 0.81 for Renal Risk Score, with better time-dependent AUC and Brier scores, especially beyond 1 year after diagnosis) (Fig. 1B). Among these 4 transcripts, the expression level of the CLU transcript had the highest correlation with glomerular involvement, kidney function at diagnosis, and kidney survival. Serum CLU levels were associated with kidney survival, especially when assessed at 6 months from diagnosis (Fig. 1C, P = .023). Conclusion Transcriptomic analysis of kidney biopsies of AAV-GN identified potential transcripts that may improve prediction of kidney survival. This transcriptomic signature may help us gain a deeper understanding of the AAV-GN pathogenesis and provide insights for developing new therapeutic options.

Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-β-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-d-ADP-heptose and β-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.

Comparative RNA-Seq Analyses of Solenopsis japonica (Hymenoptera: Formicidae) Reveal Gene in Response to Cold Stress.

Solenopsis japonica, as a fire ant species, shows some predatory behavior towards earthworms and woodlice, and preys on the larvae of other ant species by tunneling into a neighboring colony’s brood chamber. This study focused on the molecular response process and gene expression profiles of S. japonica to low (9 °C)-temperature stress in comparison with normal temperature (25 °C) conditions. A total of 89,657 unigenes (the clustered non-redundant transcripts that are filtered from the longest assembled contigs) were obtained, of which 32,782 were annotated in the NR (nonredundant protein) database with gene ontology (GO) terms, gene descriptions, and metabolic pathways. The results were 81 GO subgroups and 18 EggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) keywords. Differentially expressed genes (DEGs) with log2fold change (FC) > 1 and log2FC < −1 with p-value ≤ 0.05 were screened for cold stress temperature. We found 215 unigenes up-regulated and 115 unigenes down-regulated. Comparing transcriptome profiles for differential gene expression resulted in various DE proteins and genes, including fatty acid synthases and lipid metabolism, which have previously been reported to be involved in cold resistance. We verified the RNA-seq data by qPCR on 20 up- and down-regulated DEGs. These findings facilitate the basis for the future understanding of the adaptation mechanisms of S. japonica and the molecular mechanisms underlying the response to low temperatures.

35. CELL-TYPE-SPECIFIC PATTERNS OF DNA METHYLATION IN THE DEVELOPING HUMAN BRAIN

Matrix attachment region (MAR)-binding protein (MARBP) has profound influence on gene transcriptional control by tethering genes to the nuclear scaffold. MARBP SATB2 is recently known as a versatile regulator functioning in the differentiation of multiple cell types including embryonic stem cells, osteoblasts and immunocytes. Roles of SATB2 in erythroid cells and its working mechanism in orchestrating target gene expression are largely unexplored. We show here that SATB2 is expressed in erythroid cells and activates γ-globin genes by binding to MARs in their promoters and recruiting histone acetylase PCAF. Further analysis in higher-order chromatin structure shows that SATB2 affects physical proximity of human Gγ- and Aγ-globin promoters via self-association. We also found that SATB2 interacts with SATB1, which specifically activates ϵ-globin gene expression. Our results establish SATB2 as a novel γ-globin gene regulator and provide a glimpse of the differential and cooperative roles of SATB family proteins in modulating clustered genes transcription and mediating higher-order chromatin structures.

Comparison of gene expression in the red imported fire ant (Solenopsis invicta) under different temperature conditions

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and is known as a global problematic invasive species. This study focused on the molecular response of RIFA by comparing gene expression profiles after exposing ants to low (10 °C) and high (40 °C) temperature stress and comparing them to untreated controls (30 °C). A total of 99,085 unigenes (the clustered non-redundant transcripts that are filtered from the longest assembled contigs) were obtained, of which 19,154 were annotated with gene descriptions, gene ontology terms, and metabolic pathways. 86 gene ontology (GO) functional sub-groups and 23 EggNOG terms resulted. Differentially expressed genes (DEGs) with log2FC ≥ 10 were screened and were compared at different temperatures. We found 203, 48, and 66 specific DEGs co-regulated at 10, 20, and 40 °C. Comparing transcriptome profiles for differential gene expression resulted in various DE genes, including cytochrome P450, NADH dehydrogenase subunit 1, cuticle protein and heat shock protein (HSP), which have previously been reported to be involved in cold and high temperature resistance. GO analysis revealed that antioxidant activity is up-regulated under high temperature stress. We verified the RNA-seq data by qPCR on 20 up- and down-regulated DEGs. These findings provide a basis for future understanding of the adaptation mechanisms of RIFA and the molecular mechanisms underlying the response to low and high temperatures.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis.

Clusterin (CLU), a multifunctional chaperonic glycoprotein associated with diverse cellular functions has been shown to act as an oncogene or tumour suppressor gene in different cancers, implying a dual role in tumorigenesis. Here, we investigated the expression of CLU isoforms, their subcellular localization and functional significance in oral cancer cells. Significant downregulation of secretory CLU (sCLU) transcripts was observed in oral cancer cell lines and tumours versus normal cells while the nuclear CLU (nCLU) transcripts were undetectable. We demonstrated for the first time the nucleolar localization of sCLU, its response to different nucleolar stresses and association with cajal bodies post nucleolar stress. Functionally, knockdown of CLU revealed its negative association with ribosome biogenesis implying a possible tumour suppressor like role in oral cancers. Further, loss of sCLU in these cells also resulted in altered nuclear morphology and shrunken tubulin filaments. In addition, the levels of nucleolar Nucleophosmin 1(NPM1) and Fibrillarin, known to regulate nuclear morphology were downregulated indicating a possible role of sCLU in their stabilization. Further, an in silico docking approach to gain insights into the interaction of sCLU with nucleolar proteins NPM1, Fibrillarin, UBF and Nucleolin, revealed the involvement of a conserved region comprising of amino acid residues 140-155 of sCLU β-chain, specifically via the Phe152 residue in hydrophobic interactions with these client nucleolar proteins indicating a possible stabilizing or regulatory role of sCLU. SIGNIFICANCE OF THE STUDY: This is the first study to demonstrate the nucleolar localization of sCLU and its associated functions in oral cancer cells. Downregulation of sCLU in oral cancer tissues and cell lines, and its negative association with ribogenesis suggest its tumour suppressor like role in oral cancers. The possible role of sCLU in stabilization or regulation of different nucleolar proteins thereby impacting their functions is also implicated.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Rapid Cellular Perception of Gravitational Forces in Human Jurkat T Cells and Transduction into Gene Expression Regulation.

Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.

The piRNA pathway in Drosophila ovarian germ and somatic cells.

RNA silencing refers to gene silencing pathways mediated by small non-coding RNAs, including microRNAs. Piwi-interacting RNAs (piRNAs) constitute the largest class of small non-coding RNAs in animal gonads, which repress transposons to protect the germline genome from the selfish invasion of transposons. Deterioration of the system causes DNA damage, leading to severe defects in gametogenesis and infertility. Studies using Drosophila ovaries show that piRNAs originate from specific genomic loci, termed piRNA clusters, and that in piRNA biogenesis, cluster transcripts are processed into mature piRNAs via three distinct pathways: initiator or responder for ping-pong piRNAs and trailing for phased piRNAs. piRNAs then assemble with PIWI members of the Argonaute family of proteins to form piRNA-induced RNA silencing complexes (piRISCs), the core engine of the piRNA-mediated silencing pathway. Upon piRISC assembly, the PIWI member, Piwi, is translocated to the nucleus and represses transposons co-transcriptionally by inducing local heterochromatin formation at target transposon loci.

ASXL1 Mutations Are Associated with Widespread and Distinct DNA Methylation Alterations

Background: ASXL1 is frequently mutated in a range of myeloid malignancies, including MDS, CMML, and AML, and is strongly associated with a poor prognosis in these myeloid disorders. ASXL1 is known to play a central role in epigenetic regulation, activating or repressing the transcription of genes involved in either differentiation or proliferation through its effects on histone methylation marks. In particular, ASXL1 is thought to be involved in recruiting PRC2 for its role in epigenetic modification. Even though DNA methylation is critically important in regulating gene expression in hematopoiesis, the effect of ASXL1 mutations on cytosine methylation is not well elucidated. Methods: We have previously studied the effects of the ASXL1 mutation through the use of the KBM5 CML cell line, which carries a homozygous nonsense ASXL1G710X mutation, resulting in loss of ASXL1 protein expression (Valetta et al, Oncotarget, 2015) . In particular, we saw that the ASXL1 mutation results in failure of PRC2 to recruit the HOXA cluster, resulting in increased expression of HOXA genes and resultant myeloid transformation. We used CRISPR/Cas9 mediated correction of the ASXL1 mutation in the leukemic KBM5 cell line to generate isogenic cell lines for methylome, expression, and chromatin accessibility analysis. Results: We have previously shown that correction of the ASXL1G710X mutation in KBM5 cells by CRISPR gene editing restored ASXL1 protein expression, with restored function of PRC2, including HOXA gene down-regulation with resultant reduced cell growth and increased myeloid differentiation, as evidenced by increased expression of CD11b, CD14 and CD15 by flow cytometry. In this current study, RNA-seq performed on the isogenic KBM5 cells confirmed the expression changes and CRISPR mediated correction of the ASXL1 mutation. Methylation DNA immunoprecipitation demonstrated widespread increased cytosine methylation in the KBM5 ASXL1 mutated cell line when compared to the corrected wild type cells. Evaluating this data alongside the RNA sequencing data, a clear difference in expression was noted between the genes that have methylation peaks in the gene promoter versus those that have peaks in the gene bodies. Specifically, increased methylation of the promoter region of genes was associated with decreased gene expression, while increased methylation of the gene body was associated with increased gene expression. Promoter methylation was found to be associated with down-regulation of differentiation associated CD14 . Gene body methylation was seen in widely over-expressed HOXA cluster transcripts and CD15. ATAC-seq demonstrated corresponding changes in chromatin accessibility. Due to the increased methylation in the mutant cells, we next treated the isogenic KBM5 cells with the DNMT inhibitor 5-Azacytidine. Proliferation assays of the ASXL1 mutated and corrected cells revealed significantly decreased cell viability of the KBM5 mutated cells with concordant increase in differentiation. To validate the results in primary samples, we assessed DNA methylation in a cohort of MDS ASXL1 mutant and wild type peripheral blood mononuclear samples. The HELP assay revealed increased methylation in ASXL1 mutant samples particularly in the gene bodies, consistent with the results seen in the isogenic cell line. Conclusions: It has been hypothesized that the ASXL1 mutation results in hypomethylation with resultant increased expression of genes, as previously seen with increased expression of the HOXA gene cluster in ASXL1 mutated cells. Our current study demonstrates increased methylation of the ASXL1 mutant versus the corrected cell line and reveals a significant amount of gene body methylation in the mutant cells. Specifically, we demonstrate that aberrant methylation in mutant cells provides a therapeutic window for DNMT inhibitors and is associated with site specific associations with chromatin accessibility and gene expression. Our study reinforces that high resolution whole genome methylation studies are needed to dissect the specific effects of mutations in epigenetic regulators such as ASXL1 in MDS and AML. Disclosures Verma: Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

Single cell and single molecule resolution of herpesvirus genome transport, condensation state and transcriptional output

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.

De novo root transcriptome of a medicinally important rare tree Oroxylum indicum for characterization of the flavonoid biosynthesis pathway

Oroxylum indicum (L.) Kurz is a medicinally important and rare tree species of the family Bignoniaceae. It is rich in flavonoid content and its mature roots are extensively used in Ayurvedic formulations. O. indicum specific flavonoids like oroxylin B, prunetin and oroxindin possess antibacterial, antiproliferative, antioxidant and anticancerous properties, signifying its importance in modern medicine. In the present study, de novo transcriptome analysis of O. indicum root was performed to elucidate the genes involved in flavonoid metabolism. A total of 24,625,398 high quality reads were assembled into 121,286 transcripts with N50 value 1783. The BLASTx search of 81,002 clustered transcripts against Viridiplantae Uniprot database led to annotation of 46,517 transcripts. Furthermore, Gene ontology (GO) revealed that 34,231 transcripts mapped to 3049 GO terms and KEGG analysis demonstrated that 4570 transcripts plausibly involved in 132 biosynthetic pathways. The transcriptome data indicated that cinnamyl-alcohol dehydrogenase (OinCAD) was abundant in phenylpropanoid pathway genes while; naringenin chalcone synthase (OinCHS), flavone synthase (OinFNS) and flavonoid 3‘, 5‘-methyltransferase (OinF35 MT) were abundant in flavonoid, isoflavonoid, flavone and flavonol biosynthesis pathways, respectively. Transcription factor analysis demonstrated the abundance of MYB, bHLH and WD40 transcription factor families, which regulate the flavonoid biosynthesis. Flavonoid pathway genes displayed differential expression in young and old roots of O. indicum. The transcriptome led to the identification of 31 diverse full length Cytochrome P450 (CYP450) genes which may be involved in biosynthesis of specialized metabolites and flavonoids like baicalein and baicalin. Thus, the information obtained in this study will be a valuable tool for identifying genes and developing system biology approaches for in vitro synthesis of specialized O. indicum metabolites.

Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56sz15 and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56sz15 allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.

Abstract 2354: Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes

Abstract A comprehensive understanding of the mutations that drive tumorigenesis and disease progression is essential to understanding tumor biology and designing precision therapies. The landscape of driver mutations in the protein-coding genome has been well-characterized by large exome-sequencing studies. Surprisingly, many tumors do not have mutations in any known protein-coding driver. Non-coding driver mutations are hypothesized to explain many of these cases, but aside from a few regions, like the TERT promoter, our understanding of drivers in the complex regulatory genome remains limited. To fill this gap, we analyzed 150,000 cis-regulatory modules with clustered transcription factor binding sites in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using a new statistical method, ActiveDriverWGS, we identified dozens of frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR&amp;lt;0.05) with many structural rearrangements and focal copy number alterations in additional samples. The FMREs were enriched in super-enhancers, long-range chromatin interactions and H3K27ac marks derived from primary tumors, suggesting a gene regulatory role of these mutations through three-dimensional genome organization. The interaction network of chromatin loops and FMREs revealed putative target genes located dozens to hundreds of kilobases away from the mutated regulatory elements. We found known and putative oncogenes and tumor suppressors whose expression significantly correlated with mutations in FMREs, suggesting novel oncogenic mechanisms. Most of the FMREs were also confirmed by additional driver discovery methods, lending confidence to our statistical approach. We also validated ActiveDriverWGS on protein-coding sequence and accurately recovered known driver genes. The non-coding regulatory genome is characterized by diverse mutational processes, regional hypermutations and technically challenging areas with suboptimal sequencing coverage. Thus our findings, most of which are reported for the first time, should be carefully vetted and experimentally validated in future studies. Our integrative analysis of somatic mutations, cis-regulatory regions and long-range chromatin interaction networks is a novel framework for cancer discovery and reveals the currently largest set of potential non-coding drivers in a pan-cancer cohort. Citation Format: Juri Reimand. Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes [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 2354.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.

Co-Dependent Assembly of &lt;i&gt;Drosophila&lt;/I&gt; piRNA Precursor Complexes and piRNA Cluster Heterochromatin

In Drosophila, the piRNAs that direct germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes assembly of complexes containing cluster transcripts, UAP56, and the THO complex, and that these complexes are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA processing factors, and null alleles of uap56 and the core THO subunit gene tho2 are lethal. However, the uap56sz15 point mutation, and mutations in the THO subunit genes thoc5 and thoc7, are viable but sterile, and disrupt piRNA biogenesis. We show that the uap56sz15 mutation disrupts UAP56 binding to THO, and thoc5 and thoc7 mutations block THO complex assembly and subunit co-localization with UAP56. Significantly, these mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino to cluster heterochromatin.