Day 2 :
- Track 1:Nucleic Acids | Track 2:RNA Interference | Track 3: Nucleic Acid Therapeutics
Location:
Session Introduction
Woojin An
University of Southern California, USA
Title: MacroH2A acts as a key regulator of osteolytic bone metastasis of cancer cells
Time : 12:30-13:00
Biography:
Woojin An has completed his PhD from Oregion State University and Post-doctoral studies from the Rockefeller University. He is an Associate Professor of University of Southern California Keck School of Medicine. He has published more than 50 research papers in reputed journals and has been serving as a reviewer for multiple journals and grant study sections.
Abstract:
Breast cancer tends to metastasize to bone, and more than half of all patients dying of breast cancer have evidence of osteolytic bone metastasis. Breast cancer-derived factors facilitate this bone metastatic process by generating a permissive microenvironment in the target organs for the engraftment of cancer cells as well as the establishment of metastatic foci. Histone modifications and variants play a crucial role in remodelling chromatin organization and modulating gene expression in various cancer cells including breast cancer cells. Altered expression of metastasis- associated genes by distinct combinations of histone modifications and variants is linked to multiple stages of breast cancer metastasis to bone. Yet, how this epigenetic resetting of gene expression in breast cancer cells contributes to osteolytic metastasis and bone lesions remains unclear. We show that histone variant macroH2A is capable of governing the early events of osteolytic lesion formation by inhibiting the expression of lysyl oxidase (LOX), which is a key factor for osteoclastogenesis. We demonstrate that macroH2A, in complex with EZH2 histone methyltransferase and Mybbp1a transcription repressor, occupies the promoter and coding regions of LOX gene, elevates the levels of histone H3 lysine 27 trimethylation, and thereby keeps the gene in a repressed state. Our data unravel an unexpected role for macroH2A in regulating the pathogenesis and progression of breast cancer bone metastasis and illustrate the power of combined biochemical and cellular approaches for mechanistic analyses.
Biography:
Yuhong Wang has completed her PhD in 2002 from the Johns Hopkins University and postdoctoral studies from Caltech and UPenn. She is an associate professor in University of Houston. She developes new biophysical tools, such as single molecule FRET and mechanomagnetic force spectroscopies. Her research interests are ribosome mechanism and non-invasive detection of microRNAs.
Abstract:
The ribosomal frameshifting is a rare but ubiquitous process. The putative "-1" frameshifting motif includes a slippery sequence, a spacer, and a secondary mRNA structure. We report a new force-based method to direct observation of “-1” and “-2” frameshiftings with single nucleotide resolution. We reveal that EF-G•GTP is indispensable to frameshifting. The biological relevance of the in vitro results is verified by protein translations in the cell. The mechanistic insights provided by our assay demonstrated the application of this method to study the ribosome system. The ribosome pre- and post-translocation complexes are tethered to the surface by biotinylated mRNA. The 3'-mRNA uncovered by the ribosome forms duplexes with DNA probes with known sequences. The probes are labeled with magnetic beads. Under external forces, the duplexes dissociate step-wisely according to the base pairs in the duplexes. The dissociation is detected by an atomic magnetometer and reflects the ribosome position with single nucleotide resolution. Three consecutive translocation steps were tracked to unambiguously identify the total of nine possible ribosome positions on the mRNA under in vitro conditions. Mechanistic studies were carried out by modifying the motif, introducing a secondary structure, and varying other experimental conditions. Meanwhile, in vivo and in vitro protein synthesis experiments were performed to demonstrate the biological significance of the frameshifting results.
Jonathan Perreault
INRS - Institut Armand-Frappier, Canada
Title: Design and selection of hammerhead ribozymes
Time : 14:30-15:00
Biography:
Jonathan Perreault has completed his PhD at the University of Sherbrooke in Canada and postdoctoral studies at Yale University in the laboratory of Ronald Breaker. He joined INRS – Institut Armand-Frappier in 2011. His work on functional nucleic acids has been published in reputed journals such as Nucleic Acids Research and Nature. It encompasses bioinformatics, biochemistry, molecular biology and microbiology approaches aiming at discovering and elucidating ncRNAs as well as developing applications.
Abstract:
Since the 1990s, hammerhead ribozymes have been studied in regards to gene therapy because of their simple RNA-cleaving catalytic property and the relative ease with which they could be designed to target mRNAs through sequence modifications permitting base complementarity with the RNA to be cleaved. Since then, the discovery of RNA interference and CRISPR have relegated ribozymes behind, while spurring a renewed interest in noncoding RNA-mediated gene therapy. However, meanwhile advances in design and major discoveries on hammerhead ribozymes, such as better activity when stem I and II interact, have opened new avenues. We have demonstrated the high efficiency of hammerhead ribozymez by using combinations that target the same mRNA. Moreover, the automated design software, RiboSoft, streamlines the use of ribozymes for gene knockdowns. Results of RNA targeting against PABPN1, a gene involved in hereditary diseases, and other RNAs will be shown. Ribozymes have many advantages over RNAi and CRISPR: they are inherently active and do not rely on any accessory protein or component, making them easily portable to any organism; since they do not require processing and rely on structure for their activity, other modules can be added for their function; and they can be assayed in vitro. In short, the revived interest in using RNA for gene therapy is likely to also help ribozymes make a come back by stimulating research for general problems such as gene delivery.
Eylon Yavin
The Hebrew University of Jerusalem, Israel
Title: RNA detection in living cancer cells by far-red emitting PNA-FIT probes
Time : 15:00-15:30
Biography:
Dr. Yavin completed his PhD at the Weizmann Institute of Science (Israel). He did his postoctoral work at the laboratory of Prof. Jacqueline Barton at Caltech (CA, USA). In 2006 he joined the School of Pharmacy at Jerusalem as a faculty member. Eylon is currently a senoir lecturer and has an active research lab in the field of Nucleic Acids. He has published more than 40 papers in reputed journals and has been recently elected as the president of The Medicinal Chemistry Section of the Israel Chemical Society.
Abstract:
To diagnose cancer early on has no doubt a huge impact on the success rate of anticancer therapy in almost all clinical settings. One promising approach is based on the detection of RNA biomarkers; particularly, mutated RNA that gives a distinct signature and allows a better choice of treatment. One attractive approach is based on the use of FIT-PNA (forced intercalation – peptide nucleic acid) probes. These PNA molecules hybridize to complementary RNA and gain fluorescence only after binding to their RNA target. We have previously shown that such FIT-PNAs with TO (thiazole orange) as a surrogate base, fluoresce in living pancreatic cancer cells (Panc-1) that express mutated kRAS mRNA (G to A single point mutation at codon 12) but not so in colon cancer cells (HT-29) that express wild-type kRAS mRNA. Herein we report on the design and synthesis of a new surrogate base (BisQ) with the unique feature of far-red emission. This surrogate base was introduced into PNAs that target the mutated kRAS oncogene. PNAs with a short cell penetrating peptide (CPP) consisting of 4 D-Lysines were shown to readily penetrate living cancer cells and fluoresce in the far-red region (ï¬max = 609 nm) exclusively in pancreatic cancer cells (Panc-1) that express the mutated form of kRAS but not in pancreatic cancer cells that are non-mutated (wild type) in kRAS (BxPC-3). We are currently developing new FIT-PNA probes targeting other RNA biomarkers and exploring new surrogate bases with unique spectral properties.
Thenmalarchelvi Rathinavelan
Indian Institute of Technology Hyderabad, India
Title: Mechanism of B-to-Z transition induced by A…A mismatch in a DNA duplex comprising of CAG trinucleotide repeat expansions
Time : 15:30-16:00
Biography:
Rathinavelan has completed her PhD from Department of Crystallography and Biophysics, University of Madras. Subsequently, she did her postdoctoral studies from Center for Bioinformatics/Department of Molecular Biosciences, The University of Kansas, US. Currently, she is working as an Assistant Professor in IIT Hyderabad and she has published more than 10 research papers in reputed journals.
Abstract:
Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders (TREDs) that arise from overexpansion of TREs located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Hairpin structure formed during CAG repeat overexpansion contains periodic occurrence of A…A mismatches and hijacks the mismatch repair proteins (MSH2-MSH3) through tighter binding. Although DNA hairpin stem structure generally embraces B-form with canonical base pairs, it is poorly understood in the context of periodic non-canonical A…A mismatch in a CAG overexpansion. Molecular dynamics simulations on a DNA hairpin stem containing A…A mismatches as in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. B-to-Z transition occurs through ‘zipper mechanism’ facilitated by base extrusion, backbone and/or base flipping. Root cause for such B-to-Z transition is due to the mechanistic effect that originates from the pronounced non-isostericity exhibited by A…A mismatch with flanking canonical base pairs. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A...A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.
- Track 1: Nucleic Acids
Location:
Session Introduction
Eylon Yavin
The Hebrew University of Jerusalem, Israel
Title: Peptide Nucleic Acids (PNAs) as antimalaria agents
Time : 10:30-11:00
Biography:
Eylon Yavin has completed his PhD at the Weizmann Institute of Science (Israel). He did his Post-doctoral work at the laboratory of Prof. Jacqueline Barton at Caltech (CA, USA). In 2006, he joined the School of Pharmacy at Jerusalem as a faculty member. He is currently a Senior Lecturer and has an active research lab in the field of Nucleic Acids. He has published more than 40 papers in reputed journals and has been recently elected as the President of The Medicinal Chemistry Section of the Israel Chemical Society.
Abstract:
Specific silencing of essential genes by antisense oligonucleotides (ASOs) has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. Here, we present the use of peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum; the most lethal parasite in malaria. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP) to allow facile internalization into Plasmodium falciparum infected red blood cells. PNAs simply added to cultures were found exclusively in infected erythrocytes. We show that these PNAs specifically down regulated both stably expressed transgene as well as an endogenous gene, which significantly reduced parasites viability. In addition, we show that PNA targeting an antisense lncRNA in Plasmodium falciparum affects the expression profile of Var genes; an approach that could lead to more effective clearance of the parasite by the human immune system
Thenmalarchelvi Rathinavelan
Indian Institute of Technology Hyderabad, India
Title: Give and take: ‘Z-philic’ A...A mismatch in a GAC repeat promotes interaction with Zα binding domain of human ADAR1 protein
Time : 11:00-11:30
Biography:
Rathinavelan has completed her PhD from Department of Crystallography and Biophysics, University of Madras. Subsequently, she did her postdoctoral studies from Center for Bioinformatics/Department of Molecular Biosciences, The University of Kansas, US. Currently, she is working as an Assistant Professor in IIT Hyderabad and she has published more than 10 research papers in reputed journals.
Abstract:
Tandem repeats or microsatellites are abundant in eukaryotic genomes and are polymorphic in nature. Abnormal expansion of such tandem repeats in different sequence contexts cause many incurable genetic diseases. These over expansions lead to genome instability irrespective of its location in the genome by forming unusual secondary structures comprising of non-canonical base pairs. By employing molecular dynamics simulation technique, here we investigate the structural traits of DNA d(GAC)6.d(GAC)6 hairpin containing A...A mismatch that is associated with pseudoachondroplasia and multiple epiphysial displasias. Results show local B-to-Z DNA formation akin to d(CAG)6.d(CAG)6 repeat. This finding is further corroborated by titrating d(GAC)5.d(GAC)5 with different concentrations of salt (NaCl) and Zα binding domain of human ADAR1 proteinusing circular dichroism studies. Comparison of canonical d(GAC)5.d(GTC)5 with non-canonical d(GAC)5.d(GAC)5 duplexes confirms that non-isosterisity of A...A mismatch impels Z-DNA formation in the latter, thereby, facilitating the binding with Zα binding domain of human ADAR1 protein. As this is the first study that shows the binding of A…A mismatch containing d(GAC)5.d(GAC)5 duplex with an Z-DNA binding protein, it opens up a new avenue to investigate the role of Z-DNA binding proteins in trinucleotide repeat expansion disorders. Further, even a single A...A mismatch that intervenes canonical base pairs in the following sequence contexts: 5’GAA-3’CAT, 5’GAG-3’CAC, 5’AAC-3’TAG, 5’AAG-3’TAC, 5’TAA-3’AAT, 5’TAT-3’AAA, 5’CAA-3’GAT and 5’AAT-3’TAA (A…A mismatch underlined) also forms B-Z junction at the mismatch site. Such B-Z transition imposed by non-canonical A...A mismatch irrespective of the flanking sequence may have an impact on binding with mismatch repair or regulatory proteins and the accompanying biological processes.
Pinar Tulay Vehit
Near East University, Cyprus
Title: Differential expression of parental alleles of BRCA1 in human preimplantation embryos
Biography:
Dr. Pinar Tulay was born in Nicosia, 8th October 1985. She completed her high school education in Turk Maarif Koleji in Nicosia Cyprus. She graduated from University of Missouri in 2006 with double degree in Chemistry and Mathematics (B.Sc.).In 2007, she joined the Chemical Biology Department at Imperial College for her Master’s in Research (MRes) degree in Biomedical Physical Chemistry. Her work focused on investigating the role of N-myristole enzyme in cancer. In 2009, she joined the University College London (UCL) Centre for Preimplantation Genetic Diagnosis (PGD) team.Dr. Tulay is a member of international societies of Preimplantation Genetic Diagnosis International Society (PGDIS) and European Society of Human Reproduction and Embryology (ESHRE).
Abstract:
Introduction: The expression of parental genomes is required for completion of embryogenesis. Differential methylation of each parental genome has been observed in mouse and human preimplantation embryos. It is possible that differences in methylation affect the level of gene transcripts from each parental genome in early developing embryos. The aim of this study was to investigate if there is a parent specific pattern of BRCA1 expression in human embryos and to examine if this affects embryo development when the embryo carries a BRCA mutation. Materials and Methods: Differential parental expression of ACTB, SNRPN, H19 and BRCA1 was semi-quantitatively analysed by mini-sequencing in 95 human preimplantation embryos obtained from couples undergoing preimplantation genetic diagnosis (PGD). Results: BRCA1 was shown to be differentially expressed favouring the paternal transcript in early developing embryos. Methylation specific PCR showed a variable methylation profile of BRCA1 promoter region at different stages of embryonic development. Embryos carrying paternally inherited BRCA mutations were shown to develop more slowly compared to the embryos with maternally inherited BRCA mutations. Conclusions: The results of this study suggest that differential gene expression can influence the early development of preimplantation embryos. When the paternal BRCA1 transcript present in the embryo carries a mutation, the embryo may become more vulnerable to stress due to rapid demethylation of the paternal genome and the gradual demethylation of the maternal genome. Further extrapolation of this data suggests that the risk of transmitting a BRCA mutation may be modulated by the parental origin of the mutation.