Læknablaðið : fylgirit - 03.01.2017, Blaðsíða 51

X V I I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 9 1
LÆKNAblaðið/Fylgirit 91 2017/103 51
to silence the expression of USPL1. The effect of this silencing on DNA
damage signaling and repair were analyzed using high content imaging
techniques, complemented with standard biochemical methods.
Results: USPL1 knockdown cells showed strong signs of genomic insta-
bility, including spontaneous DNA damage and abnormal nuclear morp-
hology. When challenged with DNA damaging agents, USPL1 knock-
down cells failed to efficiently accumulate key repair factors involved in
homologous recombination (HR) repair to sites of DNA damage.
Conclusion: In this project we identify USPL1, a protein with poorly und-
erstood functions, as a guardian of genomic stability. Our findings suggest
that USPL1 plays a potential role in the regulation of HR-repair, a key
tumour suppressor pathway in humans. Our future goal is to characterize
this observation in more detail.
E 148 Microphthalmia associated transcription factor (MITF)
regulates potassium channels in the olfactory bulb
Diahann A. M. Atacho1, Hallur Reynisson1, Þór Eysteinsson2, Eiríkur
Steingrímsson3, Pétur H. Petersen1
1Anatomy, Biomedical Center, 2Physiology, Biomedical Center, 3Biochemistry and Molecular
Biology, Biomedical Center
dam9@hi.is
Introduction: Microphthalmia associated transcription factor (MITF) is a
basic helix-loop-helix-leucine zipper transcription factor essential for the
development of melanocytes and mast cells. Mitf is also expressed in the
glutamatergic neurons of the mouse olfactory bulb, but its role in these
neurons is unknown. As the nervous system is shaped and regulated by
glutamate signaling and the appropriate response to neuronal activity
is required for proper functioning of a healthy neuron, a key point of
our study is to determine whether MITF determines activity-induced
responses at the transcriptional level.
Methods: We have employed several methods including RNAScope,
qPCR, ChIP, luciferase assays, immunofluorescence, electrophysiology
and behavior studies to characterize the role of MITF in olfactory neurons.
Results: We show that Mitfmi-vga9 mutant mice have increased abil-
ity of distinguishing between odors as compared to wild type mice.
Furthermore, expression of RNAs encoding for potassium channels
sub-units is decreased in the mutants as compared to wild type mice.
Using ChIP-qPCR, we show that MITF binds to the promoter of some of
the genes encoding these sub-units. Interestingly, we show a decrease in
A-current in the mitral and tufted cells of the Mitfmi-vga9 mutant mice.
Conclusion: We propose a model where MITF regulates activity in the
mitral and tufted cells of the olfactory bulb by regulating expression of
potassium channels, which control the the neuronal firing in these cells.
Further studies are aimed at deciphering the mechanism by which MITF
regulates activity-dependent responses in the olfactory bulb and how this
relates to the olfactory phenotype.
E 149 Fast Fusion Detection, Assembly, and Quantification Using
kallisto
Páll Melsted1, Shannon Hateley2, Isaac Charles Joseph3, Harold Pimentel4, Nicolas
Bray5, Lior Pachter6
1IVT, HÍ, 2Department of Molecular and Cell Biology, University of California at Berkeley,
3Graduate Program in Computational Biolog, University of California at Berkeley, 4Department
of Computer Science, University of California at Berkeley, 5Innovative Genomics Initiative,
University of California at Berkeley, 6Department of Mathematics, University of California at
Berkeley
pmelsted@hi.is
Introduction: RNA sequencing of cancer cells is a powerful approach to
detect fusions of genes in cancer cells, allowing for de novo discovery
of fused genes that are actively expressed. We focus on the problem of
detecting gene fusions from raw sequencing data, assembling the reads to
identify the breakpoints of fusions, recovering the fused transcripts and
quantifying their abundances.
Methods/Results: Building on the recently published pseudoalignment
idea, we introduce a novel approach to fusion detection based on inspect-
ing paired reads that cannot be pseudoaligned due to conflicting matches.
All potential read pairs that overlap a fusion event are reported and
further processed by our new tool pizzly, which builds on the transcript
quantification tool kallisto. Pizzly also filters false positives, reports
fusions and assembles new transcripts from the fusion reads. With pizzly,
fusion detection from raw RNA-Seq reads can be performed with the
same accuracy on 90M read datasets in under 10 minutes compared to 8
hours for previous methods.
Conclusions: The kallisto and pizzly framework is accurate and fast and
thus suitable for the analysis of large databases of cancer RNA-Seq and
for clinical use.
E 150 Northern Lights Assay in cancer theragnostics
Hans G. Þormar1, Bjarki Guðmundsson2, Olaf Hammerlund2, Joakim Lindblad2,
María Lind Sigurðardóttir2, Salvör Rafnsdóttir2, Davíð Ólafsson2, Anna M.
Halldórsdóttir2, Hlíf Steingrímsdóttir2, Helgi Sigurðsson2, Jón J. Jónsson2
1 Department of Biochemistry and Molecular Biology, UI, 2Faculty of Medicine, Genetics and
Molecular Medicine, Lanspitali University Hospital
hans@hi.is
Introduction: Cancer is treated with various medications many of which
directly affect DNA or DNA metabolism. These medications include
crosslinking agents, alkylating agents, topoisomerase II inhibitors and
chain terminators in DNA replication. The Northern Lights Assay (NLA)
can detect most major types of structural DNA damage and has the
potential to detect the effects of these medications in easily obtained body
fluids of patients undergoing chemotherapy. We have tested if NLA can
detect the effect of cancer medications on DNA which could potentially be
used to determine therapeutic response as well as to predict risk of side
effects.
Materials and Methods: Purified DNA in solution and human genomic
fibroblasts were treated with cancer medications. DNA from body fluids
(blood, plasma, urine) was isolated from patients undergoing chemother-
apy. Damage in isolated DNA was assessed with the NLA, which is based
on Two-Dimensional Strandness-Dependent Electrophoresis (2D-SDE) in
microgels.
Results: DNA in solution and DNA from cell cultures treated with cross-
linking agents (cisplatin, melphalan, mitomycin C) showed formation of
interstrand and intrastrand crosslinks. We detected DNA damage that
corresponded to the effects of the medications.in DNA from body fluids
of patients undergoing treatment with e.g. carboplatin, anthramycin and
cytarabin.
Discussion: The effects of different cancer medications on DNA have
been detected with NLA. The analysis could potentially to be used in
theranostics to measure therapy resonse or for side-effects. Studies on
larger group of patients are ongoing in order to determine the sensitivity
and thereputic prediction of this analysis.