Identification of novel exomic regions for targeted exome analysis using RNA-sequencing
Heritable retinal degenerative diseases are rare and exhibit great genetic heterogeneity. Consequently, identification of disease-causing variants in a patient’s exome may prove challenging, particularly when one or more of the true variants lie outside of the profiled regions in targeted exome sequencing. Presented is a method using RNA-seq in healthy tissues for identifying such regions towards the purpose of constructing augmented exome capture kits for disease variant identification. Results from this method in a patient cohort demonstrate its utility in identifying genetic variants causative of retinal dystrophies.
A novel post-synaptic signaling system involved in synaptic homeostasis at the Drosophila neuromuscular junction
Forms of homeostatic neuroplasticity stabilize synaptic outputs in spite of challenges to synaptic function. Aberrant synaptic activity may underlie a number of neurological disorders including epilepsy. As such, it is important to develop an understanding of the mechanisms that stabilize neuronal function. We utilize the Drosophila neuromuscular junction (NMJ) as a model synapse for studying homeostatic plasticity. At the NMJ, impaired postsynaptic glutamate receptor activity is offset by an increase in presynaptic glutamate release, allowing muscle depolarization to be maintained at wild-type levels. However, our understanding of the signaling systems that drive this process is minimal.
In a recent screen, we uncovered that C-terminal Src kinase (Csk) and the fibroblast growth factor Heartless (Htl) are required for synaptic homeostasis. Examination of loss-of-function Csk and Htl mutant alleles confirmed these result. Both Csk and Htl are known to regulate the activity of Src family kinases (SFKs). As such, we examined the roles of Src64B and Src42A, the SFK homologs in Drosophila, at the NMJ. This analysis has revealed a role for both Src64B and Src42A in homeostatic compensation, and led us to analyze downstream targets of SFK signaling. One potential target of SFK signaling at the NMJ is Fasciclin II (FasII), a homolog of mammalian neural cell adhesion molecule (NCAM) that forms trans-synaptic complexes. We have shown that misexpression of FasII at the NMJ blocks homeostatic compensation and that Csk, Src64B, and Htl mutant NMJs have altered FasII localization. This has led us to a model in which a signaling system consisting of Csk, SFKs, and Htl regulates synaptic homeostasis, perhaps through FasII.
|1/16/2014||Xitiz Chamling||2-501 BSB|
|2/20/2014||Alex Wagner||2-501 BSB|
|3/20/2014||Bing He||2-501 BSB|
|4/17/2014||Katie Weihbrecht||2-501 BSB|
|5/15/2014||Changya Chen||2-501 BSB|
NAD+ METABOLOMICS AND ITS APPLICATION TO NICOTINAMIDE RIBOSIDE THERAPIES IN RODENTS
To support experiments that range from in vitro biochemistry to yeast, vertebrate cell culture, mouse, rat and human trials, we have improved upon our earlier LC-MS/MS assay of the NAD+ metabolome (1). The new assay utilizes separations on porous graphitic carbon and two sets of internal standards to overcome ion suppression effects plaguing external calibration based quantitation methods (2). This presentation will review newly optimized methods for separation and quantification of closely related analytes. Moreover, we will present evidence that the novel NAD+ precursor nicotinamide riboside supplementation prevents diabetic neuropathy in a Type 1 diabetic rat model.
RNAi therapy in a spinocerebellar ataxia type 7 mouse model
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and vision loss with no effective treatments currently in the clinic. SCA7 is one of nine known polyglutamine (polyQ) diseases and is caused by an expansion of >37 CAG repeats in exon 1 of ATXN7.
Mutant polyQ ATXN7 gains a dominant negative function disrupting the normal function of ATXN7. Reducing the levels of mutant polyQ ATXN7 could thus reduce the downstream toxic effects of mutant ATXN7 that lead to the disease. However, for therapy in human patients, identifying methods to reduce only the mutant allele expression will be challenging. To overcome this hurdle, we hypothesized silencing of ATXN7 by RNA interference (RNAi) would alleviate phenotypes in a SCA7 mouse model.
We tested our hypothesis in a SCA7 mouse model, which expresses human ataxin-7 cDNA containing 92 pathogenic CAG repeats. The onset of PC degeneration is ~20 weeks of age, following which motor abnormalities start to develop. Unfortunately retinal degeneration is not pronounced in this model even though the transgene is expressed as seen by the presence of nuclear inclusions and QPCR. We thus tested the therapeutic efficacy and safety of reducing ATXN7 expression in the SCA7 cerebellum and evaluated the safety of reducing ATXN7 in the SCA7 retina.
How Good Are Population Genetics models to Estimate Recombination in DROSOPHILA?
Recombination is a crucial biological process that shapes evolutionary change within and between species. Developing whole-genome genetic maps is important to understand the molecular mechanism of recombination variation across genomes but also essential for accurate inferences in phenotype-genotype analyses. The generation of high-resolution, whole-genome genetic maps based on experimental crosses is cost- and labor-intensive. Therefore, it is favorable to have more practical approaches to study recombination variability across genomes that, ideally, could be applied to model and non-model species. Population genetics analyses use coalescent models and incorporate patterns of Linkage Disequilibrium (LD) to estimate recombination rates. These LD-based genetic maps provide a quick way to survey recombination variability across the whole genome based on (now cheap) genomic sequences. We sought to generate whole-genome genetic maps for populations of Drosophila melanogaster using LD mapping and sought to test their accuracy. We tested the reliability of two population genetics methods (LDhat and LDhelmet) to construct whole-genome recombination maps in Drosophila. Our preliminary results show that both LDhat and LDhelmet are good approaches to estimate the ancestral recombination rate in D. melanogaster, with LDhat presenting a more accurate model. Our data suggest there have been recent, and significant, changes in the recombination rate landscape across the D. melanogaster genome.
ABSENCE OF THE DROSOPHILA MYB ONCOPROTEIN, BUT NOT ITS INTERACTING PARTNER NURF, RESULTS IN CELL CYCLE PROGRESSION DEFECTS
c-Myb is a proto-oncogene which when mutated causes leukemias and lymphomas in birds and mammals. Vertebrates contain three representatives of the Myb gene family consisting of A-, B- and c-Myb, all of which encode DNA-binding factors that are important for the proper expression of large numbers of genes including those that regulate cell cycle progression. Drosophila melanogaster contains a single Myb gene (Dm-Myb), mutants of which die before reaching adulthood. Dm-Myb protein is present in a complex which includes the nucleosome remodeling factor NURF. Through yeast two-hybrid experiments andgenetic screens, we have shown that Dm-Myb is directly interacting with the major subunit of NURF (NURF301). In light of these results, we performed gene expression analyses in wing discs of Dm-Myb and Nurf301 mutant animals under the assumption that a significant number of genes are co-regulated by both proteins. As expected but nonetheless striking, there is a strong overlap of the genes regulated by these two proteins. We now show that in vivo, as previously reported in cell lines, Dm-Myb is necessary for the activation of cell cycle genes, specifically those involved in the G2/M transition. Interestingly, despite the strong overlap of genes co-regulated by Dm-Myb and NURF, the latter is not required for the regulation of this class of genes, suggesting that Dm-Myb and NURF function together in some contexts but independently in others. Consistent with these data, Dm-Myb, but not Nurf301, mutants have an increased mitotic index due to cells arrested in G2/M, which translates into a significant developmental delay in only the Dm-Myb mutants.
RlpA is a peptidoglycan hydrolase that facilitates daughter cell separation in Pseudomonas aeruginosa
RlpA is a protein of heretofore unknown function found in many Gram-negative bacteria. In Escherichia coli RlpA localizes to the septal ring, suggesting the protein is involved in cell division, but mutants of rlpA have no obvious phenotype (Gerding et al, J. Bacteriol. 2009; Arends et al, J. Bacteriol. 2010). The sequence of RlpA indicates it is an outer membrane lipoprotein with a double-psi beta barrel (DPBB) domain and a peptidoglycan-binding SPOR domain. We report here that E. coli RlpA is present at about 600 molecules per cell and traffics to the outer membrane, as predicted. Because we could not find an rlpA-related phenotype in E. coli, we turned to Pseudomonas aeruginosa, for which we had access to an ordered library of transposon mutants (Liberati et al, PNAS 2006). Interestingly, an rlpA::Tn mutant formed chains of 4-8 cells when grown in media of low osmotic strength. We confirmed the chaining phenotype by constructing an in-frame deletion (ΔrlpA). Both mutants could be rescued by complementation with rlpA or an rlpA-mCherry fusion. The RlpA-mCherry fusion protein localized to the midcell during cell division and localization required the SPOR domain. Analysis of purified peptidoglycan from the ΔrlpA mutant by HPLC revealed an increase in a muropeptide whose structure was determined to be a tetrasaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc) by amino acid/amino sugar analysis and mass spectrometry. Purified RlpA protein hydrolyzed the tetrasaccharide moiety from intact peptidoglycan purified from the ΔrlpA mutant, but did not hydrolyze the purified tetrasaccharide fragment. RlpA proteins with amino acid substitutions in the DPBB had little or no detectable cell wall hydrolysis activity and were not very effective at rescuing daughter cell separation in vivo. We conclude that RlpA is peptidoglycan hydrolase that cleaves the glycosidic linkage between GlcNAc and MurNAc in the peptidoglycan strands to facilitate daughter cell separation.
Drosophila DopEcR, a dual receptor for ecdysteroids and dopamine, modulates ethanol-induced sedation behavior
Steroid hormones exhibit profound effects on behavior through both genomic and non-genomic signaling. Unlike the classic genomic mechanism where steroids act via cognate nuclear hormone receptors, the significance and molecular underpinnings of rapid non-genomic steroid signaling that occurs independently of new mRNA synthesis remains poorly understood. Recently, a Drosophila G-protein coupled receptor named DopEcR was identified as a putative non-genomic steroid receptor. Interestingly, DopEcR activates rapid intracellular signaling cascades in response to both dopamine and the major insect steroid hormone ecdysone when expressed in heterologous cell culture. Our goal is identify the function of DopEcR in vivo to elucidate the role of non-genomic steroid signaling in the nervous system. Since DopEcR is primarily expressed in the fly brain and uses signaling components associated with evolutionarily conserved alcohol-induced behaviors, we examined the responses of DopEcR mutants to ethanol. We found that DopEcR mutants are significantly resistant to ethanol-induced sedation – a phenotype that can predispose animals to alcohol addiction. To further understand DopEcR-dependent regulation of alcohol-induced behavior, we examined the potential genetic interactions of DopEcR with EGFR and cAMP signaling since both pathways have previously been shown to affect ethanol-induced sedation in flies and rodents. Our experiments strongly suggest that DopEcR functions to inhibit EGFR/Erk and activate cAMP signaling following ethanol exposure. Further observation of ethanol-induced behaviors following genetic and pharmacologic manipulations will continue to help elucidate the function and mechanism of DopEcR. Our findings offer important insight into how behavioral response to alcohol is controlled by dopamine and non-canonical steroid signaling.
Heterozygous triplication of regulatory elements is responsible for cavitary optic disc anomalies
Purpose: To identify and characterize the gene that causes autosomal dominant, congenital malformations of the optic nerve known as cavitary optic disc anomaly (CODA) in a multiplex family with 17 affected members. Features of the nerve disease in CODA closely resemble that of glaucoma (excavated optic nerve head appearance) that occurs in the absence of elevated IOP, suggesting that the gene that causes CODA may also contribute to the pathophysiology of glaucoma.
Methods: The gene that causes CODA was previously mapped to a 13.5Mb locus on chromosome 12q14. The proband of the CODA pedigree was tested for copy number variations (CNVs) in the chromosome 12q14 region with custom comparative genomic hybridization using the NimbleGen platform (Madison, WI). CNVs were confirmed with quantitative PCR in the proband and in the remaining 16 affected family members as well as in a panel of 78 controls. Confirmed CNVs were analyzed for their effect on downstream genes using a luciferase reporter gene construct (pGL3, Promega, Madison, WI) in HEK293T cells.
Results: CGH experiments identified a triplication of a 6kb segment of DNA upstream of matrix metalloproteinase 19 (MMP19) in the proband of the CODA pedigree. Quantitative PCR experiments confirmed that the MMP19 promoter CNV is present in the proband as well as in the additional 16 affected family members. No control subjects carried this mutation. Furthermore there were no instances of this variant in the database of genomic variants (projects.tcag.ca/variation). The luciferase reporter gene assay showed that the 6kb sequenced spanned by the CNV in CODA patients increased luciferase activity and functioned as a transcription enhancer. Moreover, a similar analysis of overlapping subdivisions of the DNA sequence in the 6kb CNV showed that a 1kb segment had a strong positive influence (8-fold higher) on downstream gene expression. Immunohistochemical experiments identified robust expression in the optic nerve head and throughout the optic nerve extending to the retrolamniar regions consistent with microarray expression data.
Conclusions: We have identified a copy number variation mutation in the promoter sequence of the MMP19 gene that co-segregates with CODA in our large 17 member pedigree. Moreover we have shown that the CNV spans DNA sequences that powerfully enhance downstream genes (i.e. MMP19). MMP19 is also expressed in the relevant ocular tissues. These functional data strongly suggest that the genetic defects in MMP19 cause CODA and that over-activity of this gene has an important role in the pathogenesis of this optic nerve disease.
This year the Genetics Retreat will be held at Hotel Vetro, which is located on the ped mall in downtown Iowa City. The retreat will begin at 11:00 am on Friday October 11, 2013 and will conclude with a Public Presentation by our keynote speaker, Dr. Scott Keeney. Dr. Keeney is an HHMI Investigator in the Sloan-Kettering Cancer Center Molecular Biology Program who studies the mechanisms and initiation of meiotic recombination. For more on Dr. Keeney and his work follow this link: http://www.mskcc.org/research/lab/scott-keeney. We will also have talk from a Genetics Program Graduate, Dr. Kaan Certel, who is now a group leader at X-Chem Industries.
Tentative Retreat Schedule:
11:00 AM Pre-Retreat Set Up
Students must arrive early to set up posters or upload their talks
11:30 AM Lunch with Industry Speaker: Kaan Certel, PhD
12:30 PM Opening Remarks
Daniel Eberl, PhD, Genetics Program Director
12:45 PM ”Science in the Biotech World: The Start-up Experience”
Kaan Certel, PhD, Group Leader at X-Chem Inc.
1:15 PM Poster Session
3:00 PM Keynote Presentation
Welcome: Sarit Smolikove, PhD, Retreat Committee
Keynote Speaker: Scott Keeney, PhD, HHMI Investigator and Professor at Sloan-Kettering Cancer Center
4:00 PM Break
4:15 PM Student Presentations (15 min each)
5:45 PM Student Dinner with Dr. Scott Keeney
6:50 PM Announcement of Oral Presentation and Poster Awards
7:00 PM “A Dangerous Game: DNA Breaks and Chromosome Dynamics”
Scott Keeney, PhD, HHMI Investigator at Sloan-Kettering Cancer Center
Art in Science Competition
We will celebrate the artistic side of science at this year’s Genetics Retreat. Students are encouraged to submit artistic representations of their research for display at the retreat. Art in science can include anything from artistically manipulated microscopy images to paintings and anything in between. Feel free to be creative! Submissions will be voted on by the genetics student body at the Genetics Retreat, and the scientific art with the most votes will receive a prize.
Important Dates for Genetics Students:
September 20, 2013: Retreat Abstract Submission Deadline
September 23, 2013: Announcement of Abstracts Selected for Oral Presentations
September 30, 2013: ‘Art in Science’ Titles Due
October 4, 2013: Poster Printing Begins
October 10, 2013: Final Day of Poster Printing
October 11, 2013: Genetics Retreat!
Purpose: Cancer is a complex disease caused by many somatic mutations. Cancer is often the result of random mutations. Random mutations can be classified as driver (causative) and passenger (non-causative) mutations. Differentiation between the driver mutations and passenger mutations is extremely difficult in human cancers due to the complexity of the disease and mutations. In addition, understanding the mechanisms of these genes is confounded by the rarity of many cancer genes. Network biology has been used to attempt to understand the relationship between genes and the possible shared functions of these genes. However, these networks are incomplete and often only contain well-characterized connections, making it difficult to identify more distantly connected genes.
Methods: A relatedness network was created utilizing network reliability algorithms. The relatedness of genes is the reliability of the connection between two genes. This was determined using the Edge-Packing Bounds algorithm with maximum of 4 intermediate genes, maximum of 5 paths, and minimum edge weight of 50%. This network was used to determine the relatedness between genes. By combining multiple independent paths we can better visualize and identify the connections between two genes.
Discussion: By combining these independent paths, we increase the number of pairwise comparisons from ~518,000 to ~51 million. This is mainly due to the addition of indirect connections between genes. When we use this information on pathways, we see an increase of strongly connected genes.
Lily Paemka’s Abstract:
PRICKLE1 interaction with SYNAPSIN I reveals a role in Autism Spectrum Disorders
The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; and several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD, however the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1+/- mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via theSYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.
Joshua Fletcher’s Abstract:
Identification of the candidate virulence gene virK in Francisella tularensis
Francisella tularensis is a highly virulent bacterial pathogen that parasitizes the cytosol of an infected cell. Potentially fatal infections can result from inhalation of as few as 10 organisms, leading the CDC to classify Francisella tularensis as a Tier 1 Select Agent. The bacterium escapes the host cell phagosome by an unknown mechanism dependent on a genomic region known as the Francisella Pathogenicity Island (FPI) and replicates in the nutrient-rich cytosol. Data from a recent transposon mutagenesis screen performed by our lab was combined with protein homology modeling strategies to identify candidate virulence genes in the fully virulent Francisella tularensis Schu S4 strain. One such candidate is encoded by FTT0613c, which we have shown is homologous to virK from various plant pathogens and commensals, as well as the human pathogens Burkholderia and Legionella. A ΔvirK mutant in the Schu S4 strain is attenuated approximately 1,000-fold in murine intranasal infections with an LD50 of 1.15×104 CFU. Vaccination with ΔvirK provided limited protection against 280 CFU of wild type Schu S4 (~30x LD50), increasing time to death by numerous days. Infection of two relevant cell types in vitro (murine bone marrow-derived macrophages and human small airway epithelial cells) indicates that ΔvirK does not proliferate in host cells. Additionally, preliminary data from protein-protein interaction experiments suggest that VirK may interact with host cell proteins. These data support the hypothesis that VirK is a non-FPI virulence factor that may have access to host proteins in the phagosome or cytosol. Furthermore, double and triple mutants in the ΔvirK background may prove useful for the generation of a vaccine strain.