DEFICIENCY SCREENING; IDENTIFICATION OF GLUTATHIONE S-TRANSFERASE 1 AS A GENETIC MODIFIER OF THE DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL GENE, PARALYTIC
Hung-Lin Chen, Toshihiro Kitamoto
Voltage-gated sodium (Nav) channels are essential for generation and propagation of action potentials in neurons. Dysfunction of Nav channels often causes neuronal hypo- or hyper-excitability, resulting in a variety of neurological disorders, such as epilepsy. Epilepsy is one of the most common neurological disorders in the world. As estimated 50 million people worldwide suffer from this devastating pathological condition and a significant portion of patients (~30%) do not respond to anti-epileptic drugs (AEDs). Thus, identifying new target genes for AEDs is of emergent clinical need. In this study, I take advantage of Drosophila genetics to identify modifier genes that can reduce neurological phenotypes caused by mutations in the Nav channel gene, paralytic (para). Shudderer (Shu) is a mutant allele of para, displaying neuronal hyper excitability as well as behavioral and morphological abnormalities. These include seizure-like behaviors, down-turned wings, and indented thorax. Here we carried out a deficiency screen to identify modifiers of Shu. Our working hypothesis is that the Shu mutant phenotypes are enhanced or suppressed when the activity of genes functionally interacting with Shu is reduced by 50%. We systematically introduced molecularly defined deficiencies into the Shu mutant background and tested if it results in a modification of the mutant neurological phenotypes. After morphological and behavioral analyses, we identified a short genomic deletion in the second chromosome that can ameliorate Shu’s phenotypes. This deletion covers six genes. By using RNA interference and P-insertion mutants, we narrow down the modifier of Shu to glutathione s-transferase 1 (GstS1). GstS1 mutations reduced frequency of seizures in another Drosophila Nav channel mutant that carries a mutation causing genetic epilepsy with febrile seizures plus (GEFS) in humans. Immunochemistry showed a GstS1 mutation increases GABA levels in the Shu mutant brain. It indicates that deletion of GstS1 may reduce neurological phenotype of Shu by enhancing GABAergic inhibition. Since Drosophila and human share fundamental biological pathways, my study may provide a new direction for AED development.
THE ROLE OF MATERNAL WNTS AND FRIZZLEDS IN DORSO-VENTRAL AXIS SPECIFICATION
Melissa Marchal1 and Douglas Houston1
1The Interdisciplinary Program in Genetics and The Department of Biology, University of Iowa, Iowa City, IA, 52242
The proper localization of maternal mRNAs and proteins in the egg is required for many developmental processes, including dorsoventral (DV) axis determination. In Xenopus laevis in particular, maternal factors involved in patterning the DV body axis are localized during oogenesis to the oocyte vegetal cortex. Upon sperm entry, these determinants are translocated to the future dorsal side of the embryo, in a microtubule dependent process called cortical rotation. The major outcome of cortical rotation is the asymmetric activation of the Wnt/β-Catenin signaling pathway in dorsal nuclei, where β-Catenin acts to transcriptionally activate dorsal-specific genes at the midblastula transition. However, the upstream mechanisms initiating β-catenin stabilization have remained elusive. Recent evidence has suggested that secreted Wnt ligands Wnt11 and Wnt5a may act together in axis formation. However, the function of other maternally expressed Wnts and their cognate receptors (frizzleds) remains uncharacterized. We have examined the expression of wnts and frizzleds (fzds) in the oocyte and throughout early development. Through this analysis, we have found that fzds 1, 4, 6, and 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. Using a maternal depletion approach we have begun to identify the roles of these maternal factors in DV axis patterning. We present evidence that maternal fzd1-depleted embryos show a ventralization phenotype and partial defects in dorsal-specific gene expression, while fzd4-depleted embryos show a dorsalization phenotype and the expansion of dorsal-specific markers. Additionally, we show that oocytes depleted of fzds 1, 4, and 7, have significant microtubule defects, suggesting that Frizzled-dependent signaling may play a role in cortical rotation.
Genome-wide Expression Profiles in Drosophila yakuba and D. santomea.
Population genetic models predict that the X chromosome will evolve at a faster rate than autosomes (i.e., faster-X evolution). The basis for faster-X evolution is that genes are under the highest level of selection in males. Evaluations of protein-coding sequences have indeed shown an excess of divergence on the X chromosome compared to autosomes, particularly in genes expressed higher in males than in females (i.e., male-biased genes). In addition, whole-genome analyses of gene expression in Drosophila yakuba and D. santomea males indicate that X-linked genes are more differentially expressed between species (i.e., faster-X evolution of gene expression) than autosomal genes. This trend is once again strongest for male-biased genes. However, these studies utilized only males and were therefore limited in their expression profiles. Here, we will investigate the whole-genome profiles of D. yakuba and D. santomea males and females to determine the relative rates of evolution for all gene classes. We also assess the way that divergent genes are inherited in hybrid and the regulatory factors that influence their expression patterns.
Whole Exome Analysis of Individuals and Families with CRMO
For my primary research project, I am working to determine the genes and pathways involved in the development of chronic recurrent multifocal osteomyelitis (CRMO), a rare autoinflammatory bone disease presenting in infancy and childhood. We currently have whole exome sequence data from 35 individuals with CRMO. For six of the isolated cases, we have data for one or two relatives with inflammatory disease, and for three pairs of sisters and three individuals with CRMO, we have exome data for both unaffected parents, for a total of 53 exomes. Nearly all of the probands have close relatives with psoriasis or Crohn’s disease. For all of the exome data, I have processed the data from fastq to vcf format using the Burrows-Wheeler Alignment (BWA) software, SAMtools, Picard, and the Genome Analysis Toolkit (GATK). Preliminary analysis of the data suggests that variants in genes involved in IL-17 and RANK signaling are enriched in our CRMO cohort, and I am currently working on developing and analyzing a control dataset for comparison using the publicly available 1000 genomes, EVS and ExAC databases. Additionally, our laboratory will send an additional 26 samples for exome sequencing this summer – the samples are 6 individuals with CRMO and their unaffected siblings and parents. I am also performing an experiment this summer to determine the effect of a putative enhancer mutation in the first intron of a candidate gene. The variant is enriched in our CRMO cohort and likely disrupts an NR4A2 binding site.
Characterization of the NPHP10/AIMP2 interaction
Nephronophthisis (NPHP) is a recessive kidney disorder that is the leading cause of early onset, end-stage renal failure. Many proteins mutated in cystic kidney disease have been shown to localize to the primary cilia and centrosomes, providing a coalescing mechanism for NPHP-related ciliopathies (NPHP-RC). Aside from renal failure and kidney cysts, retinal degeneration and dysplasia or degeneration of the cerebellum are also seen in many NPHP-RCs. SDCCAG8 is a nephronophthisis gene (NPHP10), with patients exhibiting retinal and renal abnormalities, obesity, and learning disabilities. Mutations in SDCCAG8 were also found in several BBS patients, making SDCCAG8 the 16th BBS gene (BBS16). However, little is known about the molecular functions of NPHP10 and how loss of NPHP10 function leads to the observed phenotypes. Our research has shown that NPHP10 interacts with components of the multi-aminoacyl tRNA synthetase complex (MSC), including 8 out of 9 aminoacyl tRNA synthetases (ARS) as well as aminoacyl-tRNA-synthetase-complex interacting multifunctional protein 2 (AIMP2). Further work determined that a direct interaction likely occurs between NPHP10 and AIMP2. Our current work focuses on determining the biological significance of this interaction.
Consequences of Recombination rate variation among Drosophila Melanogaster populations
Recombination is a crucial biological process that shapes evolutionary change within and between species. At the same time, accurate estimates of recombination rates are essential for correct inferences of selection and demographic events. In this study, we use the most accurate population genetic method, LDhelmet, to estimate and compare recombination rates in three Drosophila melanogaster populations. Recombination rates not only change in total magnitude but also in their relative distribution within chromosomes (landscapes). We show that differences in recombination landscapes between populations do not accumulate at the same rate than nucleotide differences. We also show that population-specific differences in recombination landscapes play a significant role explaining population-specific differences in nucleotide diversity. Our results suggest that inter-population differences in local recombination rates and the corresponding differences in local Background Selection (BGS) need to be considered as a possible explanation for population-specific differences in nucleotide diversity at specific genomic regions.
A NATURALLY OCCURRING MUTATION IN THE FERROUS IRON UPTAKE GENE FEOB CONFERS ENHANCED RESISTANCE TO OXIDATIVE STRESS IN A FRANCISELLA TULARENSIS VACCINE STRAIN
J Fletcher1, C Bosio2, B Jones1,3
1 The Interdisciplinary Graduate Program in Genetics, University of Iowa
2 Laboratory of Intracellular Parasites, Rocky Mountain Labs, National Institute of Allergy and Infectious Diseases Department of Microbiology, Carver College of Medicine, University of Iowa
3 Department of Microbiology, Carver College of Medicine, University of Iowa
Francisella tularensis is a highly virulent bacterial pathogen with an extremely low infectious dose (~10 CFU) and high rates of mortality if left untreated (30-60%). F. tularensis has an extensive history as a bioweapon, and there is no vaccine currently licensed. For these reasons the CDC considers F. tularensis a Tier 1 Select Agent. The unlicensed Live Vaccine Strain (LVS) provides moderate protection against virulent strains; however, we have recently discovered that various lab stocks differ in their virulence and ability to confer immunity. Genome sequencing of high virulence (RML, LD50 ~200 CFU) and low virulence (ATCC, LD50 ~9,000 CFU) strains has identified nine differences, of which four are non-synonymous substitutions. One such mutation occurs in the ferrous iron uptake gene feoB in RML. While iron is required for cellular function, ferrous iron can participate in the Fenton reaction with H2O2, leading to inactivation of essential iron-sulfur cluster enzymes, and DNA damage. Part of the innate immune response involves the oxidative burst in the phagosome and mitochondria-derived ROS in the cytosol. Fully virulent strains of F. tularensis are known to be highly resistant to such host defences, and have low levels of intracellular iron. Accordingly, the RML strain was highly resistant to exogenous H2O2 in vitro relative to the ATCC strain. Overexpression of the ATCC feoB allele, but not the RML allele, leads to significantly increased sensitivity to H2O2. Furthermore, the RML strain grows poorly under conditions of iron starvation, and an iron-responsive lacZ reporter had ~3-fold higher activity in the RML strain relative to ATCC under these conditions. Overexpression of the iron-responsive transcriptional repressor fur leads to reduced growth in the RML strain, but not ATCC. These results are consistent with the hypothesis that RML has less intracellular iron, and that this may lead to increased resistance to host-mediated oxidative stress.
Fibroblast Growth Factor Receptor signaling and Src Family Kinase activity gate homeostatic synaptic plasticity
Synapses undergo many stresses and plastic changes throughout the life of an organism. Homeostatic mechanisms respond to these stresses and maintain synaptic activity within a physiologically favorable range. When faced with a reduction in postsynaptic glutamate receptor activity, the Drosophila neuromuscular junction (NMJ) homeostatically compensates by sending a retrograde signal to the presynaptic nerve. This signal triggers an increase in the number of synaptic vesicles released from the presynaptic terminal during an action potential. One of the least well understood aspects of this process is how postsynaptic systems drive production of homeostatic retrograde signals. We have identified several factors that regulate homeostatic synaptic plasticity in the postsynaptic muscle through an RNAi- and electrophysiology-based screen. This screen revealed that C-terminal Src Kinase (Csk) and the fibroblast growth factor receptor (FGFR) Heartless (Htl) are required for homeostatic compensation at the NMJ.
Work with Csk mutant alleles shows that Csk is required for the long-term maintenance of synaptic homeostasis, but not the rapid induction of this process. Csk phosphorylates and inactivates Src Family Kinases (SFKs), of which there are two in Drosophila: Src64B and Src42A. Overexpression and suppression experiments indicate that the homeostatic defects of Csk mutants are due to elevated SFK activity in the postsynaptic muscle. Immunostaining reveals that Csk mutants have altered NMJ localization of the neural cell adhesion molecule (NCAM) ortholog Fasciclin II (FasII). We examined a potential role for FasII in homeostatic plasticity and found that increasing FasII levels partially impairs this process. Additionally, reducing FasII in a Csk mutant background restores homeostatic compensation, suggesting that Csk and FasII may regulate homeostatic compensation through a common pathway.
We show that Htl is required in the postsynaptic muscle for the long term maintenance, but not the rapid induction, of homeostatic signaling. Htl is known to activate Src64B, and we show that Src64B is required for homeostasis in the postsynaptic muscle and link Src64B and Htl/FGFR signaling in the context of homeostatic compensation. FasII has been implicated as a regulator of Htl activity in Drosophila, which is supported by our observation that FasII genetically interacts with Htl during homeostatic compensation. Collectively, these data shed light on several postsynaptic factors that may work in concert to regulate the production of a homeostatic retrograde signal.
Nicotinamide Riboside is Uniquely Bioavailable In Vivo
Nicotinamide riboside is a recently discovered NAD precursor vitamin with unique activities in protection against metabolic and neurodegenerative conditions. Though nicotinamide riboside has been administered through multiple routes, it has not been established whether it achieves different or superior bioavailability in any target tissue with respect to the other NAD precursor vitamins, nicotinic acid and nicotinamide. Moreover, because enzymatic digestion of nicotinamide riboside can produce the other two NAD precursor vitamins, it is not clear whether nicotinamide riboside acts as a unique chemical entity or whether there are nicotinamide riboside-specific biomarkers. Here we show that nicotinamide riboside exhibits superior oral availability in mouse despite its metabolism to nicotinamide prior to absorption.
|Thursday 6/11/2015||Johnny Cruz Corchado||12:00-1:00 pm||106 BBE|
|Thursday 6/25/2015||Katie Weihbrecht||12:00-1:00 pm||106 BBE|
|Thursday 7/9/2015||Danielle Herrig||12:00-1:00 pm||B20 BB|
|Thursday 7/23/2015||Melissa Marchal||12:00-1:00 pm||106 BBE|
|Thursday 8/6/2015||Changya Chen||12:00-1:00 pm||106 BBE|
DM-MYB REGULATION OF CELL CYCLE GENES IS INDEPENDENT OF NURF
J F Santana1, M Parida2, A Long3, J Birdsall3, K Rogers3, M Aguilera3, S McDermott3, and J R Manak1,2,3,4
1Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA.
2Interdisciplinary Graduate Program in Informatics, University of Iowa, Iowa City, IA.
3Department of Biology, University of Iowa, Iowa City, IA.
4Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA.
c-Myb is a proto-oncogene which when mutated causes leukemia and lymphoma 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 and genetic 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 and found that there is a strong overlap of the genes regulated by these two proteins. We 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. However, 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, mutant wing discs have an increased mitotic index, with only Dm-Myb mutant animals showing a significant developmental delay presumably due to the increased time required for mitotic cells to progress through G2/M.
The Genetics Social Activities Committee would like to congratulate Dr. Ralph Hazelwood on the successful defense of his thesis and completion of his Doctorate!
Ralph is from the Fingert lab in the Visual Sciences department. His thesis seminar, entitled “Molecular genetics of optic nerve disease using patients with cavitary optic disc anomaly”, was on Monday, March 9th, 2015. Ralph has accepted a postdoctoral appointment at Vanderbilt, where we expect he will continue to excel.
We asked Ralph a few questions about his experience in the Genetics program here at Iowa:
Q. What is the most valuable/favorite thing that you have learned/experienced here at Iowa?
A. Building networks outside of my department and field was invaluable because it exposed me to different areas of research and allowed me to make great contacts and actually build friendships with well-known and senior investigators at great institutions and markets.
Q. What was your favorite class to TA?
A. Human Molecular Genetics
Q. Do you have any advice about preparing for defense?
A. Tons of advice but the most important ones are: don’t wait until the last minute to write your thesis even if your committee hasn’t given the ok. It will help you gather your thoughts and help you to start making a story which will help in the long run. The Grad college template for submission is no easy task so the earlier your start the better sanity you will have. Also, the defense is literally just that, you have to defend the work you did so be prepared to talk about why you did the particular experiment(s) and why you came to that particular conclusion(s) and the caveats based on the data. But most importantly, you pretty much know more about your project than anyone else and so your thesis committee wouldn’t allow you to schedule a defense if they didn’t feel you were ready. Finally, think about big picture impacts and/or similar genetics mechanisms or disease processes to your project which should show you have a grasp of the field and genetics in general
Again, congratulations Ralph and good luck at Vanderbilt!
Genome-wide analysis of differential DNA methylation due to social defeat stress mouse model
Early life stress is a significant risk factor for anxiety and depressive disorders, and DNA methylation (DNAm) plays an important role in the intervening pathways. The extent to which DNAm is altered across the genome in these disorders is unknown. We sought to identify the genes and regulatory sequences altered by stress in a mouse model of the disorders. Using a social defeat stress paradigm, we assessed genome-wide DNAm changes in mouse brain. We used two cohorts of stressed mice (N=7 and 7) and non-stressed controls (N=8 and 12), for discovery and validation, respectively. DNA was extracted from the dentate gyrus using a punch technique. DNA from the first cohort was examined in the discovery phase using the target enrichment system Methyl-Seq, which captures ~220,000 CpG-rich regulatory regions of the genome. DNA from the second cohort was used for validation with bisulfite pyrosequencing. Analysis of the Methyl-Seq data was done using GENESPRING and MethylKit. Thirty-three regions were significant in the discovery phase. Of these, 31 did not validate. In the remaining two regions, we detected differentially methylated loci between stressed and control groups. These loci represent an intronic region of Drosha (encoding a microRNA processing protein) and an intergenic region on chromosome X (IntchX). However when these two regions were further interrogated with a new cohort of mice (N=16 stressed and non-stressed), only IntchX replicated. IntchX included eight differentially methylated CpGs over 150 bps within an evolutionary conserved region. Because only one differentially methylated region due to social defeat stress region was replicated, further analysis of the Methyl-Seq data with follow-up bisulfite pyrosequencing will be performed to elucidate additional regions. Moreover, the nucleus accumbens, the brain region essential to reward, will be investigated for DNAm differences induced from the social defeat stress paradigm.
Sleep Abnormalities in a Drosophila Model of Human GEFS+
Despite an established link between epilepsy and sleep behavior, it remains unclear exactly how specific epileptogenic mutations affect sleep and how sleep influences epileptic seizures. Drosophila is an attractive model for studying the underlying mechanisms of this seizure/sleep relationship as it is routinely used to examine the genetic basis of seizure susceptibility and sleep behavior. Sun et al (2012) recently created a knock-in fly model of human Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a wide spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a mutation in the voltage-gated sodium channel (Nav) gene, mimicking a disease-causing human Nav mutation (SCN1AK1270T) and display a semidominant heat-induced seizure phenotype as a result of abnormal electrophysiology in inhibitory GABAergic neurons. We found that GEFS+ mutation also dominantly modifies sleep behavior, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. This sleep profile was observed regardless of sex, mating status, and genetic background. Mutants’ exaggerated sleep was more resistant to carbamazepine (CBZ), a drug that reduces Drosophila GABAA receptor activity, and could be suppressed by either constant or acute scotophase light. We further observed that GEFS+ flies have normal circadian rhythm in free-running dark conditions, but significantly lack homeostatic rebound following sleep deprivation. Intriguingly, sleep deprivation treatment increased the heat-induced seizure susceptibility of control flies, but reduced the seizure severity of GEFS+ mutants. Ongoing experiments are addressing the potential significance of GABAergic inhibition on wake-promoting PDF+ neurons in GEFS+ mutant sleep and the impact of seizing on subsequent sleep behavior. Our findings thus far have characterized the sleep architecture of Drosophila harboring a human GEFS+ mutation and provided unique insight into the relationship between sleep and epilepsy.
DISCOVERY OF DRUGS TO RESCUE ΔF508-CFTR USING A GENOMIC SIGNATURE APPROACH
Matthew D. Strub and Paul B. McCray, Jr.
Background: Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CFTR gene. The most common CFTR mutation, ΔF508, causes protein misfolding, resulting in proteosomal degradation. However, if ΔF508-CFTR is allowed to traffick to the cell membrane, anion channel function may be partially restored. The McCray Lab previously reported that transfection with a miR-138 mimic or knockdown of SIN3A in primary CF airway epithelia increases ΔF508-CFTR mRNA and protein levels, and partially restores cAMP-stimulated Cl- conductance.
Objective: We hypothesized that a genomic signature approach can be used to identify new bioactive molecules affecting ΔF508-CFTR rescue.
The Connectivity Map (CMAP): CMAP is a catalog of gene expression profiles from cells treated with a variety of bioactive molecules and has pattern-matching software to mine data. CMAP queries using miR-138 mimic and SIN3A DsiRNA gene expression signatures identified 27 molecules that mimicked miR-138 and SIN3A DsiRNA treatments. The molecules were screened in vitro for efficacy in improving ΔF508-CFTR trafficking, maturation, and Cl- current. The McCray Lab reported the identification of 4 molecules that partially restored ΔF508-CFTR, highlighting the utility of a genomic signature approach in drug discovery.
LINCS: CMAP has greatly expanded into the Library of Integrated Network-based Cellular Signatures (LINCS). Previously generated gene sets were used to iteratively query LINCS and 125 candidate molecules were selected for further testing. Functional screens performed in CFBE-(ΔF508/ΔF508) cells identified 7/125 compounds that partially rescued ΔF508.
Conclusion: Querying LINCS with relevant genomic signatures offers a novel method to identify new candidates for rescuing ΔF508-CFTR. Further analysis of these molecules and their derivatives are ongoing. We are also generating additional genomic signatures representing ΔF508 rescue for use in LINCS queries. These results represent an important step forward from our proof-of-concept CMAP studies and highlight the utility of LINCS in drug discovery for CF.