Learning the Epilepsy Alphabet from A (syne1a&b) to Z (zfhx3)
Epilepsy, which affects ~1% of the population, is caused by abnormal synchronous neural activity in the central nervous system (CNS). While there is a significant genetic contribution to epilepsy, the underlying causes for the majority of genetic cases remain unknown. The NIH Undiagnosed Diseases Project (UDP) utilized exome sequencing to identify genetic variants in patients affected by various conditions with undefined etiology, including epilepsy. Confirming the functional relevance of the candidate genes identified by exome sequencing in a timely manner is crucial to translating exome data into clinically useful information. To this end, I developed a high throughput version of a seizure-sensitivity assay in zebrafish (Danio rerio) to rapidly evaluate candidate genes found by exome sequencing. This assay uses pentylenetetrazol (PTZ) to induce seizures in zebrafish larvae and the motility tracking software of the Zebrabox (Viewpoint Life Sciences) is utilized to record each larva’s total movement (cm). This generates massive data sets. Therefore, I developed an open access software, SEIZR (Studying Epilepsy In Zebrafish using R), to rapidly and efficiently analyze the data. My project focuses on characterizing the functional role of fifteen genes in the NIH UDP for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Using SEIZR, I characterized all fifteen candidate genes in the context of seizure sensitization. Here, I show the findings of two genes, syne1b and zfhx3, both of which result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in regions of the brain involved in motor control during critical times of neural development. Further, I find syne1b knockdown results in axon defects in the retina. Reagents to test for rescue and localization are in progress.
Filling the Gaps in the Drosophila Yakuba Genome
Most ‘reference’ genomes are incomplete, with sequence gaps throughout the euchromatic genomic regions of model organisms such as Drosophila melanogaster, zebrafish, mice and even humans. These sequence gaps are the result of difficulties in cloning and/or sequence assembling across simple repeats and repetitive elements. Because these gap regions can contain genes and add uncertainty to physical and genetic distances, the presence of gaps can hinder mapping strategies (QTL, GWAS, etc.) as well as evolutionary studies. The genome of our model organism of interest, D. yakuba, is completely sequenced but it also contains genomic gaps. Most short-read sequencing technologies (i.e., Illumina) are usually unable to address the gaps but the more recent long-read Pacific Biosciences (PacBio) SMRT sequencing approach provides a solution to this problem because individual reads can be 5-20kb long and hence span the entirety of challenging genomic regions. On the negative side, PacBio reads have an error rate that is much higher than (classic) Sanger and Illumina sequencing. The ideal strategy is, therefore, a ‘hybrid’ sequencing approach where PacBio sequences are used to fill in the gaps to later take advantage of deep-coverage Illumina sequencing to ensure high quality sequences for these new genomic regions. We used this hybrid method to improve the D. yakuba genome. We fill more than 60% of the gaps and we add approximately 6.2 million bp of high quality sequence to the D. yakuba reference genome, creating an improved version of the original reference genome with an additional 6% of genomic data. The new D. yakuba genome will be useful in several downstream applications such as identifying novel genes and transcripts. In particular, studies that use genomic distance, such as the calculation of recombination frequency per nucleotide as well as the prediction of the impact of selection on diversity will be much more accurate.
The role of gut microbiota and the antioxidant system in the seizure-like behavior of Shudderer, a Drosophila voltage-gated sodium channel
It is widely recognized that mutations in genes encoding voltage-gated sodium (Nav) channels contribute to the etiology underlying various seizure disorders. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, exhibits neuronal hyperexcitability and severe seizure-like behavioral defects, including spontaneous jerking and heat-induced convulsion. Results of microarray analyses indicated that Shu mutants have increased innate immune response and reduced insulin signaling. Because the endogenous gut microbiota significantly impacts the host immune system and insulin-mediated metabolic regulation, we hypothesized a role for the gut microbiota in Shu phenotypes. Raising Shu and wild-type (WT) flies in food containing antibiotics was sufficient to eliminate the gut microbiota. Intriguingly, the treatment significantly suppressed Shu behavioral phenotypes while having no effect on WT behavior.
We also identified changes in the gut microbiota composition of Shu mutants. In WT flies we found the microbiota consisted primarily of Acetobacter and Lactobacillus species, in agreement with the published literature. Shu mutants showed a decrease in Lactobacillus species and a drastic increase in Rhodanobacter taxa, a bacterial genus present at low abundance in our WT flies. These differences were present in both larvae and adults. Because changes in the microbiota composition can influence the gut environment we also examined the acidity and alkalinity of the gut using a pH-sensitive dye and calculated the proportion of different midgut cell populations in both WT and Shu flies. Future experiments seek to confirm these changes in microbiota composition using 16S rDNA sequencing.
ER stress phenotype in iPSC-derived RGCs of patients with a hypomorphic mutation in WFS1
Mutations in wolframin ER transmembrane glycoprotein 1 (WFS1) gene have been associated with optic atrophy and Wolfram syndrome. The purpose of this study was to use patient specific iPSCs to determine how mutations in WFS1 cause retinal ganglion cell (RGC) death. Dermal fibroblasts were obtained and expanded from two patients with suspected WFS1 associated optic atrophy and one patient with molecularly confirmed Wolfram syndrome. Fibroblasts were targeted for induced pluripotent stem cell (iPSC) generation using Sendai viruses driving expression of OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed using rt-PCR, immunocytochemistry and the TaqMan Scorecard Assay. Immunofluorescence, quantitative rt-PCR, and Western blot analyses were used to characterize expression of RGC and ER stress associated markers. Cells generated from an individual with no ocular history and a patient with molecularly confirmed Wolfram syndrome were used as controls. Homozygous Arg558Cys WFS1 variants were identified in two patients with non-syndromic recessive optic atrophy using exome sequencing. As determined by a TaqMan ER stress assay, patient-specific iPSC-derived RGCs generated from these two individuals were found to have milder activation of key ER stress genes such as HSPA5, CALR, CANX and ERO1LB compared to control and an elevated ER stress phenotype in the Wolfram syndrome patient. Likewise, ER-stress mediated RGC dysfunction, was evident by increased levels of HSPA5, ERO1LB and CANX in iPSC-derived RGCs. We have successfully demonstrated that mutations in WFS1 lead to activation of the ER-stress pathway in patient specific iPSC-derived RGCs.
Examining the role of ARHGAP29 in cutaneous wound healing
Despite every person incurring acute wounds frequently, the genetics and molecular mechanisms regulating wound healing remain to be elucidated. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration. Interferon Regulatory Factor 6 (IRF6) has been shown to regulate all of these processes, with IRF6-deficient keratinocytes displaying both a decrease in the expression of Rho GTPase Activating Protein 29 (ARHGAP29) as well as an increase in stress fibers. ARHGAP29 is a Rho GTPase Activating Protein with a high affinity for the small GTPase RhoA. However, despite our current knowledge about IRF6 and RHOA, little is known about how IRF6 regulates ARHGAP29 and the role of ARHGAP29 in cellular migration, adhesion, and wound healing. We hypothesize that ARHGAP29 is transcriptionally regulated by IRF6 and perturbation of this system will result in impaired wound healing. To start testing our hypothesis, we evaluated the expression of ARHGAP29 in adult murine skin before and up to 7 days post excisional wounding. As previously reported, ARHGAP29 was detected in the cytoplasm of keratinocytes in epidermis and in dermal fibroblasts. One day after wounding, ARHGAP29 was observed at the wound margin in keratinocytes and in inflammatory cells in the granulation tissue. Taking advantage of a murine allele harboring a K326X orofacial cleft patient-derived mutation in ARHGAP29, we performed full thickness excisional wounds on heterozygotes and harvested tissues 4 and 7 days post-injury. Preliminary morphometric analyses revealed no significant differences at 7 days. Investigation of d4 time point is currently under way. Together, these results suggest that reduced level of ARHGAP29 is still sufficient to heal a cutaneous wound. Further strategies include the use of a keratinocyte specific knockout, as well as the generation of an “allelic series” of ARHGAP29 for in vitro assessment of keratinocyte migration.
Chemogenomic meta-analysis of ΔF508-CFTR rescue identifies novel therapeutic targets and compounds
Background: The common ΔF508-CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including over-expression of miR-138. A challenge remains to translate such interventions into therapies and to understand their mechanisms. One technique for connecting such interventions to small molecule therapies is via mRNA expression profiling. The idea is to identify small molecule-induced expression responses (catalogued in CMAP/ LINCS) that are similar to the expression induced by interventions. In a complementary strategy, one can identify expression that is reversed compared with profiles characterizing diseased vs. healthy individuals.
Objective: To leverage and expand upon previous genomic signature approaches to prioritize and test compounds for rescue of ΔF508-CFTR trafficking and function.
Strategy: We developed gene sets for CMAP/LINCS queries by: 1) performing meta-analyses of rescue and disease signatures, and 2) extracting gene sets from MetaCore-curated pathways related to CFTR processing. All gene sets were scored against each CMAP/LINCS drug profile. To prioritize compounds for screening, overall scores were calculated for each drug by combining the top signature and pathway scores. We selected 120 candidate small molecules for functional testing. These molecules were administered to ΔF508/ΔF508-CFBE cells and transepithelial chloride conductance was measured.
Results: In initial studies, 38 molecules partially restored ΔF508 function, as assessed by cAMP-activated chloride conductance. Over half of these compounds showed significant cooperativity when administered with a known corrector. We have extensively validated five of these compounds in primary ΔF508/ΔF508-HAE cells.
Conclusion: This integrative drug prioritization approach offers a novel method to both identify small molecules that rescue ΔF508-CFTR function and identify gene networks underlying such rescue. Ongoing work includes informatic analyses to identify common themes and targets among the set of active compounds.
Identification of Isthmin1 as a novel gene associated with craniofacial dysmorphologies.
Clefts of the lip and/or palate (CL/P) occur about every 1 in 700 live births as isolated, non-syndromic (NSCL/P) cases or in the presence of additional syndromic (SCL/P) structural or cognitive abnormalities. We performed the largest array-based comparative genomic hybridization study to date to assess the involvement of copy number variation (CNV) in NSCL/P in 889 probands from the Philippines and 250 probands of European descent. We filtered the CNV calls to identify genes that were deleted in greater than one individual at a frequency higher in cases than controls (yet were still rare in the disease cohort) in order to identify potentially high effect size genomic aberrations driving the disease phenotype. The majority of the 42 loci identified by this strategy were novel candidates with no known association with clefting. One such gene, Isthmin1 (ISM1), was selected for functional follow-up. In situ hybridization analysis in frogs confirmed ism1 is expressed in the developing face and branchial arches, with concentrated expression around the mouth by stage 35. Morpholino (MO) knockdown or CRISPR-Cas9 deletion of ism1 in frogs resulted in a variety of strong craniofacial defects, which were rescued in MO knockdowns by ism1 mRNA co-injection. Intriguingly, “cleft-like” faces were observed in 5 of 26 stage 45 MO knockdown embryos, providing evidence that ism1 is required for proper craniofacial development.
Intra-specific variation in recombination rates as explanation for differences in levels of diversity among D. melanogaster populations
In our study, we estimated recombination rates in six Drosophila melanogaster populations (South Afirca, Zambia, Rwanda, Cameroon, France, and USA) based on population genomics data and determined the inter population variation in recombination rates across the genome. We observe that recombination rates not only change in total magnitude but also, and significantly, in their relative distribution within chromosomes (recombination landscapes). Our quantitative analysis reveals scale-specific patterns of variation with higher correlations at broad scales and lower correlations at fine scales. We then analyze if the observed differences in recombination landscapes play a significant role explaining population-specific differences in nucleotide diversity under a linked selection scenario that considers only purifying selection (i.e., Background Selection). Our results suggest that population-specific differences in nucleotide diversity at specific genomic regions can be explained by differences in population-specific recombination rates and the corresponding linked selection effects associated with the inevitable and frequent input of deleterious mutations.
Targeted Sequencing and Functional Assessment of the 2p25 Region in Suicide Attempters with Bipolar Disorder
Our lab previously conducted a genome-wide association study (GWAS) of the attempted suicide phenotype in individuals with bipolar disorder. The GWAS implicated common variation in the 2p25 region (p = 5.07 x 10-8). The most significant variants in this region localized to an intergenic and putative regulatory region. In the current study, we conducted a targeted next-generation sequencing study of the entire 2p25 region (350kb total) in 476 bipolar suicide attempters and 473 bipolar non-attempters. Our goal was to better characterize this region by assessing both common and rare variation contributing to the attempted suicide phenotype. Our sequencing effort allowed us to narrow the associated region from 350kb to 80kb based on a clustering of our top variants within an intergenic 80kb linkage disequilibrium block in 2p25. The top results from the current study also localized with our best variants from the attempted suicide GWAS. In order to determine the regulatory potential of our region, we identified and deleted the most promising 10kb section (chr2:103,500 to chr2:113,500) of our 80kb LD block in HEK293 cell lines using CRISPR-Cas9. Deletion of this region significantly altered the expression of 37 genes across the genome, including genes involved in apoptosis and DNA structure. In addition, we used RNA-sequencing to identify genes that were differentially expressed in HEK293 cells following lithium treatment and determined that a number of these genes interact with the differentially expressed genes identified through our 2p25 CRISPR experiment. These implicated genes and pathways warrant further investigation in more relevant cell types and animal models as candidates for the biological basis of suicidal behavior.
Wes Talks WES
Building and optimizing a new and improved sequencing pipeline to analyze over 2000 exomes.
A negative feedback loop involving Kctd15 and Tfap2 paralogs regulates melanocyte differentiation in zebrafish
The gene regulatory network governing melanocyte differentiation is relevant to the pathogenesis of pigmentation disorders and melanoma. We have recently shown that transcription factor TFAP2A binds 70% of active enhancers in melanocytes, including many bound by the melanocyte “master regulator” MITF. It is therefore surprising that Tfap2a-/- mice and zebrafish have only mild pigmentation phenotypes, and relatively few melanocyte genes show altered expression in mouse melanocytes depleted of Tfap2a. We hypothesize that additional TFAP2 paralogs compensate for depletion of TFAP2A. Supporting this view, we recently showed that mice with double conditional knockout of Tfap2a/b in the neural crest exhibit a far more severe reduction in melanocytes than either single mutant. To further examine the redundant activity of paralogs, we are 1) generating zebrafish lines triple mutant for tfap2a/c/e and 2) artificially expressing Kctd15a, a potent inhibitor of all Tfap2 paralogs, in zebrafish melanocytes. Towards the first approach, we introduced a 157bp deletion into zebrafish tfap2e using zinc-finger nucleases. Homozygous tfap2eΔ157 mutants do not display a notable pigmentation phenotype, and tfap2alow/tfap2eΔ157 double mutants largely retain the tfap2alow mutant phenotype, suggesting that tfap2c may compensate for loss of both paralogs. Towards the second approach, we find that melanocytes expressing mitfa:kctd15a appear smaller, paler, and abnormally shaped, suggesting that inhibition of Tfap2 paralogs impairs melanocyte differentiation. To determine a mechanism for the regulation of kctd15a expression by Tfap2 paralogs, we conducted ATAC-seq on tfap2a/c double-mutant zebrafish and identified a potential Tfap2-dependent enhancer adjacent to kctd15a. This enhancer is conserved from zebrafish to humans and comparison to ChIP-seq profiles indicated that it is bound by both TFAP2A and MITF in human melanocytes. We cloned the candidate enhancer sequence into a GFP-reporter vector and observed reporter activity in zebrafish melanocytes at 36hpf. These findings suggest that Tfap2 is regulated in part by Kctd15a via a negative feedback loop, and identify Kctd15a as a potential modulator of Tfap2 activity in melanocytes and melanoma.
Using MaxGel to study complement regulation on extracellular matrix
Extracellular matrix (ECM) is a collection of various glycoproteins, playing important roles in different organs. In kidney glomeruli, ECM covers basement membrane and acts as anchors for many proteins to regulate complement activity on cell surface. In previous studies of complement activity on cell surface, titer plates were broadly used, while by lacking the ECM, this system is different from the real situation. In this study, we used MaxGel to mimic ECM on glomerular basement membrane, therefore to reveal the complement regulation on cell surface. We found that complement components, like C3 and FB bind to MaxGel. By incubating complement components, C3b, FB, FD on the MaxGel, C3 convertase formed. Moreover, the C3 convertase formed on MaxGel was regulated by positive complement regulator, FP, negative complement regulator, FH and also C3 convertase autoantibodies found in patients’ circulation. Our study used a new system to modulate the environment on glomerular basement membrane and illustrate the complement activity on cell surface.
Involvement of a unique G-protein coupled steroid receptor in febrile seizures in Drosophila
Our lab is broadly interested in understanding the genetic and environmental factors that influence the severity of epileptic seizures. Steroid hormones are signaling molecules that have long been speculated to have a role in epilepsy. A subset of women epilepsy patients experience exacerbated seizures during certain periods of the menstrual cycle, concomitant with increases in particular sex hormones. Estrogens are generally proconvulsant while progesterone and its metabolites have anticonvulsant effects. The mechanisms by which particular steroid hormones may have pro- or anti-convulsive effects are not well understood. Thus, in order to better understand how steroid signaling influences epileptic severity, our lab studies a fruit fly gene called Dopamine Ecdysone Receptor (DopEcR) that encodes an interesting G-protein coupled receptor that responds to both ecdysone, the major steroid hormone in the fly and the catecholamine dopamine. Canonical steroid receptors typically bind steroid and function as transcription factors, leading to transcription-dependent cellular responses that are delayed but long-lived. DopEcR mediates unconventional ”non-genomic” steroid responses, which occur independently of transcriptional regulation and generally involve intracellular signaling cascades to rapidly and transiently influence cellular physiology, for example, through cAMP or MAPK signaling. I report that Drosophila DopEcR mutants show a febrile seizure phenotype, where heatshock at 40⁰C immediately causes a significant portion of flies to exhibit seizure-like behaviors. Furthermore, RNAi-mediated knockdown of DopEcR in neurons but not glia is effective at phenocopying the mutant febrile seizure behavior. The DopEcR seizure-like behaviors are also insensitive to a dietary intervention our lab has shown to be effective at reducing the severity of the neurological phenotypes of certain voltage-gated sodium channel mutations in flies. The study of DopEcR is a valuable entry point for a fuller understanding of how steroid signaling may influence the severity of seizure-like behaviors.
Assessment of daily rhythms of activity in Drosophila circadian pacemaker neurons
Endogenous circadian pacemakers drive many biological processes allowing animals to advantageously align daily behaviors to the external environment. In Drosophila, these molecular clocks reside in specialized pacemaker neurons in the brain. Our lab focuses on understanding the relationship between circadian neuron activity patterns and behavioral output. We use both the ArcLight voltage sensor and the GCaMP calcium indicator to assess spontaneous pacemaker neuron physiology as well as the neuronal response to pharmacological manipulations. Using these techniques, we have investigated the daily rhythms of excitability in two of the three major pacemaker neuronal subsets. We are currently working on examining the excitability of the third major group, the dorsolateral neurons (LNd).
Investigation of the Mechanism of BIR and ALT
Mentor: Anna Malkova
Break induced replication (BIR) is a homologous recombination-dependent mechanism that repairs double strand DNA breaks (DSBs) that are made in such a way that only one end of the break can find homology in the genome for repair. A natural situation where BIR was suggested to occur is in a process called alternative lengthening of telomeres (ALT) that is responsible for the telomere maintenance in the absence of telomerase. Overall, fifteen percent of malignant tumors maintain their telomeres by ALT, and is especially prevalent in osteosarcomas and malignant fibrous histiocytomas where it occurs in more than half of all cases. It has been shown that BIR begins with resection of the broken end that then invades homology forming a D-loop. This mechanism is unusual because at this D-loop, synthesis begins and progresses by a migrating bubble until the end of the chromosome resulting in conservative inheritance of the newly synthesized DNA. Though, BIR results in a repaired chromosome, it comes with deleterious effects that may be the result of accumulations of long stretches of ssDNA. These long stretches of ssDNA can lead to tangled toxic intermediates and may facilitate gross chromosomal rearrangements, as well as an increase mutagenesis, which was previously demonstrated to be 1000 times more as compared to S-phase replication. All these deleterious effects of BIR make it important for us to understand how BIR is carried out, and regulated.
Genes Implicated in Neurodevelopment Enriched for FOXP2 Binding Sites Associated with Language Ability
Tanner Koomar, Jacob Michaelson, PhD.
Specific Language Impairment (SLI) is a neurodevelopmental condition which causes linguistic deficits in children with otherwise normal development. SLI is relatively common (occurring in ~7% of the population) and demonstrably heritable (h2 ~ 0.6). Linkage, GWAS, and twin studies of SLI have produced mixed results with inconsistent replication, necessitating the integration of other forms of molecular data related to language ability. The transcription factor FOXP2 is robustly associated with language ability, with perturbations to the coding region of the gene resulting in severe language deficits. However, such coding changes in FOXP2 are exceedingly rare. Variation in FOXP2’s thousands of DNA binding sites is plentiful, on the other hand, making them attractive targets for interrogation in a common condition like SLI. In this work, genetic variants overlapping FOXP2 ChIP peak sites were extracted from the whole genome sequences of a cohort of ~280 children (140 SLI and 140 control), and ranked based on association with overall language ability. Variation in the FOXP2 locus was also tested for association with language ability. Some genes co-expressed with FOXP2 in the developing human brain and implicated in neurodevelopment, were enriched for high-ranking FOXP2 binding sites. Despite these intriguing results, this variation – and that in the FOXP2 locus itself – was only able to explain a fraction of differences in total language ability.
Prostaglandins regulate collective cell migration in the Drosophila ovary
Emily Toombs, Tina Tootle PhD, Anatomy and Cell Biology Department University of Iowa
Collective cell migration – the coordinated movement of tightly or loosely associated cells – is important for both normal development and tumor invasion. While prostaglandins (PGs), short-range lipid signaling molecules, are known to be important for single cell migration, their mechanism of action is poorly understood. One potential mechanism is via regulation of mechanotransduction. Mechanotransduction, or the transfer of physical force into electrical/chemical signals, is essential to proper cellular migration. Mechanotransduction is mediated by the direct connection of the cytoskeleton to the nucleoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Our lab has shown that PGs control actin remodeling via multiple mechanisms, including via regulating Fascin. Fascin is an actin binding protein essential to cell migration and known to interact directly with the LINC complex. Despite this, PG signaling has not been previously implicated in mechanotransduction. Here we present the first evidence that PGs may regulate invasive collective cell migration and propose that this is via regulation of the LINC Complex. We assess this using a collective, invasive, epithelial migration during Drosophila oogenesis. In Drosophila, the ovary contains chains of developing follicles composed of 15 germline derived nurse cells, 1 oocyte, and a surrounding layer of somatic epithelial cells. Within these follicles, a cluster of 6-8 somatic cells delaminate from the outer epithelium and migrate invasively between the nurse cells to the oocyte border. We hypothesize that PG signaling modulates border cell migration by regulating perinuclear Fascin localization to control LINC complex function. Here we show that loss of PGs results in delayed border cell migration, as does loss of LINC complex components. Current efforts focus on determining cell specific roles of PGs in this migration. This research is expected to provide the mechanistic insight into how PGs regulate 3D cellular migration. These findings will improve our understanding of the functions of PGs, Fascin, and the LINC complex, both developmentally and during tumor progression.
“Exploring Meiotic DNA double-strand break repair in Caenohabditis elegans”
Adam Hefel, Sarit Smolikove, PhD, Biology Department University of Iowa
DNA double-strand breaks (DSBs) are a major source of genome instability, with the potential to cause mutations which can lead to cancer development. In meiosis, DSBs are programmed to form in order to produce crossovers that will promote proper chromosome segregation. Programmed DSBs are produced by the topoisomerase-like Spo-11, which requires the MRN (MRE11, RAD50, and Nbs1) complex. MRN is needed to both create DSBs and to resect the broken ends, producing single-stranded DNA to facilitate homologous recombination. Our lab has discovered a separation of function allele for mre-11 (mre-1(iow1)) in which DSBs are created but ends are not resected resulting in repair by non-homologous end-joining (NHEJ). When NHEJ is ablated in this strain, these breaks are then repaired by an unknown mechanism which results in the formation of crossovers. Using RNA-seq and inducing DSBs with gamma irradiation we expect to find differentially expressed genes representing DSB repair genes in wild-type and mre-1(iow1) mutant lines that will provide insight into this unknown repair mechanism. In order to determine the pattern of repair in this mutant, we will also generate a strain that expresses a heat-shock promoter driven Isce-I endonuclease, the 24 base-pair recognition sequence of which is not found in the C. elegans genome. By creating an engineered locus with the Isce-I cut-site and inducing expression of Isce-I in the germline, we aim to create a system that can be used to address the patterns of repair in our mre-11(iow1) mutant as well as other mutants of DNA damage repair.
CRISPR-Cas9 Gene Editing Yields a Novel Rat Model of the Metabolic Syndrome
Karen C Clark BS, Janette M Pettus BS, Justin L Grobe, PhD Anne E Kwitek, PhD
Department of Pharmacology, University of Iowa Carver College of Medicine; Interdisciplinary Graduate Program in Genetics, University of Iowa Carver College of Medicine
Metabolic Syndrome (MetS) is the clinical presentation of three or more risk factors—central obesity, dyslipidemia (elevated triglycerides, low HDL), hyperglycemia and hypertension—each of which contributes to increased risk of heart disease, diabetes and stroke in more than 20% of U.S. adults. There is strong evidence that MetS and its symptoms are highly heritable, yet identification of causative genes remains elusive, likely due to the complexity of the syndrome. Genome-wide association studies in human populations have fallen short in determining the causative loci; therefore, we employ the genetically tractable inbred Lyon Hypertensive rat model to tease apart the complex etiology of MetS.
Using a genome-wide approach, we previously identified a completely novel gene on rat chromosome 17 (RNO17) using a combination of QTL and eQTL mapping and gene network analysis, and found that RGD1562963 (RGD) has genetic effects on components of MetS.
CRISPR-Cas9 gene editing was used to introduce insertion and deletion mutations (indels) in exon 2 of RGD, and we are currently studying the mutations’ effects in male and female LH-derived rats. Though experiments are ongoing, preliminary data indicates homozygous RGD mutant females have increased resting aerobic metabolic rate (RMR) compared to wild-type controls—as measured by respirometry and core body temperature—and are hypertensive, especially when challenged with a high salt diet.
Our studies suggest RGD exerts pleiotropic effects on various components of MetS, and inhibition of this gene at the whole body level is tolerated. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate a completely uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of components of the Metabolic Syndrome.
Missing the Marx: Groucho function in Wnt Signaled Asymmetric Cell Division
Kimberly N. Bekas; Bryan T. Phillips
Genetics Graduate Program, University of Iowa, Iowa City, IAAsymmetric cellular divisions (ACDs) are a fundamental component of developmental processes that result in two daughter cells with differential cell fate at birth. C. elegans uses a modified version of the conserved Wnt/beta-catenin signaling pathway to regulate its many ACDs in embryonic and larval development. The DNA binding protein TCF/POP-1 functions in the Wnt/beta-catenin asymmetry pathway to differentially regulate gene expression in the daughter cells resulting from an ACD. The ability of POP-1, to repress or activate gene expression relies on interactions with Groucho family corepressors or the coactivator beta-catenin/SYS-1, respectively. The Groucho corepressors function in fate determination and are expressed in asymmetrically dividing tissues, such as the seam cells, yet a role for these corepressors in ACD has not been demonstrated. For this reason, we investigated the function of Groucho in the seam cell lineage, which divides asymmetrically to produce a pluripotent seam cell and terminally differentiated hypodermal cell. Seam cell fate appears to heavily rely on repression rather than activation since knockdown of the POP-1 increases seam cell number while knockdown SYS-1 does not affect seam cell number. If Groucho were required for POP-1 repression in the unsignaled daughter after ACD, we expect to see duplication of the signaled fate following Groucho depletion, similar to the POP-1 RNAi phenotype. Surprisingly, no defects in seam cell fates following corepressor depletion were seen. However, we sensitized cells via POP-1 RNAi and determined that, at low levels of POP-1 knockdown, additional knockdown of Groucho results in an increase in seam cell number that resembles the full POP-1 knockdown phenotype. This enhancement provides evidence that Groucho functions in terminal differentiation of the dividing seam cells to confer hypodermal cell fate. Current efforts include testing the role of POP-1 domains that interact with SYS-1 and DNA, using specific pop-1 alleles in combination with Groucho loss-of-function. Preliminary evaluation of a transgenic strain encoding a POP-1 deficient in SYS-1 binding, pop-1(q645) coupled with a sys-1 hypomorph (q544), shows no significant defects in seam cell ACD. These data indicate that differential transcriptional repression between the two daughters provided by Grouchos, rather than activation SYS-1, may be the critical effect of Wnt signaling in the unsignaled daughter.