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.
HIGH-THROUGHPUT BEHAVIORAL ASSAY TO INVESTIGATE SEIZURE SENSITIVITY IN ZEBRAFISH IMPLICATES SYNE1 AND ZFHX3 IN EPILEPSY
TD Fuller1,2, AN Marsden1,2, Das T1, JM Hermosillo1, TA Westfall1, DC Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
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.
EVALUATING RECOMBINATION REGULATION: LESSONS FROM HYBRIDS OF DROSOPHILA
N Pettie1, J Comeron1,2 , and A Llopart1,2
1 Interdisciplinary Graduate Program in Genetics, University of Iowa, 2 Department of Biology, University of Iowa
Meiotic recombination is a polygenic, highly regulated, evolutionarily conserved process that increases genetic diversity and ensures proper chromosome segregation. Despite the almost universal presence of recombination among eukaryotes, the molecular processes responsible for the number and distribution of recombination events across genomes are highly variable. The mechanisms responsible for hotspot localization of meiotic recombination, for instance, are not conserved. Recombination localization in most mammals including humans and mice is regulated by PRDM9, which targets specific sequences of DNA motifs. In canids, however, there is no active PRDM9 and recombination is highly correlated with CpG content, which is also the case in yeast. Recombination in Drosophila appears to be controlled differently than in mammals or yeast. Recombination rates and distribution in Drosophila are influenced by many epigenetic factors, such as age, nutrition, temperature, and transcription. Here, we use the term epigenetic to describe any factor that changes the ‘phenotype’ of recombination, including gene expression changes, while maintaining equivalent genetic material. We will generate high-resolution recombination maps from two closely related species of Drosophila, D. yakuba and D. santomea, to study short-term evolutionary differences between species. We will also generate high-resolution recombination maps in their hybrids to evaluate the hypothesis that the independent evolution of this polygenic ‘phenotype’ of recombination in the two lineages has led to incompatible interactions in hybrids and ultimately distorted genetic maps.
Genetic therapeutic strategies for the BBS1 M390R mutation
Bardet-Biedl syndrome (BBS) is a rare ciliopathy caused by mutations in a number of cilia related genes. Common features of BBS include retinopathy, male infertility, polycystic kidney disease, hypogonadism, polydactyly, obesity, and several others. Currently, there are no efficacious treatments for BBS, illustrating a critical need to develop therapeutic strategies. The most common genetic cause of BBS is the M390R mutation in BBS1, accounting for approximately 20% of all genetically diagnosed BBS cases. BBS1 is normally part of a multi-protein complex called the BBsome, a complex that is important for trafficking proteins to cilia and the cell membrane. Due to the accessibility of the eyes and testes, the retinal degeneration and male infertility phenotypes of BBS are strong candidates for treatment via gene therapy and gene correction. Previous work in mice focused at using gene therapy for the treatment of retinal degeneration in BBS mice has met some challenges, as overexpression of BBS1 causes toxicity and further loss of photoreceptor cells. Our lab has previously shown that postnatal correction of BBsome proteins can halt photoreceptor death and restore male fertility in mouse models of BBS. Continued work is underway using naked DNA, viral vectors, and CRISPR-Cas9 mediated gene correction to ameliorate the phenotypes of BBS in mouse models.
The role of the anti-sigma factor RsiV in lysozyme sensing and stress response in Bacillus subtilis and Clostridium difficile
*Ana N Castro1, Lincoln T Lewerke1, Ben D Cortes2, Jessica L Hastie1, Craig D Ellermeier1
1Microbiology Department, College of Medicine, University of Iowa, Iowa City, IA; 2Biology Department, Benedictine College, Atchison, KS. *Anafirstname.lastname@example.org
ECF σ factors are alternative σ factors that allow many bacteria to sense and respond to changes in the environment. σV, an ECF σ factor, is found in low GC Gram-positive bacteria, induces resistance to lysozyme and is important for virulence in pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded by regulated intramembrane proteolysis (RIP). RIP is initiated by signal peptidase cleavage of RsiV at site-1 resulting in the release and activation of σV. We demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme. By altering the signal peptide, we find that the spacing between the cleavage site and the transmembrane is critical to RsiV avoiding signal peptidase cleavage in the absence of lysozyme. The lab previously determined the X-ray crystal structure of the extracellular domain of RsiV in complex with lysozyme. This structure revealed that RsiV does not bind near the signal peptidase cleavage site. This has led to our model in which binding of lysozyme to RsiV triggers a conformational change, which allows signal peptidase to recognize the cleavage site. Currently, we are determining the structure of full-length RsiV in both the absence and presence of lysozyme. Together this will provide information on the changes that occur to RsiV upon binding lysozyme that lead to σV activation. This will broaden our knowledge on a novel role for signal peptidase and could lead to novel drug targets.
Patient-specific iPSCs to investigate pathophysiology and develop treatments for RPGR-associated XLRP
Giacalone J.C. 1, Burnight E.R. 1,Sharma T.P., Wiley L.A.1, Ochoa D.1, Collins, M.M.1, Mullins R.F.1, Tucker B.A.1, and Stone E.M.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA
Purpose: Retinitis Pigmentosa (RP) is a heterogeneous disease that causes death of the light sensing photoreceptor cells of the outer neural retina and affects as many as 80,000 individuals in the US alone. X-linked RP (XLRP) is responsible for some of the most severe and earliest onset cases. The majority of XLRP cases are caused by mutations in the gene RPGR. The retinal-predominant isoform of RPGR, known as ORF15, contains a long repetitive sequence, which harbors the majority of the pathogenic mutations that cause RPGR-associated XLRP. The purpose of this study was to: 1) model RPGR-associated XLRP using patient-specific induced pluripotent stem cells (iPSCs) and 2) develop CRISPR/Cas9-based genome editing strategies for correction of disease-causing mutations.
Methods: Patient-specific iPSCs were generated from dermal fibroblasts ofpatients with molecularly confirmed RPGR-associated XLRP. Pluripotency was confirmed using the TaqMan Scorecard Assay. CRISPR/Cas9 constructs were generated to target patient-specific mutations. Gene targeting constructs and homology directed repair constructs were introduced to iPSCs via NEON transfection. Correction was confirmed via T7E1 assay and Sanger sequencing. Patient-specific iPSC-derived 3D retinal eyecups were generated and characterized via Western blot, immunocytochemistry and confocal microscopy.
Results: Seven iPSC lines were generated with varying mutations, disease severity, and disease phenotypes. Genome editing of patient-specific iPSCs was achieved with transfection efficiencies of 30 percent, and the resulting modified iPSC clones were isolated and expanded via reporter selection. Patient-specific iPSC-derived retinal eyecups were generated and displayed the retinal progenitor and photoreceptor-specific markers PAX6, OTX2, RCVRN, CRX and NRL.
Conclusions: With the advent of iPSC technology, we are now able to generate retinal cells from male patients with mutations in RPGR ORF15. We have shown that genome editing via the CRISPR/Cas9 system can successfully correct patient-specific mutations in iPSCs, which will serve as a valuable tool for characterizing the observed disease phenotype.
Time heals all wounds, but not without IRF6 and ARHGAP29
T Reeb, M Dunnwald
Chronic wounds affect 6.5 million people in the US, yet little is known about the genetic and molecular mechanisms regulating wound healing. 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 murine embryos deficient for Irf6 displaying impaired wound healing. Additionally, IRF6-deficient keratinocytes have been shown to display 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 RhoA. RhoA is a Rho GTPase which has been shown to play key roles in wound healing and the regulation of stress fibers. However, despite all that we know about IRF6 and RHOA, little is known about how IRF6 regulates ARHGAP29 and the role of ARHGAP29 in cellular migration, cellular adhesion, and wound healing. We hypothesize that ARHGAP29 is transcriptionally regulated by IRF6 and functionally regulates Rho GTPases. Perturbing this system will result in impaired wound healing. We plan to test this hypothesis by performing full thickness excisional wounds on Arhgap29 mutant mice. To test whether Irf6 regulatesArhgap29, a rescue experiments will be performed to determine if overexpression of Arhgap29 can alleviate the phenotypes observed in Irf6-deficient keratinocytes. By further understanding the roles of IRF6 and ARHGAP29 in wound healing, it would provide positive impacts including the ability to predict wound healing complications, the generation of novel therapies as a means preventing such complications, the potential to gain insights into the role of ARHGAP29 and IRF6 in other disorders (such as cleft lip and palate), and ultimately, the improvement of patient outcomes.
DUAL ROLES OF CYCLIN C IN HEART DISEASE
Ponce1,2, D. Hall2, I. Martin2, C. Grueter1,
Interdisciplinary Graduate Program in Genetics, Department of Internal Medicine, University of Iowa.
Cardiovascular disease is the leading cause of death worldwide. The damage inflicted on the myocardium during myocardial infarction (MI) results from (1) hypoxia during ischemia and (2) oxidative damage upon subsequent reperfusion. Despite extensive investigation, the pathophysiology of myocardial injury in response to ischemia is not fully understood. Cyclin C is a coactivator of the Mediator kinase subcomplex which regulates transcription of genes involved in cardiac metabolism, energy homeostasis and responsiveness of the heart to stress. Recent studies have shown Cyclin C to function independent of mediator in regulating stress-induced mitochondrial hyper-fission in yeast in response to oxidative damage. In humans, the constant electrical and mechanical activities of the heart require a continuous energy supply met by a rich stockpile of mitochondria. Additionally, mitochondrial dysfunction increases the pathogenesis in response to ischemia injury. Although studies have shown the effects of mitochondrial dysfunction in heart disease, there is a current gap in knowledge to understand the functional role of Cyclin C in cardiac mitochondria. We hypothesize that injury in response to IR depends on the translocation of Cylinc C from the nucleus to mitochondria where it regulates mitochondrial dynamics. Preliminary data demonstrates Cyclin C translocation in response to stress in cardiomyocytes isolated from adult mouse and neonatal rats. The overall goal of this project is to define the mechanisms whereby Cyclin C regulates metabolism, energy homeostasis in heart disease via two functions: regulating mitochondrial dynamics, as well as regulating transcription of crucial mitochondrial genes. These studies will provide new insights into the regulation of cardiac energy metabolism and may yield novel therapeutic strategies for modulating these processes in the settings of heart disease.