LINCing prostaglandin signaling and the regulation of collective cell migration.
Emily Toombs, Tina Tootle
Collective cell migration – the coordinated movement of tightly or loosely associated cells – is important for development and tumor invasion. Many signals are involved in collective cell migration including mechanotransduction, or the transfer of physical force into electrical or chemical signals. While there are many ways in which cells can respond to force, a key mechanism important for cellular migration is the direct connection of the cytoskeleton to the nucleoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Alterations in the LINC complex affect nuclear shape, nuclear position, and transcription; however, not much is known about its regulation. Another mechanism regulating cell migration is prostaglandin (PG) signaling. PGs are short-range lipid signals that are implicated in many processes, but, at the same time, little is known about their downstream targets. Our lab has shown that PGs have a large affect on actin remodeling via regulating actin binding proteins. Importantly, actin binding proteins play key roles in mechanotransduction; however, PG signaling has not been previously implicated in regulating mechanotransduction. Using the robust genetic model system of Drosophila, and the well characterized process of oogenesis or follicle development, we present the first evidence that PGs regulate the LINC Complex during the collective and invasive cell migration of the border cells. We hypothesize that PG signaling regulates perinuclear Fascin to control LINC complex function. This hypothesis is based on our prior studies that revealed PG signaling regulates Fascin, an actin bundling protein widely implicated in cell migration. This regulation occurs, in part, by PGs modulating the localization of Fascin, including Fascin’s new perinuclear localization. In both PG synthesis and LINC complex mutant follicles Fascin’s localization to the nuclear periphery is lost. Furthermore, our collaborators have found that in cancer cells, Fascin interacts directly with the LINC Complex. Here we present that loss of PGs or the LINC complex results in delayed and aberrant border cell migration; importantly, Fascin is highly expressed in the border cells. We have identified several tools and approaches that will allow us to quantitatively assess the connection between PG signaling and the LINC complex, as well as the role of Fascin in LINC complex regulation. This research is expected to provide the mechanistic insight into how PGs regulate cellular migration by controlling actin binding proteins to modulate the LINC complex, and, therefore, affect mechanotransduction. These findings will improve our understanding of the functions of PGs, Fascin, and the LINC complex both developmentally and during tumor progression.
Defining the Mediator CDK8 Module in Cardiac Stress
The Mediator complex plays key roles in regulating the transcription of nearly all RNA PolII transcribed genes. Mediator is comprised of four modules: the head, middle, tail (collectively known as the core Mediator), and the CDK8 module which is known to transiently interact with the core Mediator. Classically, it was believed that the composition of Mediator was largely invariant and that the CDK8 module was largely repressive in function. However, recent evidence suggests that during development and cellular differentiation, the composition of core Mediator and CDK8-Mediator changes. This compositional change may account for the large scale transcriptional reprogramming observed in differentiated cells. One component of the CDK8 module is Med13. Cardiac Med13 has been previously shown to regulate cardiac and whole body metabolism, with cardiac expression of Med13 being inversely proportional to susceptibility to metabolic syndromes. In murine cardiomyocytes, the expression of most CDK8 module proteins (including MED13) is decreased following birth. However, recent findings suggest that in cardiomyocytes isolated from mice exposed to cardiac stress, the expression of the CDK8 module proteins is increased. This change in expression may result in a change in composition of CDK8-Mediator, resulting in a modified transcriptome in the cardiomyocytes exposed to cardiac stress. This research aims to define the composition of the CDK8 module as well as components of core Mediator during disease progression and in CDK8 submodule mutants as a means of elucidating the etiology of cardiovascular disease.
Ana Castro’s Abstract
The role of the anti-sigma factor RsiV in lysozyme sensing and stress response
A Castro1, Jessica Hastie1, Craig Ellermeier1
Microbiology Department, College of Medicine, University of Iowa
Bacteria respond to changes in their environment by altering gene expression. Understanding the mechanism by which information is transmitted from outside the cells across the membrane is critical, as it will provide insight into how cells regulate resistance mechanisms. The goal is to gain insight into stress response mechanisms that may contribute to microbial survival in rapidly changing conditions. Extra cytoplasmic function (ECF) s factors are a subset of s factors that allow many organisms to sense and respond to changes in the environment. Most ECF s factors are held in an inactive state by an anti-s factor that prevents the ECF s from interacting with RNA polymerase. Certain envelope stresses in bacteria include the regulated intramembrane proteolysis (RIP), a mechanisms which results in the sequential cleavage of membrane-bound proteins, including s factors. In Bacillus subtilis, the ECF s factor, sV, belongs to the ECF30 subfamily of ECF s factors, members which are primarily found in firmicutes.sV is activated specifically by lysozyme, a critical component of the host innate immune system. sV induces resistance to lysozyme in organisms such as Clostridium difficile. In the absence of lysozyme sV is inhibited by the anti-s factor RsiV. In response to lysozyme, RsiV is cleaved by signal peptidase at site-1 leading to the release and activation of sV. We have shown that RIP dependent mechanism of sV activation is dependent on the anti-s, RsiV, binding to the inducing signal, lysozyme. My project involves using B. subtilis as a model to determine RsiV factors that allow signal peptidase cleavage in the presence of lysozyme. We will determine how σV is important for pathogenesis and virulence in B. subtilis. The objective of this project is to determine the mechanisms by which RsiV binding to lysozyme allow for signal peptidase cleavage at site-1 in the extracellular domain.
Joseph Giacalone’s Abstract
Disease modeling using patient-specific photoreceptor precursor cells
Induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells can be used to study biological processes and have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. Biopsies were obtained from patients with inherited retinal degeneration and fibroblast lines were established. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise 3D differentiation protocol. This method will serve as a platform for studying RPGR-associated XLRP.
Matt Strub’s Abstract
Genomic Signature-Based Approaches to Drug Repositioning for ΔF508-CFTR Rescue
Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common CFTR mutation, termed ΔF508, is a 3 base deletion resulting in the loss of a phenylalanine residue. This causes protein misfolding, resulting in proteosomal degradation. However, if CFTR-ΔF508 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 siRNA knockdown of SIN3A in primary cultures of CF airway epithelia increases CFTR mRNA and protein levels, and partially restores cAMP-stimulated Cl– conductance. The Connectivity Map (CMAP) is a catalog of gene expression profiles from cultured human cells treated with a variety of bioactive chemical compounds and has pattern-matching software to mine data. A CMAP query using previously generated gene expression signatures identified 27 candidate small molecules that mimicked miR-138 and DsiRNA SIN3A treatments. Our lab reported the identification of 4 small molecules that partially restored CFTR-ΔF508 function in primary CF airway epithelia, highlighting the utility of a genomic signature approach in drug discovery. Currently, the NIH is greatly expanding the CMAP dataset into the Library of Integrated Network-based Cellular Signatures (LINCS). Here we query the LINCS database using a meta-analysis of rescue signatures to identify candidate compounds for rescue of CFTR-ΔF508 function.
In collaboration with the Broad Institute, we used previously generated gene sets to iteratively query the Library of Integrated Network-based Cellular Signatures (LINCS). 125 candidate small molecules were selected for further testing. Functional screens performed in CFBE (ΔF508/ΔF508) cells identified 7/125 compounds that partially rescued ΔF508 function, as assessed by cAMP-activated Cl– conductance. Additional experiments performed to assess their activity in primary human CF epithelial cells confirmed the ability of these seven compounds to partially rescue ΔF508 function. Interestingly, some of these compounds showed significant cooperativity when administered with C18. We then obtained 70 congeners, derivatives, or related compounds of the seven validated LINCS hits and identified 18/70 compounds that increased cAMP-activated chloride conductance by at least 50% compared to a DMSO control. We also tested compounds in the presence of C18 and identified four compounds that increased conductance by at least 40% compared to a C18 control.
We recently used a meta-analytic approach to analyze multiple rescue signatures and relevant pathways to create a prioritized candidate drug list for functional screens. Transcriptomic profiles of RNAi (siRNA knockdown of SIN3A, SYVN1, and NEDD8, and miR-138 overexpression) and temperature (27°C for 24 hours, 40°C for 24 hours, and 27°C for 24 hours followed by 40°C for 24 hours) treatments were used to represent a rescue signature. Profiles of primary cells (both human and pig) from CF and healthy donors were used to represent a disease signature. The rescue and disease signatures were then analyzed together to create representative lists of up- and down-regulated genes. Meanwhile, gene sets were extracted from relevant curated pathways related to CFTR trafficking (from Thomson Reuters MetaCore). Gene sets from both strategies were then scored against all drug profiles contained in the CMAP and LINCS databases. The rescue signature- and disease signature-based scores were combined to form an overall score that was used to identify and prioritize candidate molecules. We have tested 115 compounds from the resulting meta-analysis-based candidate drug list and 36 compounds have been shown to increase cAMP-activated chloride conductance by at least 30% compared to DMSO. Additionally, 21 compounds in the presence of C18 increased conductance by at least 30% compared to a C18 control. Further analysis of preliminary hits will include validation in primary cells. We also have the ability to test the most efficacious drugs in our ΔF508/ΔF508 porcine model. Lastly, we are performing chemogenomic enrichment analysis using the results of all tested compounds to elucidate possible classification, structural, or transcriptomic similarities between efficacious drugs. Such analysis may help us to identify and prioritize additional candidates.
Tricia Braun’s Abstract
Genome-wide DNA methylation analysis of glucocorticoid treatment in human Blood and saliva and the correlation of DNA methylation between peripheral tissues and brain
Patricia Braun1, Yasunori Nagahama2, Marie Hafner1, Lauren O’Sullivan1, Melissa McKane1, Tanner D. Gardiner1, Andrew Grossbach2, Matthew A Howard III2, Hiroto Kawasaki2, James B. Potash1, Gen Shinozaki1
1Department of Psychiatry, University of Iowa Carver College of Medicine
2Department of Neurosurgery, University of Iowa Carver College of Medicine
Glucocorticoids help regulate the stress response, and an imbalance of glucocorticoids has been implicated in depression. Within mouse models, candidate genes have been shown to be differentially methylated in response to glucocorticoid treatment. Using the Infinium HumanMethylation 450K Array, which covers over 450,000 CpGs, we performed preliminary studies on genome-wide DNA methylation (DNAm) changes that occur within saliva samples from 10 subjects and blood samples from 6 subjects before and after treatment with dexamethasone, a corticosteroid, in the context of neurosurgery. Within saliva samples, average DNAm differences of >10% were observed for 84 CpGs. One CpG in a long intergenic non-coding RNA (LINC00871) attained near genome-wide level of significance (average DNAm: pre-dexamethasone 49%, post-dexamethasone 38%; p=5×10-7). Within blood samples, over 200 CpGs had >20% difference; however, none were statistically significant. These findings provide initial evidence for an influence of glucocorticoids on DNAm within humans. To understand the relevance DNAm in saliva and blood to brain DNAm, we also examined the correlation of DNAm between peripheral tissues and brain tissues. This is an essential issue for not only our analysis, but for the field more broadly. We obtained saliva, blood, and live brain tissue samples from 13 patients undergoing neurosurgery and analyzed genome-wide DNAm with the 450K Array. Blood and saliva showed a high degree of correlation for DNAm (r2=0.97), and saliva DNAm was revealed to be more similar to brain DNAm (r2=0.84) than blood (r2=0.81; p<1×10-4). As we have ongoing access to samples from neurosurgery patients, we will expand these studies to understand the extent to which peripheral tissues can be used as surrogate tissues for DNAm in the brain. Furthermore, we will collect samples from oral surgery patients given a higher dosage of glucocorticoids to more fully ascertain the global effects of glucocorticoids on DNAm.
Jessica Ponce’s Abstract
Stephanie Haase’s Abstract
The Social Activities Committee would like to remind you all that the second Genetics Winter Party is THIS FRIDAY, December 11 from 7-10p.m. at the North Ridge Pavilion in Coralville (2250 Holiday Rd.). This will be a BYOB (beer and wine only) and potluck-style event, so we request but do not require that people bring an appetizer or dessert option to share. Additionally, this will be an ugly sweater party, so we would love to see everyone’s most garish, gaudy, and hideous holiday wear. Prizes will be awarded for the ugliest sweaters!
The entrance to the North Ridge Pavilion can be difficult to see in the dark. If you are driving in from the East, you will see a blue street sign on the right side of the road for the pavilion and the turn will be on your left. There is a sign for the pavilion next to a row of pine trees. If you are driving in from the West, you will see a blue street sign on the right side of the road for the pavilion and it will be the next right turn.
Please feel free to contact anyone on the Social Activities Committee with any questions.
We look forward to seeing you there!
The Genetics Social Activities Committee would like to congratulate Dr. Fengxiao Bu on the successful defense of his thesis and completion of his Doctorate!
Bu is from the Smith lab in the Otolaryngology department. His thesis seminar, entitled “Exploring the Genetics of a Complex Disease – Atypical Hemolytic Uremic Syndrome”, was on Friday, December 4th, 2015. Bu has accepted a postdoctoral appointment here at the University of Iowa to continue his research, and we expect that he will continue to excel.
We asked Bu a few questions about his experience in the Genetics program here at Iowa:
Q: What is your favorite thing that you have experienced here at Iowa?
A: The best thing is that I had a great advisors and met so many awesome people here.
Q: What was your favorite class to take?
Q: What was your favorite class to TA?
A: Bioinformatics techniques.
Q: Do you have any advice about preparing for defense?
A: Better to start early. I would say 6 months is a good number for prepare everything.
Again, congratulations Bu and good luck on your future research!
A Targeted Sequencing Study of Glutamatergic Candidate Genes in Attempted Suicide
Suicidal behavior has been shown to have a heritable component that is partly driven by psychiatric disorders. However, there is also an independent factor contributing to the heritability of suicidality. We previously conducted a whole exome sequencing study of bipolar suicide attempters and bipolar non-attempters to assess this independent factor. This whole exome study implicated glutamatergic neurotransmission in attempted suicide, as did our genome-wide association study (GWAS) of the attempted suicide phenotype. In the current study, we have conducted a targeted next-generation sequencing study of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, neurexin, and neuroligin gene families in 476 bipolar suicide attempters and 473 bipolar non-attempters. The goal of this study was to gather sequence information from coding and regulatory regions of these glutamatergic genes to identify variants associated with attempted suicide. We identified 186 coding variants and 4,298 regulatory variants predicted to be functional in these genes. No individual variants were overrepresented in cases or controls to a degree that was statistically significant after correction for multiple testing. Additionally, none of the gene-level results were statistically significant following correction. While this study provides no direct support for a role of the examined glutamatergic candidate genes, further sequencing in expanded gene sets will be required to understand the role of glutamatergic signaling in the risk for suicidal behavior.
Soluble CR1 Gene therapy rescues renal phenotypes in a murine model of C3G
Xue Xiao1,2, Yuzhou Zhang1, Janice Staber3, Sanjeev Sethi5, Paul B. McCray, Jr.2,3,
Carla M. Nester1,3,4, Richard JH Smith1,2,3,4
1Molecular Otolaryngology and Renal Research Laboratories, Caver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 2Interdepartmental PhD Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 3, 4Departments of Pediatrics and Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 5Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
C3 glomerulopathy (C3G) encompasses two prototypical diseases, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Both diseases are characterized by fluid-phase dysregulation of the alternative pathway (AP) of complement that leads to C3 deposition in the renal glomerulus. Unknown disease triggers and lacks of specific treatments lead to end-stage renal failure in 50% of patients. Soluble complement receptor 1 (sCR1) is a soluble form of a membrane bound regulator of complement. Short-term studies show that sCR1 is capable of restoring complement control in a murine model of C3G, the Cfh-/-/huCR1-Tg mouse. However, within days of terminating treatment, complement dysregulation is again evident. In this study, we sought to determine whether continuous presence of sCR1 could provide long-term complement control in the C3G murine model. Using the piggyBac transposon system coupled with hydrodynamic tail vein injection, we delivered a construct of sCR1 (LHR A-C) to the C3G murine model to provide constitutive sCR1 expression in mouse circulation. Animals were followed for 6 months. sCR1 expression was detected by real time PCR and ELISA in mouse liver and circulation respectively in 6 months. C3 levels approximately doubled and clearance of glomerular C3 and C3 fragments deposition was documented by immunofluorescence. Electronic microscopy showed a reduction in dense deposits in injected as compared to control animals. There were no changes by light microscopy. Renal function improvements had been revealed from stabilized 24-hr urine albumin creatinine ratio. In this study, we proved that long-term expression of sCR1 could rescue the renal phenotype in C3G mice and may be a viable treatment for patients with this disease.
THE ROLE OF ENDOGENOUS MICROBIOTA IN SEIZURE-LIKE BEHAVIOR OF SHUDDERER, A DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL MUTANT
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 seizure-like behavioral defects, including spontaneous leg jerking, twitching, and heat-induced convulsion. Intriguingly, we have recently discovered that food supplemented with milk whey acts as a nutritional therapy, drastically suppressing these behavioral phenotypes. Microarray analysis revealed high levels of insulin receptor (InR) expression in Shu mutants relative to wild-type (WT) flies, indicating Shu has reduced insulin signaling. Following milk whey treatment, InR expression in Shu mutants returned to wild-type levels, suggesting milk whey increases insulin signaling. Because the endogenous gut microbiota are known to impact metabolic and developmental homeostasis through insulin signaling, we hypothesized that the microbiome plays a role in Shu phenotypes and their diet-dependent modification. Raising Shu mutants and WT flies in either antibiotic-containing or sterile food was sufficient to eliminate the gut microbiota. Further, both treatments were found to significantly suppress Shu behavioral phenotypes while having no obvious effect on WT behavior. Culturing extracts of homogenized flies on LB agar plates revealed drastic differences in the number and possibly the species of bacteria found in Shu and WT flies raised on conventional or milk whey-supplemented food. To confirm these results, we plan to perform high-throughput sequencing of the bacterial 16S ribosomal gene to identify differences in the gut microbiome composition of Shu and WT flies in the context of both conventional and milk whey-containing diets. This and future experiments are expected to provide us with a better understanding of the interplay between dietary therapy and the microbiome in the context of seizure disorders.
Homophilic protocadherin cell-cell interactions drive dendrite complexity
Growth of a properly complex dendrite arbor is a key step in neuronal differentiation and a prerequisite for neural circuit formation. Diverse cell surface molecules, such as the clustered protocadherins (Pcdhs), have long been proposed to regulate circuit formation through specific cell-cell interactions. Here, using transgenic and conditional knockout mice to manipulate g-Pcdh repertoire in the cerebral cortex, we show that the complexity of a neuron’s dendritic arbor is determined by homophilic interactions with other cells. Neurons expressing only one of the 22 g-Pcdhs can exhibit either exuberant, or minimal, dendrite complexity depending only on whether surrounding cells express the same isoform. Furthermore, loss of astrocytic g-Pcdhs, or disruption of astrocyte-neuron homophilic matching, reduces dendrite complexity cell non-autonomously. Our data indicate that g-Pcdhs act locally to promote dendrite arborization via homophilic matching and confirm that connectivity in vivo depends on molecular interactions between neurons, and between neurons and astrocytes.
The Genetics Social Activities Committee would like to congratulate Dr. Emily Petruccelli on the successful defense of her thesis and completion of her Doctorate!
Emily is from the Kitamoto lab in the Anesthesia department. Her thesis seminar, entitled “A Tale of Two Genes Controlling Behavior in Drosophila: Role of DopEcR in Alcohol-Induced Behavior and Effects of Epilepsy Mutations on Sleep”, was on Thursday, October 15th, 2015. Emily has accepted a postdoctoral appointment at Brown University, where we expect she will continue to excel.
We asked Emily a few questions about her experience in the Genetics program here at Iowa:
Q: What is the most valuable thing that you have learned here at Iowa?
A: If I can make it through grad school, I can make it through anything.
Q: What was your favorite class to take?
A: GABS, great way to learn about the breadth of research and be introduced to a variety of faculty.
Q: What was your favorite class to TA?
A: Biology of the Brain, a non-major undergrad course.
Q: Do you have any advice about preparing for defense?
A: Think of everything you could possibly be asked and prepare rational answers.
Again, congratulations Emily and good luck at Brown!
The role of copy number variation in cleft lip and palate.
L. A. Harney1,2,3, B. W. Darbro1,3, A. Long2, J. Standley1, A.M. Hulstrand2,3, H. Liu4, R.A Cornell3,4, D.W. Houston2,3, J. C. Murray1,3, J. R. Manak1,2,3
1) Department of Pediatrics, University of Iowa, Iowa City, IA; 2) Department of Biology, University of Iowa, Iowa City, IA; 3) Interdisciplinary Genetics Program, University of Iowa, Iowa City, IA; 4) Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA
Clefts of the lip and/or palate (CL/P) occur in about 1 in 700 live births. Categorized as non-syndromic (NSCL/P) or syndromic (SCL/P), individuals with NSCL/P have isolated clefts and account for about 70% of clefting cases whereas syndromic occurrences include additional cognitive or structural anomalies. Although genome-wide association, candidate gene, and animal model studies have been used to study CL/P, a largescale analysis to determine the contribution of copy number variation (CNV) to CL/P has yet to be performed. We performed the largest high resolution array-based comparative genomic hybridization study to date to identify copy number variants associated with NSCL/P in a cohort of 868 cases from the Philippines and 212 individuals with SCL/P of mixed ethnicities. A preliminary analysis is underway which prioritizes likely causative CN events for follow-up in zebrafish and frogs. Focusing on rare copy number losses, we identified 196 genes that were deleted in greater than one individual while 735 genes were deleted in a single case; collectively, the majority of genes were not previously implicated in clefting. After comparing the list of deleted genes to OMIM, DECIPHER, NCBI, and MGI databases, four were selected for functional follow-up in zebrafish. These genes, ISM1, PKP2, MYO5C and ULK4, are all novel clefting candidates, are overlapped by a CNV loss in greater than one individual, and appear in less than 1% of the cohort. Six additional genes identified have been previously implicated in clefting through association studies (NTN1, PCYT1A), variant analyses (ZNF750, CDH1, OFD1), or chromosomal microarrays (IMMP2L).Together, these studies will define the contribution of copy number variants to disease incidence of CL/P.