Katie Weihbrecht and Fengxiao Bu will present their research on May 16th 2013
Katie’s Research Abstract:
Nephronophthisis (NPHP) is a recessive kidney disorder that is the leading cause of early onset, end-stage renal failure. Localization to the ciliary-centrosomal complex of many proteins mutated in cystic kidney disease provided a coalescing mechanism for NPHP-related ciliopathies (NPHP-RC). Aside from renal failure and kidney cysts, the two other main phenotypes are retinal and cerebellar degeneration. One such disease with NPHP-RC phenotypes is Bardet-Biedl Syndrome (BBS), with cardinal phenotypes of retinal degeneration, renal abnormalities, obesity, polydactyl, and learning disabilities. An NPHP gene associated with BBS is serologically defined colon cancer antigen 8 (SDCCAG8) later defined as NPHP10. However, the association with this disease was only recently made and little is known about the molecular function and how it leads to the observed phenotypes. BBS patients with NPHP10 mutations exhibit retinal and renal abnormalities, obesity, and learning disabilities. The purpose of this study is to identify and characterize the interactions of NPHP10 and determine how these interactions lead the different phenotypes seen in patients. NPHP10 has not been shown to be involved in the pathway that the other NPHP genes are involved in nor has it been shown as an interactor with the other current BBS genes. Thus, the pathway through which NPHP10 leads to BBS phenotypes is novel and will provide further insight into potential BBS candidate genes. Along with a biochemical approach to the characterization of this gene, a knockout mouse model has recently become available. Current work involves characterizing the phenotypes of this mouse and how the biochemical data we currently have correlate with the phenotypes seen in an organism model.
Bu’s Research Abstract:
Targeted mass parallel sequencing for gene screening in atypical hemolytic uremic syndrome
Atypical hemolytic uremic syndrome (aHUS) is a rare renal disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 50% of aHUS cases progress to end-stage renal failure, with the death rate as high as 25%. Previous studies of aHUS have implicated genetic variation in multiple genes in complement pathway in disease pathogenesis, defining the causative mechanism as complement dysregulation at the cell surface level. This understanding improved the disease treatment, and significantly changed the prognosis of aHUS patients. However, it still remains major challenges in understanding the uncovered detailed picture of aHUS at the genetic level. Evidences suggest, beside of the complement pathway, the coagulation pathway may also paly important role in the disease. We are using an optimized platform called CasCADE (Capture and Sequencing of Complement-Associated Disease Exons), which is based on targeted sequence capture and mass parallel sequencing, to screen affected subjects for genetic variation in 85 candidate genes prioritized and selected from the complement pathway and coagulation pathway. Data analysis, including sequencing results qualification control, reads alignment, variant calling, annotation, and filtering, will be conducted on the University of Iowa Galaxy Bioinformatics Server using CLCG Illumina Paired-End Workflow. The on-going project would reveal more genetic abnormalities in aHUS subjects; improve understanding of disease mechanism; and response to therapy strategies.
Ashlyn Thorn and Alex Wagner will present their research on April 18th 2013
Ashlyn’s Research Abstract
The Roles of Csk, Src Family Kinases, and the FGF Receptor Heartless in Synaptic Homeostasis
Synaptic homeostasis is the process by which synapses respond to perturbation of normal neurotransmission to maintain set point function and stabilize neuronal signaling. Aberrant neuronal signaling and activity is associated with a number of neurological disorders including epilepsy, migraine, schizophrenia, ataxia, and myasthenia gravis. As such, understanding the mechanisms that regulate synaptic homeostasis may provide insight into the underpinnings of these disorders. To examine these mechanisms, the Frank lab studies synaptic homeostasis at the larval neuromuscular junction of D. melanogaster. In this system, postsynaptic blockade of glutamate receptors triggers an increase in presynaptic glutamate release that allows the synapse to maintain wild type neurotransmission. This phenomenon and the signaling networks that regulate it have only recently begun to be characterized. This work has revealed a presynaptic pathway composed of the Eph receptor, the guanine exchange factor Ephexin, the GTPase Cdc42, and voltage-gated Cav2 calcium channels. In order to identify additional members of this pathway, we undertook and RNAi-based candidate screen. This screen provided the first evidence that C-terminal Src kinase (Csk) and the fibroblast growth factor receptor Heartless (Htl) may regulate synaptic homeostasis. Verification of these candidates with mutant alleles has confirmed that Csk and Htl are required for synaptic homeostasis. Csk functions in many cellular contexts to phosphorylate and down-regulate the activity of Src family kinases (SFKs). As such, we examined the homeostatic roles of src64B and src42A, the SFK homologs in flies, and have shown that both of these SFKs regulate synaptic homeostasis. Additionally, we have determined that src64B is required in the post-synaptic muscle for homeostatic regulation. Htl is also required solely in to the muscle, where it may be signaling through src64B. The tissues in which Csk and src42A function and the downstream targets of the SFKs remain to be determined.
Alex’s Research Abstract
Computational Approaches for Characterizing Inherited Retinal Degenerative Disease
Heritable retinal degenerative diseases such as Retinitis Pigmentosa exhibit extreme genetic and locus heterogeneity, and are observed in only a small percentage of the population. Despite our current knowledge of disease alleles in a large number of genes, only ~50% of observed cases can be explained by mutations observed in these genes. In this talk, I discuss computational approaches towards identifying novel disease alleles that may cause retinal degenerative disorders, and using phenotypic information to constrain causative variant hypotheses in a clinical setting.
Joshua Fletcher and Bing He will present their research on March 21 2013
Bing He’s research abstract
Identify enhancer-promoter associations in human
Transcriptional enhancers play an essential role in establishing cell type and developmental stage specific gene expression patterns in metazoans. They typically comprise multiple transcription factor (TF) binding sites (TFBS) located in DNA regions of a few hundred base pairs. To date, mechanisms of enhancer action are still poorly understood and a key gap in our knowledge is how enhancers select specific promoters for activation. To fill this gap we aim to develop an integrative approach to computationally predict enhancer-promoter associations followed by experimental validation in human. Our preliminary results based on a set of experimentally supported enhancer-promoter pairs show that several features can be extracted from ENCODE data and are effective in discriminating between real and random enhancer-promoter associations. These features include activity profile correlation, TF-target expression correlation, evolutionary constraint, and distance constraint between enhancers and target promoters. They can be integrated to construct a computational framework for robust prediction of enhancer-promoter pairs. This work will have significant impact on multiple aspects of enhancer research such as the basic mode of action of enhancers and their involvement in disease etiology.
Joshua Fletcher’s research abstract
Functional Genomics and Immunological Approaches to Understanding Francisella tularensis Virulence Mechanisms
Bacterial pathogens often subvert host immune function by effector proteins that are delivered through sophisticated secretion systems. Unlike other intracellular pathogens such asLegionella and Salmonella, the Francisella genome does not encode common secretion systems (Type III, Type IV, etc.) or their associated effectors, although evidence exists for a putative Type VI apparatus encoded in the Francisella Pathogenicity Island. Subversion of the host response (inhibition of oxidative burst, degradation of the phagosome, etc.) is an active process requiring live bacteria, indicating that while LPS and capsule contribute to immune evasion, other undiscovered Francisella factors may also be important. To identify these factors a combined bioinformatics and transposon mutagenesis approach was used to generate a list of candidate virulence factors. The amino acid sequences of the proteins encoded by the Schu S4 genome were analyzed for functional domains. We sought to identify eukaryotic-like domains, prokaryotic domains associated with virulence, and domains of unknown function. Candidate genes were compared to previously published TraSH screen data generated by our lab. Site-directed mutants for FTL0881c, FTL0784, and FTL0177 were generated for three candidates in the Live Vaccine Strain (LVS) strain of Francisella tularensis subsp. holartica. FTL0881c is predicted to be a homolog of a secreted protein found primarily in plant pathogens and Legionella; FTL0784 has numerous SEL1 domains, originally identified in C. elegans and thought to mediate protein-protein interactions; FTL0177 is predicted to be a member of the alpha hemolysin family of proteins. The ability of the mutants to survive and replicate intracellularly was tested by infecting the murine macrophage-like J774 cell line. Preliminary data indicates that none of the mutants are defective for intracellular growth over 24 hours, however subtle decreases in fitness are detectable for FTL0881c and FTL0177 in competition infections. Future work includes in vitro infections with these and other candidate mutants generated in the fully virulent Schu S4 strain.
Emily Beck and Colleen Campbell to present on 21 Feb 2013
Emily Beck’s research abstract
Genome-wide assessment of introgression and hybrid sterility factors in Drosophila yakuba and D. santomea
Introgression, also called introgressive hybridization, is the exchange of genetic information between different species through natural hybridization. These events occur when reproductive isolation is not complete, and can lead to the production of hybrids which are often sterile or inviable; this is referred to as hybrid dysfunction. More recently, there is evidence that introgression can lead to the acquisition of new adaptations, called adaptive introgression. Novelty also arises when unique genetic combinations, resulting from the acquisition of genetic material from another species, produce new phenotypes and serve as sources for novel adaptations. Hybrid zones, areas where two distinct species meet and hybridize, provide biologists with “natural experiments” and thus constitute ideal settings to study introgression. Sadly, occurrences of these natural hybrid zones in genetically amenable biological systems, such as Drosophila, are very infrequent. In 2000, however, a new unique hybrid zone formed by two species in the melanogaster subgroup, Drosophila yakuba and D. santomea, was discovered in a small African island of the Gulf of Guinea. Previous studies in our lab showed that the mitochondrial genome of the former species had introgressed into the latter and replaced completely the native form. Additionally, a series of nuclear proteins, which act intimately with the mitochondrial genome, co-introgressed with the mitochondrial genome. Utilizing Next Generation sequencing technology, we aim to create genome-wide maps identifying alternate regions of introgression based on DNA from single flies.
Juan Santana and Samuel Trammell will present their research on January 17th 2013
Juan Santana’s Research abstract
Drosophila Myb represses retrotransposition and regulates DNA copy number
c-Myb is a proto-oncogene associated with leukemias and lymphomas in birds and mammals. Vertebrates have three representatives of the Myb gene family consisting of A-, B- and c-Myb, all of which encode DNA-binding factors that are important for the proper expression of large numbers of genes including those that regulate cell cycle progression and cell differentiation. Drosophila melanogaster contains a single Myb gene (Dm-Myb), mutants of which die before reaching adulthood. Small interfering RNA (siRNA) knockdown of Dm-Myb in an embryonic Drosophila cell line was shown to prevent the proper expression of genes with prominent roles in coordinating cell division. Along the same lines, Dm-Myb mutant flies display cell cycle defects such as aneuploidy, aberrant spindle formation, and abnormal numbers of centrosomes. Dm-Myb protein is present in a complex which includes the nucleosome remodeling factor NURF. Through yeast two-hybrid experiments and genetic screens, we show that Dm-Myb is specifically interacting with the major subunit of NURF (NURF301) and that these proteins are co-regulating a large number of genes, including those involved in cell cycle control. More surprising, we show that Dm-Myb and NURF301 are working in concert to repress transcription of repeat elements. Finally, we show that both proteins are required to repress DNA replication of a multitude of genomic regions in polytene tissue nuclei. Collectively, these data have allowed us to identify two new roles for Dm-Myb in controlling large-scale genomic processes which, when compromised, lead to deleterious effects on genome function.
Samuel Trammell’s Research Abstract
Replicative life-span extension by calorie restriction in yeast is non-cell autonomous
Wild-type Saccharomyces cerevisiae are calorie restricted by lowering glucose from 2.0% (v/v) to 0.2% or 0.5%. Under calorie restriction (CR), replicative life-span is extended in a manner depending upon protein deacetylase Sir2p and biogenesis of the Sir2p substrate, NAD+. Given the role of NAD+ in glycolysis, many postulated reduction of glucose concentration alters the abundance of NAD+ metabolites and consequentially increases Sir2p activity. CR does not significantly alter NAD+ metabolite abundances. To further dissect the relationship of CR to NAD+ and Sir2p, we measured the copy number of NAD+ biosynthetic, consuming, and requiring enzymes. Only Sir2p and Pnc1p, which would convert NAD+ to nicotinic acid (NA) as a final product, are increased in expression with CR. Given intracellular NA did not change, we hypothesized extracellular NA increased with CR and was responsible for increased replicative life-span (RLS). We will present data acquired through a novel bioassay that CR depends upon the extracellular environment, NA is sufficient to extend RLS, and extracellular NA does not increase with CR. We will reveal evidence of a molecule of a known molecular weight but unknown identity that increases extracellularly as a function of CR that is strain specific.
Presentation Schedule – Summer 2013
| Summer 2013 | ||
| 6-Jun | Changya Chen | TBD |
| Danielle Herrig | ||
| 20-Jun | Crystal Maki | TBD |
| Melissa Marchal | ||
| 11-Jul | salleh-ehaideb | TBD |
| Sara Hanson | ||
| 25-Jul | Man Chun John Ma | TBD |
| Danielle Beekman | ||
| 8-Aug | Yan Zhang | TBD |
| Heather Brockway | ||
| 22-Aug | Tryphena Cuffy | TBD |
| Matthew Faron |
Emily Petruccelli and Pavitra Ramachandran will present their research on 13 December 2012
Emily’s research abstract:
Drunk DopEcR Drosophila Mutants
Steroid hormones exhibit profound effects on behavior by regulating transcription through the action of nuclear hormone receptors. However, many steroids also elicit rapid responses independent of new mRNA synthesis via “non-genomic” steroid actions, the mechanisms of which remain poorly understood. Recently, Drosophila DopEcR, a G-protein coupled receptor with homology to vertebrate β-adrenergic receptors, was identified as a potential non-genomic steroid receptor. DopEcR is primarily expressed in the nervous system and activates intracellular signaling cascades in response to both the catecholamine dopamine and the major insect steroid hormone ecdysone. Since both dopamine and steroid hormones are known to affect evolutionarily conserved alcohol-induced behaviors, we examined the responses of DopEcR mutants to alcohol. Interestingly, we found that DopEcR mutants are more resistant to ethanol-induced sedation and more hyperactive during intoxication. Since knockdown of the EGF receptor (EGFR) or increase of cAMP levels restores sensitivity of DopEcR mutants to sedation, DopEcR may normally activate cAMP signaling and inhibit MAPK/Erk signaling in response to alcohol. Further observation of ethanol-induced behaviors following genetic and pharmacologic manipulations will continue to help elucidate the function and mechanism of DopEcR. Additionally, our findings offer important insight into how behavioral response to alcohol is controlled by dopamine and non-canonical steroid signaling.
Pavi’s research abstract:
RNAi therapy for polyglutamine expansion disorders
RNA interference (RNAi) therapy for autosomal dominant polyglutamine (polyQ) expansion disorders such as Huntingtons disease (HD) and spinocerebellar ataxia (SCA) 1, SCA2 and SCA7 are currently being explored in our lab. The goal of RNAi therapy in CNS disorders is to treat the disease directly by targeting silencing of the mutant gene using RNA interference (RNAi). This strategy has shown therapeutic benefit in SCA1 and HD mouse models. We have designed RNAi vectors to target ATXN7 in SCA7 and ATXN2 in SCA2.
SCA7 is unique among the polyQ diseases as it is characterized by retinal cone-rod dystrophy in addition to purkinje cell neurodegeneration. We designed RNAi vectors to test non-allele specific silencing in a mouse model of SCA7. RNAi triggers were designed to target coding sequences found in both human and mouse ATXN7 to facilitate non-allele specific silencing in the SCA7 mouse. The RNAi triggers and a scrambled control were tested for knockdown of human mutant ATXN7-92Q in vitro in HEK293 cells. Significant knockdown of ATXN7 protein was seen with two sequences by western blot. We are currently testing these sequences in vivo in the retina and the cerebellum using the SCA7 mouse model.
Similar to HD and SCA7, SCA2 is a neurodegenerative disorder caused by a polyQ expansion in ATXN2. RNAi sequences were designed to target coding sequences found in both human and mouse ATXN2 to facilitate non-allele specific silencing. These RNAi triggers including a scrambled control were tested for knockdown of ATXN2 in vitro in HeLA cells, which endogenously express human ATXN2. In HeLa cells, two RNAi triggers transfected showed a significant (P<0.05) knockdown efficiency of ~25%. We are currently testing many more RNAi sequences to identify more potent sequences to target ATXN2 for SCA2 therapy.
Genetics PhD Graduate Program PhD Thesis Seminar
“Computational Approaches to the Study of Human Trypanosomatid Infections”
Ralph Hazlewood and Xitiz Chamling will present their research on 15th Nov 2012
Ralph Hazlewood presentation abstract
Identification and characterization of genetic factors responsible for cavitary optic disc anomalies
Excavation of the optic disc leading to retinal ganglion cell death is a chief feature of optic nerve disease and blindness. Although risk factors for optic nerve diseases are known, such as the elevated intraocular pressure (IOP) observed in some glaucoma subjects, the molecular events of optic nerve degenerations are poorly understood. Patients with congenital malformations collectively called cavitary optic disc anomalies (CODA) have optic nerve head excavation that, in some, progressively deteriorates in the absence of elevated IOP. Interestingly, this occurs in a manner similar to that observed in normal tension glaucoma (NTG) patients. Since the only therapy is geared at lowering IOP and these patients have normal IOP, our central hypothesis that genes involved in Mendelian forms of CODA would also be involved in a subset of NTG cases because of the similar clinical phenotypes and provide insight into glaucomatous optic neuropathy. To address this clinically relevant problem, previous work in the laboratory revealed a novel locus on chromosome 12 via linkage analysis and positional cloning of a large autosomal dominant family with CODA; examination of this locus revealed a copy number variation (CNV). Comparative genomic hybridization experiments identified two extra copies of a 6kb segment of DNA that segregates with disease in this family and this copy number variation (CNV) was not detected in unaffected family members or in normal controls. This finding was confirmed by qPCR. Subsequent reporter gene analysis revealed a 774bp transactivating domain within the CNV that drives expression 8-fold higher when subcloned into HEK293T mammalian cells. Moreover, immunohistochemical experiments revealed ubiquitous expression of a matrix metalloprotease gene in retinal sections. We report a CNV within the previously linked region that is co-inherited with CODA in our family. We hypothesize that this CNV leads to dysregulation of gene expression and ultimately to the development of CODA. Ongoing research includes generating animal models.
Xitiz Chamlings presentation abstract
The centrosomal protein AZI1 interacts with BBS4 and is involved in ciliary trafficking of BBSome to the cilia
The centrosome is the site of microtubule organization and centriole nucleation, and is essential for cilium formation. Primary cilia, microtubule-based subcellular organelles protruding from the surface of cells, have been shown to be essential for tissue homeostasis, photoreceptor function, olfaction, and several signaling pathways including Hedgehog, Wnt and PDGFR. Defects in cilia or centrosome function lead to phenotypically related syndromes called ciliopathies. Bardet-Biedl syndrome (BBS) is a well-known ciliopathy and most of the BBSome (complex of seven BBS proteins) is known to localize to the centrosome as well as to cilia. In the centrosome, many BBS proteins are thought to interact with centrosomal proteins; for example, BBS4 has been shown to interact with PCM1 and CEP290. Due to the pleiotropic nature of the disease and the abundance of proteins at the centrosome and transition zone where BBS proteins localize, we hypothesized that there are other centrosomal proteins that interact with the BBSome, which have not been identified. Moreover, the functional role of the interaction between BBS and centrosomal proteins has not been fully investigated. Here, we identify a novel BBSome interacting protein, AZI1 (also known as Cep131), which directly interacts with BBS4. We show that AZI1 is involved in ciliary trafficking of the BBSome complex. We also show that AZI1 is required for cilia formation. Under conditions in which the BBSome does not traffic normally to cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, using morpholino knockdown of Azi1 in zebrafish, we were able to show that Azi1 knockdown results in zebrafish phenotypes seen in other Bbs gene knock down, which suggests the possibility that AZI1 might be a new BBS gene.
Matthew Jorgenson will present his research on 18th October 2012
TOWARD DETERMINING THE FUNCTION OF RLPA, A CELL DIVISION PROTEIN FROM PSEUDOMONAS AERUGINOSA
Cell division is an essential process, requiring the concerted efforts of at least 30 proteins in a Gram-negative bacterium like Escherichia coli. Together, these proteins form a complex structure at the midcell known as the septal ring to mediate the simultaneous inward growth of the three layers of the cell envelope: the outer membrane, the peptidoglycan cell wall, and the inner membrane. Recently, our lab identified three new septal ring proteins—DamX, DedD and RlpA—in E. coli, all of which contain a ~75 amino acid SPOR domain that is sufficient for localization to the septal ring. Loss of DamX or DedD resulted in cell division defects. Conversely, loss of rare lipoprotein A (RlpA) did not cause a cell division defect in E. coli. However, we found that a transposon mutant of rlpA in Pseudomonas aeruginosa has defects in cell separation when grown in mediaof low ionic strength, forming chains of 4-8 cells. We constructed a non-polar deletion of rlpA in P. aeruginosa and confirmed the chaining phenotype. We also constructed an RlpA-mCherry fusion and showed septal ring localization during cell division. Interestingly, the SPOR domain is necessary for RlpA localization but not for function. Utilizing the RlpA-mCherry fusion, we also show that RlpA is an outer membrane lipoprotein. Peptidoglycan analysis revealed some structural differences between wild type and the rlpA mutant, suggesting RlpA is an enzyme involved in peptidoglycan turnover or maturation. Data from an in vitro assay combining purified RlpA with isolated peptidoglycan supports this hypothesis.
