TFAP2A drives melanocyte gene expression in parallel with MITF
H E Seberg1, E Van Otterloo2, S K Loftus3, J P Lambert4, G Bonde2, R Sompallae5, J F Santana1, J R Manak1, A C Gingras4, W J Pavan3, R A Cornell1,2
1 Interdisciplinary Graduate Program in Genetics, University of Iowa
2 Department of Anatomy and Cell Biology, University of Iowa
3 Genetic Disease Research Branch, National Human Genome Research Institute, Bethesda, MD
4 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
5 Bioinformatics Division, Iowa Institute of Human Genetics, University of Iowa
Disruption of the transcription factor network governing melanocyte development contributes to the pathogenesis of pigmentation disorders and melanoma. The activity levels of an important member of this network, MITF, have been proposed to control melanoma phenotype. Mid- to high-level MITF activity drives growth and differentiation, while lower levels confer a stem cell-like, invasive quality. Transcription Factor Activator Protein 2 alpha (TFAP2A) expression is reduced in advanced stage melanoma tumors, and mutations in TFAP2A cause pigmentation phenotypes in humans, mice, and zebrafish. Because TFAP2A is widely expressed in the neural crest and skin, its specific role in melanocytes and relationship to MITF has been unclear. To determine the position of TFAP2A in the melanocyte gene regulatory network, we first used microarray analysis of wildtype and tfap2a null zebrafish to profile genes that are downregulated in the absence of TFAP2A. We then conducted anti-TFAP2A ChIP-seq to create a profile of TFAP2A-bound loci in melanocytes. Genes at the intersection of these profiles are likely direct targets of TFAP2A. These include melanin synthesis genes, such as dct, tyrp1, and trpm1, most of which are also thought to be direct targets of MITF. Comparison with published MITF ChIP-seq showed that TFAP2A peaks overlap MITF peaks more often than expected by chance. In reporter assays, deletion of TFAP2A binding sites in a minimal TRPM1 promoter decreased its activity, similar to published results for deletion of MITF binding sites from this element. Furthermore, we found that tfap2a and mitfa interact both physically in vitro and genetically in zebrafish. These results provide evidence that TFAP2A and MITF work in parallel to promote melanocyte differentiation, and show that widely-expressed TFAP2A can directly regulate expression of lineage-specific melanocyte genes. In addition, TFAP2A expression may be able to influence levels of MITF, driving cells toward differentiation and away from an invasive state.
Eric’s Talk Title
Investigating the Human Exome in Suicidal Behavior
|9/17/2015||Eric Monson||12:00-12:50 PM||ML B111|
|10/15/2015||Lisa Harney||12:00-12:50 PM||ML B111|
|11/12/2015||Michael Molumby||12:00-12:50 PM||ML B111|
|12/10/2015||Sophia Gaynor||12:00-12:50 PM||ML B111|
Thank you to all of the students, faculty, and family members who made it out in the rain to join us for the 2015 Genetics Welcome Picnic!
Program Director Dan Eberl kicked off the festivities with his annual address,
followed by the second annual Newly-Grad game!
Our contenders this year were the First year mentees and their mentors: Karen and Kellie, James and Patrick, Tanner and Nikale (standing in for Wes), Zach and Hannah, Adam and Tyson, Alyssa and Sophie, and finally, Kimberly and Lisa. The competition was intense with our MC Tanner asking the tough questions like Where is your mentee’s hometown? and What is your mentee’s favorite food? In the end, Karen and Kellie were able to blow the competition away with their in sync answers.
After the delicious catering by A Guy and a Grill, we completed the raffle with the grand prize, the board game Pandemic, going to James Mrkvicka!
CALCIUM DEPENDENT NAKED-DISHEVELLED INTERACTION MODULATES WNT SIGNALING OUTPUTS
Autumn N, Marsden1,2, Sarah W. Derry1, Trudi A. Westfall1, Diane C. Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
The Wnt signaling network plays critical roles in development and is implicated in disease. Wnt signaling gives rise to two distinct outputs: polarized cell movement (Wnt/PCP) and stabilization of β-catenin (Wnt/β-catenin). The mechanisms that determine the output is not completely understood, especially because of shared upstream effectors. My project focuses on two shared components that also bind each other, Dishevelled (Dvl) and Naked (Nkd), an EF-hand containing protein. We have demonstrated that Nkd is required for dorsal forerunner cell (DFC) migration, Kupffer’s vesicle formation and proper organ laterality. Using biochemical and functional assays, we show calcium-induced conformational changes in the Nkd-Dvl complex and identify a requirement for the Nkd EF-hand in PCP but not β-catenin outputs. We predict that Nkd and Dvl form a cooperative calcium binding pocket, which allows for conformational changes or subcellular localization to direct Wnt/PCP output. To determine the impact upon Wnt signaling output, I utilize gene knockdown and overexpression in the DFCs, a tissue that has dynamic calcium fluxes and hosts converging Wnt signals. I also determined the subcellular localization of Nkd and Dvl components within the DFCs and cells that are calcium quiescent. Our data suggests that calcium-induced secondary structure changes in the Nkd-Dvl complex serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.
The Epigenetic Landscape and Promoter-Interactome during Development of Hematopoietic Stem Cells
Hematopoietic Stem Cells (HSCs) are derived from in fetal liver (FL) undergo rapid self-renewal divisions, which lead to a massive increase in cell number of the HSC pool. In contrast, the adult bone marrow (BM) HSCs have lower self-renewal capacity. In addition, FL and BM HSCs display differences in their differentiated cell output. FL HSCs have a biased erythro-myeloid lineage output while BM HSCs have balanced lineage output. These differences of biological properties between FL and BM HSCs correlate with distinct gene expression programs. Our RNA-Seq analysis has shown that about 1600 genes are differentially expressed between FL HSCs and BM HSCs. It is also well known that gene expression is regulated by cis-regulatory element. Detailed studies for single loci have revealed some mechanisms how cis-regulatory elements regulate gene expression. However, genome-wide long-range enhancer-promoter interactomes in FL and BM HSCs are still not well studied. We have generated histone modification profilings (H3K4Me1, H3K4Me3, H3K27me3, and H3K27Ac) for both FL and BM HSCs by ChIP-Seq. Based on histone modification profilings, we predicted more than ten thousands active enhancers in both FL and BM HSCs using our computational method: CSI-ANN. Combined our RNA-Seq, we predicted Enhancer-Promoter interactions using IM-PET. To further address research question, we are using Capture-C, a chromosome conformation capture-based technology to generate genome-wide promoter interactomes in both FL and BM HSCs. We will integrate these datasets to understand how differences in the promoter interactomes contribute to the gene expression programs, and then contribute to the phenotypic difference between FL and BM HSCs.
DEFICIENCY SCREENING; IDENTIFICATION OF GLUTATHIONE S-TRANSFERASE 1 AS A GENETIC MODIFIER OF THE DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL GENE, PARALYTIC
Hung-Lin Chen, Toshihiro Kitamoto
Voltage-gated sodium (Nav) channels are essential for generation and propagation of action potentials in neurons. Dysfunction of Nav channels often causes neuronal hypo- or hyper-excitability, resulting in a variety of neurological disorders, such as epilepsy. Epilepsy is one of the most common neurological disorders in the world. As estimated 50 million people worldwide suffer from this devastating pathological condition and a significant portion of patients (~30%) do not respond to anti-epileptic drugs (AEDs). Thus, identifying new target genes for AEDs is of emergent clinical need. In this study, I take advantage of Drosophila genetics to identify modifier genes that can reduce neurological phenotypes caused by mutations in the Nav channel gene, paralytic (para). Shudderer (Shu) is a mutant allele of para, displaying neuronal hyper excitability as well as behavioral and morphological abnormalities. These include seizure-like behaviors, down-turned wings, and indented thorax. Here we carried out a deficiency screen to identify modifiers of Shu. Our working hypothesis is that the Shu mutant phenotypes are enhanced or suppressed when the activity of genes functionally interacting with Shu is reduced by 50%. We systematically introduced molecularly defined deficiencies into the Shu mutant background and tested if it results in a modification of the mutant neurological phenotypes. After morphological and behavioral analyses, we identified a short genomic deletion in the second chromosome that can ameliorate Shu’s phenotypes. This deletion covers six genes. By using RNA interference and P-insertion mutants, we narrow down the modifier of Shu to glutathione s-transferase 1 (GstS1). GstS1 mutations reduced frequency of seizures in another Drosophila Nav channel mutant that carries a mutation causing genetic epilepsy with febrile seizures plus (GEFS) in humans. Immunochemistry showed a GstS1 mutation increases GABA levels in the Shu mutant brain. It indicates that deletion of GstS1 may reduce neurological phenotype of Shu by enhancing GABAergic inhibition. Since Drosophila and human share fundamental biological pathways, my study may provide a new direction for AED development.
THE ROLE OF MATERNAL WNTS AND FRIZZLEDS IN DORSO-VENTRAL AXIS SPECIFICATION
Melissa Marchal1 and Douglas Houston1
1The Interdisciplinary Program in Genetics and The Department of Biology, University of Iowa, Iowa City, IA, 52242
The proper localization of maternal mRNAs and proteins in the egg is required for many developmental processes, including dorsoventral (DV) axis determination. In Xenopus laevis in particular, maternal factors involved in patterning the DV body axis are localized during oogenesis to the oocyte vegetal cortex. Upon sperm entry, these determinants are translocated to the future dorsal side of the embryo, in a microtubule dependent process called cortical rotation. The major outcome of cortical rotation is the asymmetric activation of the Wnt/β-Catenin signaling pathway in dorsal nuclei, where β-Catenin acts to transcriptionally activate dorsal-specific genes at the midblastula transition. However, the upstream mechanisms initiating β-catenin stabilization have remained elusive. Recent evidence has suggested that secreted Wnt ligands Wnt11 and Wnt5a may act together in axis formation. However, the function of other maternally expressed Wnts and their cognate receptors (frizzleds) remains uncharacterized. We have examined the expression of wnts and frizzleds (fzds) in the oocyte and throughout early development. Through this analysis, we have found that fzds 1, 4, 6, and 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. Using a maternal depletion approach we have begun to identify the roles of these maternal factors in DV axis patterning. We present evidence that maternal fzd1-depleted embryos show a ventralization phenotype and partial defects in dorsal-specific gene expression, while fzd4-depleted embryos show a dorsalization phenotype and the expansion of dorsal-specific markers. Additionally, we show that oocytes depleted of fzds 1, 4, and 7, have significant microtubule defects, suggesting that Frizzled-dependent signaling may play a role in cortical rotation.
Genome-wide Expression Profiles in Drosophila yakuba and D. santomea.
Population genetic models predict that the X chromosome will evolve at a faster rate than autosomes (i.e., faster-X evolution). The basis for faster-X evolution is that genes are under the highest level of selection in males. Evaluations of protein-coding sequences have indeed shown an excess of divergence on the X chromosome compared to autosomes, particularly in genes expressed higher in males than in females (i.e., male-biased genes). In addition, whole-genome analyses of gene expression in Drosophila yakuba and D. santomea males indicate that X-linked genes are more differentially expressed between species (i.e., faster-X evolution of gene expression) than autosomal genes. This trend is once again strongest for male-biased genes. However, these studies utilized only males and were therefore limited in their expression profiles. Here, we will investigate the whole-genome profiles of D. yakuba and D. santomea males and females to determine the relative rates of evolution for all gene classes. We also assess the way that divergent genes are inherited in hybrid and the regulatory factors that influence their expression patterns.
Whole Exome Analysis of Individuals and Families with CRMO
For my primary research project, I am working to determine the genes and pathways involved in the development of chronic recurrent multifocal osteomyelitis (CRMO), a rare autoinflammatory bone disease presenting in infancy and childhood. We currently have whole exome sequence data from 35 individuals with CRMO. For six of the isolated cases, we have data for one or two relatives with inflammatory disease, and for three pairs of sisters and three individuals with CRMO, we have exome data for both unaffected parents, for a total of 53 exomes. Nearly all of the probands have close relatives with psoriasis or Crohn’s disease. For all of the exome data, I have processed the data from fastq to vcf format using the Burrows-Wheeler Alignment (BWA) software, SAMtools, Picard, and the Genome Analysis Toolkit (GATK). Preliminary analysis of the data suggests that variants in genes involved in IL-17 and RANK signaling are enriched in our CRMO cohort, and I am currently working on developing and analyzing a control dataset for comparison using the publicly available 1000 genomes, EVS and ExAC databases. Additionally, our laboratory will send an additional 26 samples for exome sequencing this summer – the samples are 6 individuals with CRMO and their unaffected siblings and parents. I am also performing an experiment this summer to determine the effect of a putative enhancer mutation in the first intron of a candidate gene. The variant is enriched in our CRMO cohort and likely disrupts an NR4A2 binding site.
Characterization of the NPHP10/AIMP2 interaction
Nephronophthisis (NPHP) is a recessive kidney disorder that is the leading cause of early onset, end-stage renal failure. Many proteins mutated in cystic kidney disease have been shown to localize to the primary cilia and centrosomes, providing a coalescing mechanism for NPHP-related ciliopathies (NPHP-RC). Aside from renal failure and kidney cysts, retinal degeneration and dysplasia or degeneration of the cerebellum are also seen in many NPHP-RCs. SDCCAG8 is a nephronophthisis gene (NPHP10), with patients exhibiting retinal and renal abnormalities, obesity, and learning disabilities. Mutations in SDCCAG8 were also found in several BBS patients, making SDCCAG8 the 16th BBS gene (BBS16). However, little is known about the molecular functions of NPHP10 and how loss of NPHP10 function leads to the observed phenotypes. Our research has shown that NPHP10 interacts with components of the multi-aminoacyl tRNA synthetase complex (MSC), including 8 out of 9 aminoacyl tRNA synthetases (ARS) as well as aminoacyl-tRNA-synthetase-complex interacting multifunctional protein 2 (AIMP2). Further work determined that a direct interaction likely occurs between NPHP10 and AIMP2. Our current work focuses on determining the biological significance of this interaction.
Consequences of Recombination rate variation among Drosophila Melanogaster populations
Recombination is a crucial biological process that shapes evolutionary change within and between species. At the same time, accurate estimates of recombination rates are essential for correct inferences of selection and demographic events. In this study, we use the most accurate population genetic method, LDhelmet, to estimate and compare recombination rates in three Drosophila melanogaster populations. Recombination rates not only change in total magnitude but also in their relative distribution within chromosomes (landscapes). We show that differences in recombination landscapes between populations do not accumulate at the same rate than nucleotide differences. We also show that population-specific differences in recombination landscapes play a significant role explaining population-specific differences in nucleotide diversity. Our results suggest that inter-population differences in local recombination rates and the corresponding differences in local Background Selection (BGS) need to be considered as a possible explanation for population-specific differences in nucleotide diversity at specific genomic regions.
A NATURALLY OCCURRING MUTATION IN THE FERROUS IRON UPTAKE GENE FEOB CONFERS ENHANCED RESISTANCE TO OXIDATIVE STRESS IN A FRANCISELLA TULARENSIS VACCINE STRAIN
J Fletcher1, C Bosio2, B Jones1,3
1 The Interdisciplinary Graduate Program in Genetics, University of Iowa
2 Laboratory of Intracellular Parasites, Rocky Mountain Labs, National Institute of Allergy and Infectious Diseases Department of Microbiology, Carver College of Medicine, University of Iowa
3 Department of Microbiology, Carver College of Medicine, University of Iowa
Francisella tularensis is a highly virulent bacterial pathogen with an extremely low infectious dose (~10 CFU) and high rates of mortality if left untreated (30-60%). F. tularensis has an extensive history as a bioweapon, and there is no vaccine currently licensed. For these reasons the CDC considers F. tularensis a Tier 1 Select Agent. The unlicensed Live Vaccine Strain (LVS) provides moderate protection against virulent strains; however, we have recently discovered that various lab stocks differ in their virulence and ability to confer immunity. Genome sequencing of high virulence (RML, LD50 ~200 CFU) and low virulence (ATCC, LD50 ~9,000 CFU) strains has identified nine differences, of which four are non-synonymous substitutions. One such mutation occurs in the ferrous iron uptake gene feoB in RML. While iron is required for cellular function, ferrous iron can participate in the Fenton reaction with H2O2, leading to inactivation of essential iron-sulfur cluster enzymes, and DNA damage. Part of the innate immune response involves the oxidative burst in the phagosome and mitochondria-derived ROS in the cytosol. Fully virulent strains of F. tularensis are known to be highly resistant to such host defences, and have low levels of intracellular iron. Accordingly, the RML strain was highly resistant to exogenous H2O2 in vitro relative to the ATCC strain. Overexpression of the ATCC feoB allele, but not the RML allele, leads to significantly increased sensitivity to H2O2. Furthermore, the RML strain grows poorly under conditions of iron starvation, and an iron-responsive lacZ reporter had ~3-fold higher activity in the RML strain relative to ATCC under these conditions. Overexpression of the iron-responsive transcriptional repressor fur leads to reduced growth in the RML strain, but not ATCC. These results are consistent with the hypothesis that RML has less intracellular iron, and that this may lead to increased resistance to host-mediated oxidative stress.
Fibroblast Growth Factor Receptor signaling and Src Family Kinase activity gate homeostatic synaptic plasticity
Synapses undergo many stresses and plastic changes throughout the life of an organism. Homeostatic mechanisms respond to these stresses and maintain synaptic activity within a physiologically favorable range. When faced with a reduction in postsynaptic glutamate receptor activity, the Drosophila neuromuscular junction (NMJ) homeostatically compensates by sending a retrograde signal to the presynaptic nerve. This signal triggers an increase in the number of synaptic vesicles released from the presynaptic terminal during an action potential. One of the least well understood aspects of this process is how postsynaptic systems drive production of homeostatic retrograde signals. We have identified several factors that regulate homeostatic synaptic plasticity in the postsynaptic muscle through an RNAi- and electrophysiology-based screen. This screen revealed that C-terminal Src Kinase (Csk) and the fibroblast growth factor receptor (FGFR) Heartless (Htl) are required for homeostatic compensation at the NMJ.
Work with Csk mutant alleles shows that Csk is required for the long-term maintenance of synaptic homeostasis, but not the rapid induction of this process. Csk phosphorylates and inactivates Src Family Kinases (SFKs), of which there are two in Drosophila: Src64B and Src42A. Overexpression and suppression experiments indicate that the homeostatic defects of Csk mutants are due to elevated SFK activity in the postsynaptic muscle. Immunostaining reveals that Csk mutants have altered NMJ localization of the neural cell adhesion molecule (NCAM) ortholog Fasciclin II (FasII). We examined a potential role for FasII in homeostatic plasticity and found that increasing FasII levels partially impairs this process. Additionally, reducing FasII in a Csk mutant background restores homeostatic compensation, suggesting that Csk and FasII may regulate homeostatic compensation through a common pathway.
We show that Htl is required in the postsynaptic muscle for the long term maintenance, but not the rapid induction, of homeostatic signaling. Htl is known to activate Src64B, and we show that Src64B is required for homeostasis in the postsynaptic muscle and link Src64B and Htl/FGFR signaling in the context of homeostatic compensation. FasII has been implicated as a regulator of Htl activity in Drosophila, which is supported by our observation that FasII genetically interacts with Htl during homeostatic compensation. Collectively, these data shed light on several postsynaptic factors that may work in concert to regulate the production of a homeostatic retrograde signal.
Nicotinamide Riboside is Uniquely Bioavailable In Vivo
Nicotinamide riboside is a recently discovered NAD precursor vitamin with unique activities in protection against metabolic and neurodegenerative conditions. Though nicotinamide riboside has been administered through multiple routes, it has not been established whether it achieves different or superior bioavailability in any target tissue with respect to the other NAD precursor vitamins, nicotinic acid and nicotinamide. Moreover, because enzymatic digestion of nicotinamide riboside can produce the other two NAD precursor vitamins, it is not clear whether nicotinamide riboside acts as a unique chemical entity or whether there are nicotinamide riboside-specific biomarkers. Here we show that nicotinamide riboside exhibits superior oral availability in mouse despite its metabolism to nicotinamide prior to absorption.
|Thursday 6/11/2015||Johnny Cruz Corchado||12:00-1:00 pm||106 BBE|
|Thursday 6/25/2015||Katie Weihbrecht||12:00-1:00 pm||106 BBE|
|Thursday 7/9/2015||Danielle Herrig||12:00-1:00 pm||B20 BB|
|Thursday 7/23/2015||Melissa Marchal||12:00-1:00 pm||106 BBE|
|Thursday 8/6/2015||Changya Chen||12:00-1:00 pm||106 BBE|