Tube formation in the fruit fly (Drosophila melanogaster) egg chamber is an excellent model for vertebrate neural tube formation, as they closely resemble each other. During egg chamber development, a subset of cells, the roof cells and the floor cells, form two tubes that make eggshell structures called dorsal appendages (DAs). Roof cells, which express the protein Broad, constrict their apices (inner surfaces) to curve up and out of the epithelium. The floor cells elongate, dive under the roof cells, and zipper together to seal the floor; they are distinguished by expression of the protein Rhomboid. Defects in this process, especially in floor closure, create open tubes, similar to spina bifida. To observe cell movement in the developing egg chamber, we want to outline the shapes of cells with a fluorescent molecule. mCherry is a red fluorescent protein that has good fluorescence and a short maturation time (15 minutes), so we can observe the rapid process of tube formation. If one links the CD2 gene, which encodes a cell membrane adhesion protein, to the mCherry gene, red fluorescence will appear all around the cell surfaces. Such a fusion protein lets us observe changes in cell shape and cell movements during tube formation. When the CD2-mCherry segment is connected to the regulatory region for the rhomboid gene, fluorescence occurs in just the floor cells. I have created a rhomboid-CD2-mCherry construct that I will introduce into flies through injection with a recombinase that will integrate the construct into the fly genome. With this construct I can visualize DA tube development live by observing fluorescence in the floor cells. Analysis of these cells can give insight into conserved tube-forming processes.

Genomic engineering has a wealth of possible applications, from curing genetic disease to curbing the spread of malaria. Homing endonucleases (HEs), which cut DNA at long, specific DNA target sites, are useful for genomic engineering because of their high site specificity. However, to use HEs for engineering, we must be able to modify their target site. Though this can be done with current computational and experimental protein engineering techniques, some HEs are resistant to those approaches. Therefore, we are working to develop a general methodology to modify the site specificity of HEs, based on a new synthetic biology technique called phage-assisted continuous evolution (PACE). PACE is a continuous evolution system in which the evolution of a desired biomolecular activity is linked to the reproductive cycle of phage particles containing the evolving gene(s), and should allow us to develop proteins or nucleic acids with almost any desired activity or specificity. We have built an automated system to keep the growing host cells and phage at a constant cell density and temperature, which is required for PACE. We are also working to design a mechanism to link HE DNA binding activity to phage reproduction by engineering plasmids in the host cells and phage. In the future, we will be refining the hardware set-up and protocol even further, and looking for ways to modularize the PACE system.

RNA Polymerase V is a DNA-dependent RNA polymerase involved in RNA-directed DNA methylation (RdDM) (Matzke M et al 2007). RdDM is required for transcriptional silencing of intergenic regions, repetitive DNA, and transposons genome-wide. Arabidopsis polV mutants show increased developmental instability and aberrant root hairs. We hypothesize these phenotypes are the result of alterations in transcriptional silencing and we predict changes in transcriptional silencing are due to altered chromatin accessibility. To asses chromatin accessibility in polV mutant plants we will use a technique called DNaseI-seq that chops up chromatin with a restriction enzyme. This technique allows us to measure which regions of the genome are highly accessible to transcriptional machinery and which are not. Currently, I am developing transgenic plant strains as well as quality control assays to improve DNaseI-seq feasibility in polV mutant plants.  Thus the knowledge will enhance our understanding of developmental instabilities in Arabidopsis and plants in general.
 

Centromeres in many species are found to reside near origins of replication that are activated early in S-phase, yet the biological significance of this conserved early replication of centromeres is unknown. Previous research has shown that the activation time of an origin is affected by its chromosomal location. A recent study has found that centromeres in the yeast Saccharomyces cerevisiae advance the activation time of nearby origins, and that this effect depends on the centromere’s ability to establish a functional kinetochore. These observations lead to the hypothesis that centromeres need to be replicated early to allow sufficient time for the assembly of functional kinetochores. If kinetochores fail to be established in a timely fashion, chromosomes may not be correctly bi-orient at mitosis. Daughter cells therefore may inherit the incorrect number of chromosomes creating conditions of aneuploidy such as those found in Down syndrome and many cancers. I will test the importance of early centromere replication by deleting origins immediately flanking a yeast centromere to delay that centromere’s replication. I will then conduct an assay to test for the rate of chromosome mis-segregation events in the mutant strain compared to the wild-type strain. I anticipate that the mutant strain may have a higher rate of loss for the modified chromosome, suggesting that early replication of centromeres is important for maintaining genome stability. This project will also provide insights into the regulation of the temporal program of replication origin activation and provide the first functional test for the consequences of disrupting the temporal program of chromosome replication.

Sequencing the genome of an organism, or determining the order of the base pairs in its DNA, is one approach to understanding that organism’s chromosome structure.  Genome sequence assembly is accomplished with the help of computer programs, which often fail to include or map repetitious sequences in the final chromosome assembly.  One such unmapped sequence in the Lachancea waltii (L. waltii) yeast genome is the ribosomal DNA (rDNA), a sequence composed of hundreds of identical repeating segments that encodes the RNA component of ribosomes, the protein-producing machines in the cell.  My previous research has shown the rDNA is located in two large sequence gaps on chromosome 8, the only chromosome published incompletely as three separate segments of sequence.  My current research involves determining the order and orientation of these sequence scaffolds to verify and complete the assembly of L. waltii chromosome 8.  To investigate the arrangement of the three scaffolds, I am first performing indirect end labeling, where I am cleaving the chromosome with restriction enzymes to produce a specific fragmentation pattern.  Alignment of this pattern with that expected based on the proposed structure of chromosome 8 would validate the proposed chromosome structure and rDNA location.  In addition, I am performing a snap-back assay, a test that will indicate the orientation of the rDNA segment repeats – thereby verifying if the rDNA is present in one or two locations.  Finally, I am degrading the ends of chromosome 8 using an exonuclease – an end-specific enzyme – to determine the sequences, and thus the identity and orientation of the scaffolds, that are present at the chromosome ends.  By generating a complete, contiguous sequence of L. waltii chromosome 8, we hope to provide a more in-depth profile of this relatively uncharacterized yeast for future studies on chromosome evolution.

The process of cellular reproduction is dependent upon precise timing and coordination of regulatory events. Oscillations in cyclin-dependent kinase (Cdk) activities drive the cell cycle by regulating changes in the phosphorylation states of protein substrates. This dynamic and intricate signaling network of protein phosphorylation is a central component of the cell-cycle control system and enables the cell to modulate cellular functions and processes. Despite extraordinary advances in depth of coverage, phosphoproteomics experiments have struggled to expand in breadth, often comparing thousands of phosphorylation sites between only two moments in time. Such snapshots provide a biased view of the highly dynamic phosphoproteome and ignore important contextual information. To meet this challenge, we are developing robust and reproducible phosphoproteomic methodologies and utilizing mass spectrometry to study the changes in S. cerevisiae phosphoproteome over the course of the cell cycle. I first analyzed the reproducibility of the sample preparation techniques and data acquisition methodologies, which are key factors in the ability to distinguish true differences between different samples. Repeat analyses of 10 identical samples consisting of the entire yeast phosphoproteome were conducted, and preliminary results show that about 70% of the total identified phosphoproteins and 37% of the total identified phosphorylation sites were detected in all 10 repeat analyses with a variance in quantitation of less than 5%, indicating an accurate measurement of changes across the runs. I am now collecting global measurements of relative phosphorylation and protein changes in 5-minute intervals over the entirety of the cell cycle in a synchronously growing population to identify and quantify the proteins present at each time point of the cell cycle. This research will provide a wealth of information on the dynamics of proteome and phosphoproteome regulation throughout the cell cycle and unlock new insights into the biology of defective checkpoints in cancer.

Speciation is the process by which one species becomes two species that can no longer interbreed. A common hypothesis is that infertility between closely related species is due to the rapid evolution of DNA double-strand-break hotspots that are active in meiosis. DNA double-strand-breaks play an important role in meiosis by initiating recombination after chromosome replication and can be repaired as crossovers. However, hotspots where most of the breaks happen are preferentially lost to gene conversion during DNA repair. It is a mystery how hotspots are maintained over evolutionary time when they are constantly destroyed during meiosis. We hypothesize that hotspots rapidly appear and disappear over evolutionary time, which results in speciation. Schizosaccharomyces kambucha and Schizosaccharomyces pombe are two very closely related fission yeast species that are 99.5% identical at the DNA sequence level. Mapping out the hotspots of Schizosaccharomyces kambucha and comparing them to those of Schizosaccharomyces pombe will be a useful way to test our hypothesis. For this experiment, we have created Schizosaccharomyces kambucha mutants that can no longer repair the breaks so that we can pull out the broken DNA pieces using Chromatin Immunoprecipitation and locate where the DNA double-strand-break hotspots are in the genome. We expect to see the hotspots of Schizosaccharomyces kambucha differ from those of Schizosaccharomyces pombe, which will support the hypothesis that the difference in DNA double-strand-hotspots is indeed contributing to speciation. This research can be extended further to explain speciation in other organisms or unexplained infertility between human couples.

Understanding stem cell maintenance is crucial for delineating conditions such as tissue loss, aging, and cancer. Using the Drosophila female germ-line stem cell (GSC) model to observe stem cells in vivo, several extrinsic and intrinsic factors have been demonstrated to regulate maintenance of Drosophila GSCs; they have also been found to be conserved in mammalian systems. The search is on for novel regulatory factors of stem cell maintenance with the hope of extending our understanding to stem cells in mammalian systems. My project involves the characterization of the molecular function of Pineapple eye gene (Pie), a novel regulator of GSC maintenance. Through a loss-of-function screen in GSCs, it has been demonstrated that mutations of pie result in GSC maintenance failure. The amino acid sequence of pie revealed that it contains ring-domains suggesting possible E3 ubiquitin ligase function. Such proteins are key components of the ubiquitination pathway, which are involved in protein tagging and degradation. To test pie’s function as an E3 ligase, I am using a novel assay that employs the fact that bacteria do not have endogenous ubiquitination pathway and therefore utilizes Escheria coli as an in vivo system to express the components of the ubiquitination pathway and detect the formation of ubiquitin chains, a key indicator of E3 Ligase activity. I have demonstrated that the expression of pie’s ring-domains without a substrate is not sufficient for the detection of ubiquitin chains. I will now test whether the full length pie is necessary for proper ubiquitination. Further, I will also test whether pie’s functional domain needs a substrate in order to function effectively. Successful characterization of pie can reveal new pathways to target for conditions involving stem cell maintenance.

Embryonic stem cells are isolated from the early developing embryo and are capable of forming the different cell types found in the body. Understanding how these cells develop and maintain their specialized state is critical to understanding how they function. We hypothesize that embryonic stem cells acquire a unique metabolic state that aids them in maintaining their specialized state. Using two distinct mouse pluripotent states, embryonic stem cells (ESC’s) and epiblast stem cells (EpiSC’s) representing cells isolated from the inner cell mass of the blastocyst and the epiblast, a later stage in embryonic development, we found that EpiSC’s are much more glycolytic in terms of their energy usage compared to ESC’s as measured by oxygen consumption and extracellular acidification rates. Furthermore, using quantitative polymerase chain reaction experiments, we found that genes key to regulating and maintaining high rates of glycolysis are significantly upregulated in EpiSC’s compared to ESC’s. We showed that the overexpression of HIF1α, a known key regulator of these glycolytic genes, in ESC’s is able to shift the ESC’s towards a more glycolytic metabolic state and cellular morphology reminiscent of EpiSC’s. In addition, transition of ESC’s to EpiSC’s using the known chemical inducers Activin and Fibroblast Growth Factor 2 revealed that changes in the expression of metabolic genes occur prior to changes in expression of cell fate markers, which suggest that a metabolic transition in the ESC’s may be key in its transition to an EpiSC-like state.

Schizophrenia is a debilitating neuropsychiatric disorder with a worldwide prevalence of 1%. Although the heritability of schizophrenia is high, there is a large proportion of affected individuals with no family history. We are testing the hypothesis that many different severe mutations are the causal genetic factors for schizophrenia. In order to test our hypothesis, we sequenced the exomes of approximately 50 trios consisting of an affected individual and his/her parents who are free of mental illness. Exome sequencing is a rapid and robust method for looking at only coding portions of the genome, and allows for the detection of rare variants in those regions. Sequence data is then run through several bioinformatics filters to ensure that the variant is not common in the general population, and that the variant is probably damaging to the gene product. All variants that pass through the bioinformatics filters are validated with Sanger sequencing. De novo mutations were found in 19 different genes in 11 trios. Of the mutations 13 were single base pair changes, and 6 were truncating nonsense or insertion/deletion events. After these experiments have been carried out on approximately 100 trios, we will use the group of candidate genes to identify critical pathways that may be affected in schizophrenia. Understanding the structure of these pathways may allow for the development of targeted therapies for schizophrenia.

Schizophrenia is a devastating neurodevelopmental disorder whose genetic influences continue to be elusive. Rare, gene-disrupting genomic deletions and duplications – called copy number variants (CNVs) – have been implicated in schizophrenia; however, much remains to be understood about which genes are causative, as well as the cellular mechanisms involved. Biological follow up of individual CNVs may give insight to the origin of schizophrenia. We focused on a CNV present in a patient with early onset schizophrenia. This mutation duplicates the 5' ends of two genes that lie head-to-head on chromosome 11q22: DCUN1D5, a previously uncharacterized gene predicted to be involved in cullin neddylation of ubiquitin ligase complexes, and DYNC2H1, a dynein active in cilia. Both are expressed in brain and are plausible candidate genes for schizophrenia. Using RNAseq, we detected novel DCUN1D5 transcripts in the patient’s lymphoblasts that we predict will result in aberrant, truncated DCUN1D5 protein. To test the conformational stability of these aberrant proteins, we then expressed epitope-tagged full length and truncated DCUN1D5 in HEK293 cell lines. These transfected cells also allow us to study the functional capabilities of the aberrant proteins in the cullin neddylation pathway, and the specific cullin binding partners of DCUN1D5. Investigation of these aberrant proteins and their function may shed light on pathways that contribute to schizophrenia, potentially guiding the search for new candidate genes and the development of novel treatment strategies.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins and signatures of episodic lineage-specific positive selection suggests a function in suppressing genetic conlifcts such as segregation distortion in the male germline.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

Differentiated human embryonic stem cells (hESCs) demonstrate the potential for regenerative medicine and drug discovery utilizing rationally assigned cell fate. But hESC are limited by the technical difficulty of recapitulating an ever growing set of cellular phenotypes using a finite set of pluripotent cell lines. The resulting molecular deficiencies limit the ability of differentiated cells to integrate into host tissue, and diminish the translational potential of findings based on differentiated hESC. Induced pluripotent stem cells (iPSCs) can be derived from patients with or without genetic disorders, and have much greater use for drug discovery and basic research, and patient derived iPSCs are less prone to rejection by host tissue. However, iPSC generated by insertion of genes into the host genome, increasing risk of rejection and limiting maturation of iPSC-derived cells. RNA-induced pluripotent stem cells (RiPSC) offer a solution to problems of genomic modification by inducing pluripotency exclusively through the transfection of mRNA. MicroRNA (miRNA) exhibit widespread regulation of mRNA translation and degradation, and do not directly alter the genome. A bioinformatics study curating publicly available microarray data for components of the miRNA biogenesis pathway, pluripotency factors, and miRNA expression will generate hypotheses about the functions of miRNA and relevant proteins, with a focus on differentiation and neural cell fate. Quantification of miRNA and mRNA expression in hESC and RiPSC during retinal differentiation will support or disprove hypothesized relationships between protein and miRNA expression. Transfection of miRNA will be used to determine the ability of these miRNA to enhance differentiation and maturation of retinal cells. Additionally, the resulting retinal progenitors will be implanted into mouse retina, directly testing the potential of miRNA to improve clinical outcomes of stem cell based therapies for the nervous system.

Genetic conflicts arise between two genetic elements that compete to promote their own evolutionary success. Transposons and host defense factors are a classic example. Transposons are selfish, mobile DNA elements that threaten to disrupt other genomic sequences when active. Polycomb Group (PcG) proteins, previously thought of exclusively as developmental regulators, can also function as transposon-silencers. Several PcG proteins are rapidly evolving under positive selection in Drosophila, suggesting they might be engaged in genetic conflict with transposons. My research project seeks to determine whether transposons are the biological force driving the signature of selection observed with certain PcG proteins. In order to test this, I am in the process of creating transgenic Drosophila melanogaster flies that are homozygous mutants for a given PcG gene and harbor a diverged, transgenically introduced allele of the PcG gene instead. I created the diverged PcG constructs using stitch PCR, cloning and sequencing. I plan to measure the transposon activity levels of these transgenic flies. Elevated transposon expression in flies that harbor a diverged PcG gene would suggest that transposons are responsible for the signature of selection observed at several PcG proteins.

Understanding the genetic and molecular mechanisms behind development is necessary for identifying and treating genetic disorders. A key developmental process is tube formation (tubulogenesis), which produces structures such as the vascular system, bronchi, and the embryonic neural tube. Our lab uses the model organism Drosophila melanogaster, a fruit fly, to study the formation of a tubular organ called the dorsal appendage (DA). Two DA tubes form during oogenesis, creating extensions of the eggshell used for gas exchange once the egg is laid. DA formation resembles neural tube formation in vertebrates. To understand the molecular mechanisms that underlie DA formation, we disrupt specific genes and proteins and then analyze how their loss affects DA formation. This project focuses on tiggrin, a gene that encodes an extra-cellular matrix protein that interacts with cell surface receptors to shape tissues. A previous genetic screen identified tiggrin as a potential regulator of DA formation. To understand tiggrin’s role, we have made 24 potential deletions of the tiggrin gene by excising a P-element transposon just upstream of tiggrin. We are using the polymerase chain reaction (PCR) to determine if we have produced any actual deletions of or within tiggrin. Once we determine which lines contain deletions, we will analyze their effect on DA formation. We are breeding lines with potential tiggrin deletions to determine if they are homozygous viable or fertile. We are crossing any lines that do not result in adult tig/tig individuals to a previously established line with a large deletion that removes tiggrin (and neighboring genes) to see if the resulting flies are viable and fertile. Also, we will determine the spatial and temporal expression of tiggrin protein during oogenesis using immunostaining. Adding tiggrin to our study of DA formation will broaden our holistic understanding of the molecular mechanisms of tubulogenesis.

Chemostats are a continuous culture system in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients. Unlike batch cultures, the selective pressure imposed in a chemostat is constant. This has made them a desirable tool for evolution and competition experiments. Traditionally chemostat experiments with S. cerevisiae have been constrained due to the limited number of cultures that can be run at once. In order to solve this problem our team is working with a new culturing system that allows 64 chemostats to be run simultaneously. In our experiment we evolved 32 biological replicates of haploid MATa yeast cells under constant sulfur-limitation for 250 generations. We are now using a variety of techniques including whole genome sequencing of evolved populations to characterize mutations that have been selected for during the course of these evolutions. Changes in the genome can be correlated to changes in relative fitness, which we assayed through competition with a GFP (a green fluorescent protein that exhibits bright green fluorescence when exposed to ultraviolet blue light) expressing ancestral strain every 50 generations. Ultimately it is our hope that higher replicate number will better reveal the spectrum of genes important for adaptive growth in a sulfur-limited environment. Furthermore we would like to use these methods to understand what genes are important in a wide variety of nutrient limited environments.

The Dunham group studies the costs and benefits of changes in gene and chromosome copy number (aneuploidy) in S. cerevisiae. Gene copy number change is a common evolutionary mechanism, and is often seen in cancers. Previously, the lab carried out a series of experiments to determine the consequences of changing the copy number of every gene in the yeast genome. 4383 yeast strains, each carrying a plasmid containing a yeast gene and a unique barcode sequence, were competed in nutrient-limited continuous-culture environments. The relative success of each strain was found by performing high-throughput sequencing on the barcodes. Our project explored three assumptions of this experiment, using direct chemostat competition assays. First, we confirmed that fitness effects observed in the pooled experiment could be repeated in individual experiments. We performed competition experiments using 22 genes from the experiment that were found to either be beneficial in certain environments or potentially harmful when duplicated. We also found that the plasmid alone had a fitness effect, by competing a strain containing a plasmid without a gene insert against a wild-type strain. Finally we tested the fitness effects of Autonomously Replicating Sequences (ARSs), which are origins of DNA replication found in many of the loci we are investigating. In a competition between a strain transformed with a plasmid with two ARSs and a strain with a plasmid containing a single ARS, a fitness benefit was observed for the presence of an extra ARS on the plasmid. These data are important controls for the pooled experiments previously performed in the Dunham lab and will help to identify the precise molecular causes of fitness changes associated with aneuploidy, and, perhaps in the future, provide some new insights into cancer biology.