UC San Diego SearchMenu
2018 Research Showcase
HR Abstracts
Abstract Title : Ezh2 Loss is Associated With Aberrant Chromatin Structure in Progenitor and Mature Mouse Neutrophils
Abstract : Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), a histone methyltransferase primarily responsible for transcriptional silencing of chromatin. Ezh2 loss of function mutations are associated with poor survival in Myelodysplastic Syndromes (MDS). Prior studies identified dysfunction of neutrophils derived from Ezh2 null cell lines. We hypothesize that Ezh2 loss results in aberrant epigenetic structure in these cells. To test this hypothesis, we characterize the chromatin structure of wildtype and Ezh2 null murine derived neutrophils using Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Our results indicate that loss of Ezh2 confers significant changes in neutrophil chromatin structure, primarily loss of open chromatin, especially in immature undifferentiated neutrophils. Further, we show that significant chromatin closure occurs as these neutrophils differentiate, resulting in a more transcriptionally silent epigenome in mature cells.
Advisor : FRED GAGE
Abstract Title : Studying the role of protocadherin genes in regulation of human neuronal morphology in vitro
Abstract : Protocadherins (PCDH) are a subgroup of the cadherin superfamily of homophilic cell-adhesion proteins. It was found that PCDH alpha genes promote survival and migration of neurons, axonal targeting, and synaptogenesis in mice brains (Yagi, 2013). Hence, we sought to examine how regulation of PCDH genes found in our screen would regulate neuronal morphology. Human neurons can be generated in vitro with pluripotent stem cell culture, and we examined whether knockdown of PCDH genes regulated neuronal morphology. We observed significant alterations in neuronal morphology following knockdown of PCDH genes in human pluripotent-stem-cell-derived neurons in vitro.
Abstract Title : Uncovering the Secret Life of Genes
Abstract : The structural organization of cells is often regarded as consisting primarily of membrane-enclosed organelles. Notwithstanding, there are a number of cellular organelles that do not exhibit a delimiting membrane. These structures are still capable of assembling, maintaining their structural integrity, and compartmentalizing through the driving force of intrinsically disordered regions (IDR). While a number of intracellular structures have been identified as having this form of phase separation, many questions regarding their function and properties remain. Studying phase separation can help us better understand the mechanisms that underlie a number of protein aggregation and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). An in vitro model of HP1α in relation to chromatin will be replicated with the purpose of investigating the liquid-phase nature that has been exhibited by this chromo domain containing protein. This project seeks to address the HP1α conformation that is generated during this heterochromatin-mediated gene silencing mechanism and subsequently address some of the environmental conditions of phase-separated heterochromatin.
Abstract Title : Mutation Rate Variation within the Yeast Genome
Abstract : Mutations cause genetic and phenotypic variation upon which natural selection acts. Despite this ubiquitous role among and within living organisms, our understanding and of mutations remains incomplete. By measuring a reporter gene’s loss-of-function rate in a Yeast Knockout (YKO) library of 457 haploid Saccharomyces cerevisiae strains, we will estimate mutation rates at a 10 kb magnitude resolution across the yeast genome. We are currently developing a method to measure all strains in a high-throughput manner by sequencing DNA barcodes, unique to each strain and reporter insertion site, within a pooled culture of mutants collected by drug selection. In screening the YKO library for outliers, we have identified several strains that potentially have increased mutation rates by over one order of magnitude in their respective reporter cassette. These strains displaying locally elevated mutation rates provide insight into the role of chromosomal and genetic context in mutation rate variation.
Abstract Title : Inhibition of D1R Expressing Neurons in the Dorsal Striatum Promotes Meth Addiction-like Behavior.
Abstract : Evidence from previous studies indicates that methamphetamine (Meth) self-administration upregulates dopamine D1 receptors (D1Rs) in the dorsal striatum. It is therefore hypothesized that D1 receptor expressing medium-sized spiny neurons in the dorsal striatum may contribute to reinforcing effects of Meth and produce dependence-like behavior. Here we seek to determine if inhibiting D1R expressing neurons in the dorsal striatum alters Meth self-administration. A viral vector-mediated approach was used to overexpress the inhibitory (G i coupled-hM4Di) designer receptors exclusively activated by designer drugs (DREADDs) engineered to only respond to exogenous ligand clozapine-N- oxide (CNO). Preliminary findings from behavior data reveal that CNO treatment in animals overexpressing DREADDs increased responding for Meth compared to vehicle saline in a within subject treatment paradigm. CNO treatment in animals that did not express DREADDs did not alter responding for Meth, demonstrating specificity for DREADD- CNO interaction. Postmortem tissue analysis reveal that CNO treatment reduced neuronal activation in the dorsal striatum compared to non DREADD controls and animals that self-administered saline. Ongoing studies will determine the cellular mechanisms underlying enhanced neuronal activation in the dorsal striatum. This suggests that normal functioning and activity of D1R expressing neurons in the dorsal striatum is necessary for reducing Meth addiction-like behavior.
Abstract Title : Deregulation of miRNA profiles in MSA may alter oligodendrocyte maturation pathway and contribute to α-syn accumulation
Abstract : Multiple Systems Atrophy (MSA) is a fatal neurodegenerative disorder affecting around 100,000 people in the U.S.; however, there is still little known about the molecular basis of the disorder and thus no disease -modifying therapy available. MSA is characterized by the accumulation of α-synuclein (α-syn) in oligodendrocyte cells and an increased proportion of immature oligodendrocytes in the brain. Mature oligos, in contrast to immature oligos, clear α-syn aggregates more efficiently. Deregulation of miRNA profiles in MSA may play a role in altering oligo maturation pathways, contributing to α- syn accumulation and demyelination. We established a protocol to differentiate oligodendrocytes from adult rat hippocampal neuronal progenitor cells and used lentiviral vectors to overexpress α-syn. We identified several miRNAs deregulated at different stages of oligo maturation associated with accumulation of α-syn. Changes in miRNA levels and gene expression related to MSA were also measured in human MSA postmortem brains to identify miRNA-mRNA inverse correlations.
Abstract Title : Spatial Organization and Functional Characterization of Auditory Cortex Projection Neurons
Abstract : Sensory information processed in the primary auditory cortex (A1) is directed to a variety of spatially and functionally distinct targets, including contralateral A1, amygdala, inferior colliculus, striatum, and thalamus. These areas play distinct roles in sound-guided behavior and cognition. However, it is unclear whether the organization of A1 projections supports transmission of redundant auditory information to all target regions or transmission of specific aspects of auditory information to different target regions. Understanding the nature of these projections may shed light on neural circuit disorders such as autism and schizophrenia. To address this question, we used the retrograde tracer, cholera toxin B (CTB), conjugated to different fluorescent markers to visualize and characterize the spatial organization of A1 projections. We subsequently used two-photon calcium imaging to characterize the response properties of Layer 2/3 projection subpopulations in primary auditory cortex (A1) of awake mice. Our results suggest that individual A1 principal cells rarely target more than one downstream region and are thus organized to transmit unique channels of auditory information to functionally distinct areas of the brain. However, our findings indicate that frequency selectivity to pure tones is very similar for L2/3 corticostriatal and corticocortical projection neurons. We are currently pursuing functional characterization of A1 projection subpopulations in deeper layers.
Abstract Title : Characterizing non-catalytic domains of unconventional deadenylase TOE1 and investigating their biological importance on snRNA processing
Abstract : Deadenylases are best known for their role in degrading the poly(A) tail of mRNAs and promoting their degradation, which is a vital step of mRNA turnover. TOE1 is a deadenylase that, in addition to its deadenylase catalytic domain and unlike other deadenylases, also has other conserved domains. Previous research in Dr. Lykke-Andersen’s lab demonstrated that TOE1 trims another class of RNA called snRNAs and facilitates their maturation. This event is defective in patients with Pontocerebellar Hypoplasia 7 (PCH7), a disease caused by TOE1 mutation. I hypothesize that non-catalytic conserved domains of TOE1 are involved in trimming of snRNAs. In this project, multiple TOE1 constructs with specific domain deletions were created and incorporated into stable cell lines. To test the importance of each domain, TOE1 mutant variants were expressed at endogenous levels and the 3’ ends of snRNAs were monitored by 3’ end sequencing. Deletions of the several domains of TOE1 result in accumulation of extended snRNAs, suggesting these domains may be important for functionality. Furthermore, preliminary data suggests that deletion of Zn finger and last 30 amino acids of C-terminal, which are both highly conserved across different species, results in the inability to associate with snRNAs in vivo. Further efforts are focused on identifying mechanistically how these domains are important to functionality, with the hypothesis that they function in recruitment of TOE1 to snRNAs directly or through snRNA-specific proteins.
Abstract Title : Investigating the role of endogenous opioids in learning and motivation
Abstract : Endogenous opioid neuropeptides are distributed throughout reward and motivation circuits in the brain. To determine how endogenous opioids contribute to goal-directed actions, we systemically administered naloxone (NLX) to mice prior to performing a goal-oriented operant conditioning task. Intriguingly, we found that the NLX-treated mice made significantly less lever presses than their saline-treated littermates. Open field and rotarod experiments suggested that gross locomotion and coordination, respectively, remained intact. Free feeding and drinking tests showed that food- and water-restricted mice receiving NLX consumed slightly less food and water, respectively, compared to controls, indicating that NLX treatment did not cause satiety. These experiments suggest that in the context of a goal-directed task, although endogenous opioids do not regulate the movement required to perform the task, they may regulate general motivation irrespective of the nature of the reward. Alternatively, they may be involved in learning action-outcome associations, a possibility that is currently under investigation.
Abstract Title : What is the Role of PDLIM1 in Oxygen-Induced Retinopathy?
Abstract : The blood-retinal barrier (BRB) is a unique set of properties possessed by the endothelial cells of the retinal vasculature. Together, these properties prevent free movement of molecules and ions between the blood and retinal tissue, thereby maintaining a precise biochemical environment for proper retinal function. Many retinal diseases are characterized by the disruption of the BRB, but many of the molecular changes that drive BRB dysfunction and repair remain unknown. To address this question, we used the oxygen-induced retinopathy (OIR) mouse model of retinopathy of prematurity (ROP), a common cause of blindness in premature babies. We purified endothelial cells from healthy and dysfunctional retinal vessels and performed RNA sequencing to identify genes whose expression levels change during blood-retinal barrier dysfunction. Among other genes, PDLIM1 was found to be strongly upregulated in endothelial cells in OIR. We confirmed PDLIM1 expression in flat-mounted and sectioned hypoxic retinas from the OIR model via immunofluorescent staining; almost no signal was detected in control retinas. Our in vitro experiments show that PDLIM1 inhibits the Wnt signaling pathway in transfected HEK293 cells. As vascular development in the retina is dependent upon Wnt signaling, we postulate that PDLIM1 inhibits the abnormal neovascularization of retinal blood vessels in disease. Our final aim is to determine how PDLIM1 affects BRB function and neovascularization in the context of retinal disease.
Abstract Title : Defective Mechanism of Nerve Growth Factor Secretion in the Context of HSAN Type V
Abstract : Hereditary Sensory Autonomic Neuropathy Type V, a rare but severe peripheral sensory neuropathy, is associated with a missense mutation (R100W) in nerve growth factor (NGF). We hypothesize that the point mutation NGFR100W induces a deficit in secretion of mutant mature NGF that contributes to sensory neuropathy in the NGFR100W knockin mouse model. Our objective is to test our hypothesis using mouse embryonic fibroblasts (MEFs) cultured from wild-type, heterozygote and homozygote genotypes. We used ELISA to measure the level of NGF in the media. Our data shows NGF secretion in culture media is significantly reduced in NGFR100W cells in a gene-dosage-dependent manner. These results suggest that reduced mature NGF secretion from target tissues, either due to a reduction in cellular synthesis of NGFR100W protein or retarded secretion of NGFR100W, results in NGF deficiency that leads to peripheral sensory neuropathy in HSAN V.
Abstract Title : A maize CCAAT box binding transcription factor that regulates defense against Fusarium venenatum
Abstract : Fungus of the genus Fusarium can induce serious diseases on crops, resulting in significant economic losses. Fusarium causes ear rot, reduces yield, and even produces mycotoxins that threaten the health of both humans and livestock. In maize, Fusarium graminearum induces the production of zealexins and kauralexins. Based on a 32 hour fungus elicitation experiment, total phytoalexin levels increase in response to Fusuarium venenatum treatment when compared to a damage control. A transposon insertional mutant for ca2p6, a CCAAT box binding transcription factor, produces more total phytoalexins than wildtype. Proteomics analysis revealed that mevalonate pyrophosphate decarboxylase and HMG-CoA synthase, two enzymes in the phytoalexin synthesis pathway, had a greater than 1.5-fold change in expression upon elicitation. Expression of DXS, an early enzyme in the synthesis pathway, was notably increased upon elicitation only in the wildtype. In the mutant, HMG-CoA reductase and kaurene synthase 2 showed increase in expression upon elicitation. Ca2p6 may therefore help control phytoalexin production by regulating expression of these enzymes. We aim to identify the regulators of fungal defense mediated phytoalexin production and help characterize the pathways that ca2p6 works in to mediate phytoalexin production.
Abstract Title : The Recruitment of TRF2 to TATA-less Promoters via Transcription Factor Interactions
Abstract : Drosophila TRF2 is a TBP (TATA-binding protein)-related RNA polymerase II transcription factor that is used in place of TBP at TATA-less gene promoters containing either the TCT or the DPE promoter element. Unlike TBP, TRF2 does not bind DNA and is likely recruited to the promoter through protein-protein interactions that mostly remain unknown. Potential sites of interaction with transcription factors TFIIB, the Taf1 subunit of TFIID, Mot1, NC2, and TFIIA have been identified based on regions of homology of TRF2 to TBP. It has also been shown that transcription factors M1BP and DREF bind to TRF2. The 37 amino acid residues exposed on the surface of TRF2 have been mutated in clusters of three residues. The ability of these mutant TRF2 proteins, wtTRF2, and TBP to bind the transcription factors mentioned above will be compared to determine the mechanism of TRF2 recruitment to TATA-less promoters.
Abstract Title : Circuit and Behavioral Investigation via Optogenetic Manipulation in Target-Cell-Type-Defined Corticostriatal Pathways
Abstract : Parkinson’s disease is characterized by impaired motor movement caused by malfunctions in basal ganglia circuitry. Corticostriatal (CSTR) projection pathways connect the motor cortex and basal ganglia. However, the pathways’ function during movement is unknown. By implementing a motor skill task in which head-fixed mice walk on a motorized rotation ladder, we aim to examine the circuit and behavioral effects by activating specific pathways using optogenetics. Using a rabies-virus-mediated tracing technique, we express channelrhodopsin (ChR2), a light-gated cation channel, in CSTR pathways targeting direct pathway medium spiny neurons (dMSN), indirect pathway medium spiny neurons (iMSN), or cholinergic interneurons. We postulate that activating these pathways will not elicit movement, but instead modulate movement such that mice will speed up with dMSN-projecting neuron activation, halt with iMSN-projecting neuron activation, and not be affected by cholinergic interneuron activation.
Advisor : ROB KNIGHT
Abstract Title : The After-Antibiotics Study: Investigating the Implications of Antibiotic Treatment for Urinary Tract Infection on the Urinary Microbiome and Metabolome in Young Women
Abstract : Until recently, the urinary tract infection (UTI), a disease affecting nearly 1 in 3 women globally by the age of 24, was thought to be caused by the invasion of a single foreign pathogenic bacterial species into a sterile urinary tract. However, contemporary advances have revealed that urine is not sterile: like other environments of the human body, the urogenital tract has its own distinct community of bacteria (microbiota) that collectively play a role in maintaining a healthy urinary tract. The current UTI treatment does not take into account the effect of antibiotics on protective microbes in the bladder and contribute to antibiotic resistance. There is a need to investigate the collateral effects of UTI antibiotics and non-antibiotic means of UTI treatment on the urinary microbiome and metabolome. These therapies include cranberry products, d-mannose, NSAIDS, and urinary analgesics. Our research project will study the metabolic interactions between common clinical UTI treatments and the resident urinary microbiome during recovery after UTI. Laboratory techniques to characterize the urogenital microbiota during and after UTI will include shotgun sequencing, metabolomics, and multi-omics visualization. This work is designed to advance prevention of UTI in young women, and may provide information to develop alternative UTI therapies.
Abstract Title : Measuring Rat Impulsivity and Cognition using Delay Discounting and Visual Attentional Engagement Tasks
Abstract : The aim of this research was to investigate the effects of impulsivity on rat decision-making in different behavioral contexts. The project studied the effects of methylphenidate on behavioral measures of brain function. The open-field exploration test measured gross locomotion and risk tolerance, and the delay discounting task measured the ability to forego the opportunity for a small, immediate water reward in favor of a larger, but delayed water reward. In the visual attention task, rats discriminated between visual stimuli of varying attentional demand. We hypothesized that methylphenidate would increase tolerance of reward delay and reduce errors in the visual attention task. The effects of methylphenidate on the delay discounting task was inconclusive due to development of location-based biases. Methylphenidate did not have any significant effect on performance in the visual attention task.
Abstract Title : Investigating the Role of Zeb2 Transcription Factor in CD8+ Memory T cell Differentiation and Homeostasis
Abstract : CD8+ T cells are a key component of the host immune response specialized to target and kill malignant cells and cells infected with intracellular pathogens, including bacteria and viruses. Upon recognizing antigen, naive CD8+ T cells get activated, rapidly proliferate and differentiate to form a heterogeneous population of effector T cells able to clear the pathogen. After the pathogen is cleared, the majority of the effector cells will die by apoptosis. However, a population of long-lived T cells survive as a heterogeneous memory T cell population which can be subdivided into central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). To understand how these memory T cell subsets develop and are maintained, it is important to define the transcriptional network that facilitates CD8+ T cell differentiation. The transcription factor Zeb2 was previously identified to be important in terminal effector CD8+ T cell differentiation. Interestingly, Zeb2 is highly expressed in the TEM population at a late time point of infection; therefore, we hypothesize that Zeb2 is also required for the maintenance of the TEM population. Here, we established an inducible Zeb2 ER-Cre knock-out mouse model that is crucial to the study of how ZEB2 specifically regulates memory CD8+ T cell maintenance and homeostasis without affecting effector T cell differentiation. Following an acute lymphocytic choriomeningitis virus (LCMV), Zeb2 deletion was induced in established memory CD8+ T cells and the frequency of effector-like memory T cells was reduced. This suggests that Zeb2 is important in maintaining the phenotype of effector-like memory T cells.
Abstract Title : Oxidative Stress is a Cause of Reduced Fitness in Copepod Hybrids
Abstract : According to Dobzhansky-Muller model of hybrid incompatibility, hybridization sometimes result in genetically incompatible offspring compared to their original species. In this speciation process, hybrids show reduced fitness resulting from interactions between differentiated genes. Such fitness breakdown happens after the F1 hybrid generation when incompatibilities accumulate and become unmasked. Studies have shown a direct relationship between genetic incompatibility and reduced fitness of hybrids. Because the highest rates of mutations occur in the mitochondrial genome (mtDNA), incompatibilities may evolve between mtDNA and the nuclear genome, affecting mitochondrial performance in hybrids. Here, we compared reactive oxygen species (ROS) levels in interpopulation hybrids to parental populations in copepod Tigriopus californicus, using ROS fluorescent assay. Comparisons between three hybrid lines and their parents showed one case of significant elevation of hybrid ROS production. This result is consistent with the hypothesis that hybridization can result in incompatibilities between mtDNA and the nuclear genome.
Abstract Title : Loss of RIAM in B cell impairs T-independent immune response
Abstract : Rap1-GTP-interacting adapter molecule (RIAM) regulates integrin activation, a cellular adhesion process important in multiple stages of immune response. Previous studies have shown that germline RIAM knockout mice have defects in T-cell-dependent humoral immunity, while T Cell specific RIAM knockout mice display poor T cell activation and resultant effector functions. In this study, we utilized Cre-Lox recombination to investigate the role(s) of cell-intrinsic RIAM in B-cell-mediated immune responses. RIAM is necessary for B cell trafficking and residence in the marginal zone of the spleen, as naïve spleen B cell subset analysis showed increased number of follicular B cells and decreased number of marginal zone B cells. Loss of RIAM in B cells did not cause observable defects in a T-cell-dependent humoral response, but humoral response to both TI-1 and TI-2 antigens were impaired. We are testing autoantibody production and disease development in B cell RIAM deficient mice using the EAE model.