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Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

Polyploidy is widely acknowledged as a widespread mechanism in the evolution and speciation of the majority of flowering plants. Allopolyploid forms through interspecific hybridization and whole genome duplication. While allopolyploids may display increased vigor relative to their progenitors, they can also face challenges to fertility following hybridization. Genetic changes in allopolyploids result from recombination between the hybridized subgenomes, which can influence phenotype and ultimately determine fitness of future generations. To study dynamic changes that follow allopolyploid formation, Brassica napus lineages were derived by hybridizing Brassica oleracea and Brassica rapa. Two lineages of B. napus were analyzed for genetic and phenotypic changes in the S2, S7, and S12 generations. Although these lineages were genetically identical at the time of hybridization, divergence was apparent by the S2 generation. There was a significant increase in sequence loss across generations within both lineages. Four of six generations from both lineages displayed no significant differences to each other in sequence loss relative to the parental generation. In both lineages, there was a bias towards losing sequences from the B. olereacea subgenome. Some individual plants showed novel phenotypes; however, there was no correlation between the examined genetic changes and selected phenotypes.

Hypoxia is a significant low oxygen state that has complex and diverse impacts on organisms. In aerobes, various adaptive responses to hypoxia are observed that vary depending on the level of oxygen depletion and previous adaptation, hence the continued attention to hypoxia as an important abiotic stressor. Adaptive responses to hypoxia are primarily governed by the hypoxia-inducible factors (HIFs), which activate downstream genetic pathways responsible for oxygen transport and metabolic plasticity. In aquatic habitats, oxygen availability can vary greatly over time and space. Therefore, aquatic organisms’ adaptation to hypoxia is likely pervasive, especially in genotypes originating from waterbodies prone to hypoxia. Here we report the transcriptional response to severe hypoxia in the freshwater crustacean Daphnia magna. We observe improved survival in media containing elevated calcium ion (Ca2+) concentrations. Additionally, we observe changes in lactate and pyruvate concentrations within tissues. To elucidate the transcriptome basis of these effects, we examine transcripts with known gene ontologies indicating roles in Ca2+ homeostasis and signaling, and in pyruvate metabolism, including gluconeogenesis (GNG). We observe the up-regulation of numerous transcripts encoding GNG pathway enzymes, including the rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK-C) and fructose-1,6-bisphosphatase (FBP). In contrast, no transcripts involved in Ca2+ homeostasis or signaling showed any significant differential expression. Some GNG transcripts are more up-regulated in clones from permanent waterbodies not prone to hypoxia, inconsistent with the hypothesis about its protective effects. One exception is the FBP transcript, which has been identified to be up-regulated in some hypoxia-tolerant aquatic organisms.

Preeclampsia (PE) is a disorder affecting 2-10% of pregnancies worldwide. Clinical signs include high blood pressure (HBP) and proteinuria in the mother after the 20th week of pregnancy. Currently, the only cure for PE is delivery of the fetus, which is often necessary preterm and thus dangerous for both mother and fetus. Maternal symptoms of PE are caused by excess anti-angiogenic proteins of placental origin called soluble Flt1s (sFlt1s). sFlt1 mRNA isoforms are produced by alternative polyadenylation (APA) of full-length Flt1 (fl-Flt1) pre- mRNA. While fl-Flt1 encodes a transmembrane protein, sFlt1s encode truncated proteins that are soluble. Multiple sFlt1 isoforms exist, and their respective contribution to the pathophysiology of PE is unclear. Furthermore, it is unknown whether there is a genome-wide role for APA in PE. In my thesis research, I developed a polyadenylation site sequencing method, and used this method to simultaneously quantify transcriptome-wide polyadenylation site usage and gene expression levels in normal, early-onset PE, and late-onset PE human placentae. I observed distinct expression profiles amongst the three groups, with differential expression of genes in several functional categories, including angiogenesis. I found that three sFlt1 isoforms account for >94% of all placental FLT1 transcripts, and that increased transcription of the entire FLT1 locus drives upregulation of both fl-Flt1 and sFlt1 in PE. I found that APA does not contribute substantially to PE pathophysiology. I also identified siRNAs that knock down sFlt1 mRNA efficiently in cell lines that pave the way for further development of novel RNAi based therapeutics to alleviate PE.

MicroRNAs play an important role in protecting biological robustness during development. Biological robustness is the ability to maintain a consistent output despite variation in input, such as transcriptional noise or environmental stresses. Here, we show that the conserved microRNAs mir-34 and mir-83 promote the robust migration of the distal tip cells in Caenorhabditis elegans when stressed by changing environmental temperature. Our results show that distal tip cell migration is sensitive to temperature changes occurring within a two hour period during the first larval stage. mir-34 and mir-83 protect distal tip cell migration by regulating potential targets cdc-42, pat-3, and peb-1. cdc-42 and pat-3 are known components of the integrin signaling network controlling pathfinding during migration, while the involvement of peb-1 is a novel finding. Additionally, loss of the two microRNAs leads to a reduction in both fecundity and lifespan, suggesting that the loss of developmental robustness leads to a decrease in fitness. mir-34 and mir-83 are not only conserved in higher organisms, but duplicated. Both have been implicated as tumor suppressor genes in mammalian work. Our work has found a role for both microRNAs in integrin-regulated cell migrations that is potentially conserved in higher organisms. Additionally, our work supports the growing appreciation for the role of microRNAs in both stress response and promoting developmental robustness.

Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.

Many researchers across a wide range of disciplines have turned to gene expression anal- ysis to aid in predicting and understanding biological outcomes and mechanisms. Because genes are known to work in a dependent manner, it’s common for researchers to first group genes in biologically meaningful sets and then test each gene set for differential expression. Comparisons are made across different treatment/condition groups. The meta-analytic method for testing differential activity of gene sets, termed multi-variate gene set testing (mvGST), will be used to provide context for two persistent and problematic issues in gene set testing. These are: 1) gathering organism specific annotation for non-model organisms and 2) handling gene annotation ambiguities. The primary purpose of this thesis is to explore different gene annotation gathering methods in the building of gene set lists and to address the problem of gene annotation ambiguity. Using an example study, three different annotation gathering methods are proposed to construct GO gene set lists. These lists are directly compared, as are the subsequent results from mvGST analysis. In a separate study, an optimization algorithm is proposed as a solution for handling gene annotation ambiguities.

Multiple Sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system, characterized by axonal demyelination and multifocal inflammation. Like many autoimmune diseases, it is a sexually dimorphic disease, being 3-4 times more common in females than in males. p38α MAP kinase (MAPK) has an integral role in modulating inflammatory processes in autoimmunity. Conditionally ablating p38α MAPK in myeloid cells in B6 mice shows a sex difference in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In the absence of sex hormones, this sex difference was reversed, suggesting a role for sex hormones in modulating p38α MAPK signaling in EAE. Based on these findings, we hypothesized that pro-inflammatory functions in EAE is p38-indepdendent in the presence of androgens and p38-dependent in the presence of estrogens. For the purposes of this project, the role of androgens was evaluated. Both in vivo and in vitro techniques were used to assess how androgen receptor (AR) signaling: 1) impacts EAE pathogenesis, and 2) impacts the role of p38α in EAE pathogenesis and macrophage function. To this end, using Cre-Lox technology, we generated mice deficient in: 1) AR globally or conditionally in macrophages, as well as 2) mice doubly deficient in AR and p38α. In vivo results from p38α-sufficient global AR knockout mice show no effect of global AR deletion on EAE pathogenesis. Surprisingly, results from p38α-sufficient conditional AR knockout mice showed significant worsening in disease compared to WT counterparts, suggesting that AR signaling in myeloid cells has a protective role in EAE pathogenesis. These findings implicate a protective role for AR signaling in EAE. Studies with mice doubly deficient in p38α and AR to determine whether AR regulates the role of p38α in EAE are ongoing, but so far show no effect on AR deletion on the role of p38α MAPK. Further studies with larger cohorts of mice are needed elucidate the relationship between AR and p38α MAPK signaling in myeloid cells in EAE pathogenesis. In vitro studies using the immortalized macrophage cell line RAW 264.7 showed that pharmacologic inhibition of p38 MAPK after stimulation with LPS reduced the production of classic pro-inflammatory cytokines IL-6 and TNFα, and effect that was not affected by treatment with 5-dihydrotestosterone, suggesting that the AR does not modulate the role of p38α in cytokine production. These findings implicate no direct role of AR signaling on the functional role of p38α MAPK in the myeloid cell lineage in inflammatory and autoimmune responses.

Our understanding of genetic predisposition to inflammatory and autoimmune diseases has been enhanced by large scale quantitative trait loci (QTL) linkage mapping and genome-wide association studies (GWAS). However, the resolution and interpretation of QTL linkage mapping or GWAS findings are limited. In this work, we complement genetic predictions for several human diseases including multiple sclerosis (MS) and systemic capillary leakage syndrome (SCLS) with genetic and functional data in model organisms to associate genes with phenotypes and diseases. Focusing on MS, an autoimmune inflammatory disease of the central nervous system (CNS), we experimentally tested the effect of three of the GWAS candidate genes (SLAMF1, SLAMF2 and SLAMF7) in the experimental autoimmune encephalomyelitis (EAE) mouse model and found a male-specific locus distal to these loci regulating CNS autoimmune disease. Functional data in mouse suggests this male-specific locus modulates the frequency of immune cells including CD11b+, TCRαβ+CD4+Foxp3+, and TCRαβ+CD8+IL-17+ cells during EAE disease. Orchiectomy experiments demonstrate that this male specific phenotype is dependent on testis but not testosterone (T) or 5α-dihydrotestosterone (DHT). Using a bioinformatic approach, we identified SLAMF8 and SLAMF9 along with other differentially expressed genes in linkage with MS-GWAS predictions whose expression is testis-dependent, but not directly regulated by T or DHT, as potential positional candidates regulating CNS autoimmune disease. Further refinement of this locus is required to identify the causal gene(s) that may be targeted for prevention and/or treatment of MS in men. Using SCLS, an extremely rare disorder of unknown etiology characterized by recurrent episodes of vascular leakage, we identified and modeled this disease in an inbred mouse strain, SJL, using susceptibility to histamine- and infection-triggered vascular leak as the major phenotypic readout. This trait “Histamine hypersensitivity” (Histh/Histh) was mapped to a region on Chr 6. Remarkably, Histh is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3). Subsequent studies found that the Histh locus is not unique to SJL but additional mouse strains also exhibit Histh phenotype. Considering GWAS studies in SCLS are limited by the small number of patients, we utilized interval-specific SNP-based association testing among Histh phenotyped mouse strains to predict Histh candidates. Furthermore, to dissect the complexity of Histh QTL, we developed network-based functional prediction methods to rank genes in this locus by predicting functional association with multiple Histh-related processes. The top-ranked genes include Cxcl12, Ret, Cacna1c, and Cntn3, all of which have strong functional associations and are proximal to SNPs segregating with Histh. Lastly, we utilized the power of integrating genetic and functional approaches to understand susceptibility to Bordetella pertussis and pertussis toxin (PTX) induced histamine sensitization (Bphs/Bphs), a sub-phenotype with an established role in autoimmunity. Congenic mapping in mice had earlier linked Bphs to histamine H1 receptor gene (Hrh1/H1R) and demonstrated that H1R differs at three amino acid residues in Bphs-susceptible and -resistant mice. Our subsequent studies identified eight inbred mouse strains that were susceptible to Bphs despite carrying a resistant H1R allele. Genetic analyses mapped the locus complementing Bphs to mouse Chr 6, in linkage disequilibrium with Hrh1; we have designated this Bphs-enhancer (Bphse). Similar to the approaches used for Histh, we utilized interval-specific SNP based association testing and network-based functional enrichment to predict nine candidate loci for Bphse including Atp2b2, Atg7, Pparg, Syn2, Ift122, Raf1, Mkrn2, Timp4 and Gt(ROSA)26Sor. Overall, these studies demonstrate the power of integrating genetic and functional methods in humans and animal models to predict highly plausible loci underlying QTL/GWAS data.