RNA helicases are enzymes that bind or remodel RNA and RNA-protein complexes. They are involved in numerous cellular functions including RNA metabolism, transcription, translation, and mRNA decay. Defects in helicase function or disregulated expression, can cause diseases. DEAD-box (DDX) RNA helicases are highly conserved and are known to be involved in muscle development and disease, by interacting with muscle specific transcription factors and genes in humans. The Gupta Lab is currently studying zebrafish (an established and reliable model to study muscle diseases) with a mutation in ddx27. These fish have impaired motility behavior, skeletal muscle hypotrophy, and extensive central nucleation. They also exhibit disorganization of skeletal muscle, abnormalities in the brain, eyes, and heart. These phenotypes mimic the abnormalities seen in human myotonic dystrophy. It is known that ddx27 is necessary for regulation of rRNA maturation. Recent studies have pointed to it’s non-ribosomal roles of nucleolar genes. IGHMBP2, another RNA helicase, is known to result in spinal muscular atrophy (SMARD1) or Charcot-Marie Tooth disease when mutated. We used zebrafish and patient myoblast cells to determine the role of ddx27 in myogenesis and diseases. As a basis for future studies, the 43 known human DDX genes were outlined for their functions. Immunofluorescence studies in ddx27 mutant zebrafish showed drastic skeletal muscle and nucleolar assembly defects with large numbers of cells with transcriptionally active euchromatin, suggesting altered gene regulation. In addition, IF with Pax7 (a marker for satellite cells) and MF20 (a marker for myosin heavy chain antibodies) showed a significant increase in the number of Pax7 positive cells that suggest perturbed satellite cell regulation. Nucleolar defects were also seen in cells isolated from myotonic dystrophy patients. While the cause of these defects is not known, the results lead us to believe that ddx27 may be involved in cell cycle regulation or apoptosis events. Finally, while this study also attempted to develop a zebrafish model of IGHMBP2 deficiency in order to study and develop therapies for SMARD1, a consistent phenotype was not observed and further work is required to characterize this model. More than one million Americans suffer from neuromuscular disorders, however many of these conditions have no known treatments. By studying the molecular pathways involved we can attempt to develop therapies for these diseases.

Nemaline Myopathy is a congenital disorder that is characterized by muscle weakness and limited mobility. Clinically, Nemaline Myopathy is highly heterogeneous, ranging from severe congenital forms with neonatal death to more mild childhood and adult onset forms. Mutations in 10 different genes have been linked to Nemaline Myopathy in human patients (ACTA1, NEB, TPM3, TPM2, CFL2, TNNT1, LMOD3, KBTBD13, KLHL40, and KLHL41). Approximately 75% of Nemaline Myopathy cases with a known genetic diagnosis are caused by mutations in structural proteins within the thin filaments in skeletal muscle. It is very difficult to develop therapies that target structural proteins and there are no effective, specific therapies for Nemaline Myopathy. Recent discovery of mutations in Kelch family genes, which are non-structural genes, provide an alternative pathway for developing targeted treatments for Nemaline Myopathy. Of particular interest in this study is the protein KLHL41. Mutation of KLHL41 has shown a clear genotype-phenotype relationship in Nemaline Myopathy with frameshift mutations resulting in severe forms and missense mutations resulting in typical congenital forms. KLHL41 is found primarily in striated muscle and plays a role in protein turnover through the ubiquitin-proteasome pathway. KLHL41 interacts with Cullin 3 and serves as a substrate adapter in the E3 ligase complex, the last step in ubiquitination of proteins targeted for degradation by proteasomes. Preliminary studies demonstrate that KLHL41 interacts with nebulin, a major structural protein in muscle. Mutations in the nebulin gene are implicated in 50% of Nemaline Myopathy cases. KLHL41 has also been shown to interact with POMP, a protein that facilitates the formation of the 20S proteasome subunit. The proteasome pathway has been shown to be impaired in Nemaline Myopathy patients, regardless of genetic mutation. A better understanding of how the proteasome pathway becomes disrupted in Nemaline Myopathy will contribute to the development of specific targeted therapies. This study sought to understand the significance of KLHL41-protein interactions on disease pathology in Nemaline Myopathy. Co-transfection of increasing amounts of KLHL41 with constant levels of POMP or a nebulin fragment showed a decrease in POMP and nebulin expression, suggesting that KLHL41 is a negative regulator of POMP and nebulin. Co-transfection and immunoprecipitation were also performed to confirm the direct interaction between KLHL41 and POMP or nebulin. However, this study could not re-confirm a direct physical interaction between KLHL41 and POMP or nebulin identified by previous Y2H and HTRF studies. As a substrate adapter in the E3 ligase complex, KLHL41 plays an important role in protein turnover and disruption of this pathway may contribute to the pathology of Nemaline Myopathy. Confirming the role of KLHL41 in the development of Nemaline Myopathy is critical to development of future therapeutic strategies.

Opioid addiction is a growing epidemic with no known genetic basis. Mice represent a valuable tool that can be used to better understand the genetic components of opioid addiction by studying opioid induced behaviors such as locomotor activity. The two closely related C57BL/6 substrains, C57BL/6J (B6J) and C57BL/6NJ (B6NJ), exhibit limited genetic diversity, yet display phenotypic differences when under the influence of oxycodone. Quantitative trait locus (QTL) mapping, a discovery based approach to identifying genomic regions underlying statistical variation in complex traits, was used to identify a locus on distal chromosome 1 for oxycodone-induced locomotor activity and withdrawal. F2 offspring from a cross utilizing these substrains that are homozygous B6J across the chromosome 1 QTL compared to offspring that are heterozygous display the same phenotypic differences as the parental strains, namely oxycodone-induced locomotor activity. F2 mice were selected based on distal chromosome 1 genotypes and backcrossed to parental strain C57BL/6J to fine map the QTL interval. Through family analysis, regions proximal to 167 Mb and distal to 187 Mb have been ruled out as containing the QTL mediating oxycodone-induced locomotor activity. Future studies should employ this same technique to fine map the QTL mediating oxycodone withdrawal in order to differentiate whether it is one locus or two loci controlling these oxycodone induced behaviors.

<p> In this dissertation I addressed the problem of SNP array bias when finding runs of ho- mozygosity. I demonstrated the pitfalls of using uninformed methods for finding runs of homozygosity and provide better alternatives, including a more reliable algorithm for identi- fying runs of homozygosity than the most commonly used program. I then provide a review of Ashkenazi population genetics. Next, I developed software to efficiently run millions of whole chromosome simulations, which is publicly available through GitHub, DockerHub, and on the CyVerse Discovery Environment. I applied my computational method to use Approximate Bayesian Computation to test models of Ashkenazi Jewish demographic his- tory. I found that the Ashkenazi Jews are comprised of genetically distinct subgroups from Eastern and Western Europe, as a result of massive population growth in the Eastern Ashkenazi Jews, but not in the Western Ashkenazi Jews. I further confirmed that the Ashkenazi Jews do not primarily originate from Khazaria. Finally, I created a correction for SNP array ascertainment bias in the median and total length of runs of homozygosity, and applied this correction to world-wide human populations. However, I found that ascertainment bias plays a minor role compared to SNP array bias in human populations. </p><p>

I have studied 26 children who have a de novo rearrangement of the acrocentric chromosomes in order to understand the formation of these aberrations. The families include '25 probands ascertained for Robertsonian-type translocations, 13 between nonhomologous chromosomes and 12 between homologs, and one rea(21;21)(q22;q22). The parental origins of the de novo rearrangements were determined in 26/26 families using QFQ and NOR variants and/or RFLP analyses. While there was no overall difference in the sex distribution of the parents of origin, there were more maternally derived nonhomologous ("true" Robertsonian) translocations (8 mat: 5 pat) and more paternally derived homologous rearrangements (4 mat: 9 pat). A role of the NOR in de novo formation of acrocentric rearrangements was suggested by a significantly higher incidence of dNOR variants in the parents in whom the rearrangements originated (11/26) as compared to their normal spouses (1/26) and a control population (5/50) (p<0.0001). The dNOR variant was found both in parents in whom de novo Robertsonian.translocations and homologous rearrangements had occurred. Additionally, both the parents in whom rearrangements originated and their spouses had significantly higher NOR scores than the controls. This suggests that higher NOR scores in the parents may have contributed to the survival of their offspring with de novo acrocentric rearrangements since these rearrangements generally resulted in the loss of two NORs. However, compensation in NOR scores or satellite associations was not evident in these probands. RFLP analysis of rearrangements between homologous chromosomes resulting in secondary trisomy in 8 cases suggested that these rearrangements were isochromosomes, derived from one parental chromosome. Four of the homologous rearrangements were dicentric suggesting that these rearrangements may have resulted from U-type exchanges in the NOR or short arm.

The term `oncogene addiction' has been used to describe the phenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions. In non-small cell lung cancer (NSCLC), this principle is perhaps best exemplified with the finding that epidermal growth factor receptor (EGFR) mutations predict response to EGFR-targeted therapies and thus represent a dependency in the subset of tumors harboring these alterations. Yet while EGFR-mutant tumors often respond dramatically to EGFR inhibition, nearly 30% of cases are refractory to therapy at the outset, and all responsive patients ultimately develop resistance to therapy. A deeper understanding of the genetic underpinnings of EGFR dependence, and of the mechanisms by which EGFR-mutant cells can overcome addiction to EGFR, may improve clinical outcomes.

The human genome's strongest influences on two common diseases, systemic lupus erythematosus (SLE) and schizophrenia, arise from genetic variation in the Human Leukocyte Antigen (HLA) locus. However, the genes and functional alleles driving these genetic relationships have remained unknown. We hypothesized that a complex, multi-allelic form of structural variation in the Complement component 4 (C4) gene, within the HLA locus, underlies these relationships. Loci that exist in many structural forms and vary widely in copy number have been difficult to analyze molecularly. As a result, we know little about their population genetic properties or their influence on phenotypes. In this work, we developed molecular and statistical methods to characterize such loci and to evaluate their contribution to phenotypes. Applying these methods to the C4 locus, we found that C4 segregates in four common and at least eleven low-frequency structural forms in human populations. Although there was only partial correlation between C4 structural variation and individual single nucleotide polymorphisms (SNPs), we developed an imputation approach to enable statistical prediction of C4 structural states from flanking SNP haplotypes. C4 structural variation associated to gene expression in lymphoblastoid cell lines and human brain tissue. Applying our imputation strategy to SLE and schizophrenia case-control cohorts totaling > 75,000 individuals, we found that structural variation in C4 contributes to risk of both phenotypes in a manner predicted by its effect on gene expression in relevant tissues, and with largely opposite directions of effect - alleles that were protective for schizophrenia increased risk for SLE, and vice versa. Leveraging a natural allelic series of C4 structural forms, we developed a novel form of association testing and showed that the association to C4 is unlikely to be caused by correlation with HLA SNPs. C4 was expressed in human neurons, whereas other upstream complement pathway genes were expressed primarily by microglia. Mice lacking C4 showed a deficit in synaptic pruning that was rescued by human C4. The methods developed in this thesis enable analysis of complex structural variation, and our results identify a novel form of genome variation as making a strong contribution to phenotypes.

The Drosophila morgue gene was identified as a regulator of programmed cell death and encodes a novel ubiquitination (Ub) protein. Morgue protein contains a distinct combination of functional domains including: a zinc finger, F box, and variant Ub E2 conjugase domain. This unique combination suggests that Morgue may influence protein ubiquitination and targeting to the 26S proteasome for degradation. Morgue has been shown to promote turnover of a conserved anti-apoptotic protein, DIAP1. In the first part of this study, I present a published paper that describes the isolation and initial analysis of morgue. In the second part of this thesis, I present a published review that discusses different models for Morgue function and describes the identification of a morgue orthologue in mosquito, Anopheles gambiae. This study compares the architecture of the two Morgue proteins, as the F box and conjugase domain within the two proteins are located in similar positions, and the F box and conjugase domains of AgMorgue exhibit 54% and 66% amino acid sequence identity to Morgue. Interestingly, the glycine substitution for the active site cysteine in AgMorgue is conserved. A zinc finger motif located at the NH2-terminus was also identified in both Morgue proteins. In the third part of this study, I performed additional P-element excision screens to generate specific loss-of-function morgue alleles. Several viable alleles were obtained; however, the mutants appeared weak and were shown to exhibit decreased locomotor capabilities in adult climbing assays. Analysis of mutant embryos using a number of cell-type specific markers revealed partially penetrant disruptions in the number, position, and morphology of specific neurons and glia in both the central and peripheral nervous system. Lastly, I describe strong genetic interactions between morgue mutants and mutations in the effete gene, which encodes a conserved Ub E2 conjugase that also influences DIAP1 levels and programmed cell death. Thus, in contrast to either morgue or effete mutants alone, animals homozygous for mutations in both these genes exhibit major disruptions in life cycle progression as they arrest in the third instar larval stage. These mutant larvae come to exhibit several morphological and anatomical abnormalities.

Patients with Parkinson’s disease (PD) lose their motor controls resulting form the selective loss of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc) and have the ubiquitin-positive cytoplasmic protein aggregation, Lewy Body (LB), in the surviving DA neurons. The studies with Parkin, an E3 ubiquitin ligase, suggest that Parkin is important in DA neuron survival and that a functionally overlapping protein(s) with Parkin, whose expression is absent in a SNpc region, protects DA neurons in brain except for those in midbrain. One candidate is Ariadne whose human homologue (HHARI) shares its binding partners with Parkin, and is heterogeneously expressed in the midbrain. Our data suggested that HHARI was able to interact with proteins Parkin interacts with including α-synuclein, a toxic substrate for Parkin, and that ectopic expression of human Parkin and HHARI partially rescued the muscle defects of Parkin null flies. In order to study pathological significance of their overlapping functions, we tried to use a human α-synuclein expressing Drosophila model for PD. Unfortunately, our analysis revealed that α-synuclein expression itself did not cause loss of DA neurons in the transgenic flies and neither increased sensitivity against environmental toxins such as rotenone. The contradictory findings may result from variation in methodology. Due to these problems in the animal model, we could not answer our question. In order to study protein aggregation-related diseases, we generated a new Drosophila model for a human degenerative disease, sporadic inclusion body myositis (s-IBM) disease. The patients suffer from severe muscle weakness. Many studies suggest that intracellular accumulation of human amyloid precursor protein (APP) and its proteolytic fragments, Aβs, is sufficient for s-IBM pathogenesis. In order to understand the molecular mechanism of IBM, we drove muscle specific expression of human APP in Drosophila. The APP expressing flies showed age-dependent behavioral defects resulting from muscle degeneration and abnormal mitochondria at early stages. Interestingly, the behavioral defects were affected by not only APP expression but also culturing conditions. These findings suggest that APP expression in flies’ muscle leads to increased sensitivity to environmental factors as well as muscle weakness.

Myb is a proto-oncogene that when mutated causes leukemias and lymphomas in birds and mammals. Vertebrates contain three representatives of the Myb gene family consisting of A-, B- and c-Myb, all of which encode DNA-binding factors that are important for the proper expression of genes. Several studies have described Myb’s primary function as a factor that upregulates transcription by binding to promoter regions, thus controlling the expression level of genes adjacent to these sequences. In flies, this regulation has been shown to be accomplished epigenetically such that Myb is only required after initial activation of a gene in order to potentiate this distinct transcriptional state. Here, we further characterize the potentiator role of Myb and show that its absence leads to a reduction in H3K4me3 along promoters and increase RNA polymerase pausing at promoters leading to decrease occupancy across gene bodies, resulting in downregulation of transcription. However, up to now, no other mechanisms have been proposed that account for the thousands of genes whose expression is altered in the absence of Myb, including those that appear to be “repressed” by Myb. Here we uncover a novel and critical role of Myb in demarcating and maintaining silent chromatin domains. We observe that Myb demarcates and stabilizes H3K27me3 domains associated with silent genomic regions, and in its absence, these domains become reduced in length and less enriched for this chromatin mark, promoting an enrichment of H3K4me3 and subsequent derepression of the genes within these domains. We also identify the nucleosome remodeling factor, NURF, to be genetically and physically interacting with Myb. We show that they work in concert to regulate a subset of tissue specific genes. More specifically, they are both needed for proper maintenance of H3K4me3 and RNA polymerase levels of active genes. Finally, we describe a novel function of Myb essential for silencing retrotransposable elements. Myb binds to both 5' and 3' end long terminal repeats (LTRs) and its absence leads to reduction of H3K9me3 levels along these repeats. As a result, derepression and ectopic transcription occurs in different tissues of Myb mutant animals such as brain, salivary glands and wing discs. We observed a significant increase in copy number of retrotransposons in all these tissues indicative of retrotransposition events. In conclusion, we find that genes whose expression levels change in the absence of Myb (~2,000 genes) are directly influenced or regulated by Myb, with virtually no genes showing altered expression through secondary consequences of aberrant transcription factor upregulation, thus providing strong evidence for a chromatin “buffering” effect in cells that helps prevent misprogramming.