The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

The ground state naive pluripotency is established in the epiblast of the blastocyst and can be captured by culturing mouse embryonic stem cells (mESCs) with MEK and GSK3 inhibitors (2i). The transcription network that maintains pluripotency has been extensively studied with the indispensable core factors being Oct4, Sox2 and Nanog, together with other ancillary factors reinforcing the network. However, how this network is dissolved at the onset of differentiation is still not fully understood. To identify genes required for differentiation in an unbiased fashion, I conducted a genome-wide CRISPR-Cas9-mediated screen in Rex1GFPd2 mESCs. This cell line expresses GFP specifically in the naive state and rapidly down-regulate upon differentiation. I differentiated mutagenised mESCs for two days and sorted mutants that kept higher GFP expression. gRNA representation was subsequently analysed by sequencing. I identified 563 and 8 genes whose mutants showed delayed and accelerated differentiation, respectively, at a false discovery rate (FDR) cutoff of 10%. The majority of the previously known genes were identified in my screen, suggesting faithful representation of genes regulating differentiation. Detailed screening result analysis revealed a comprehensive picture of pathways involved in the dissolution of naive pluripotency. Amongst the genes identified are 19 mTORC1 regulators and components of the mTORC2 complex. Deficiency in the TSC and GATOR complexes resulted in mTORC1 upregulation in consistent with previous studies. However, they showed opposite phenotype during ESC differentiation: TSC complex knockout cells showed delayed differentiation, whereas GATOR1 deficiency accelerated differentiation I found that the pattern of GSK3b phosphorylation is highly correlated with differentiation phenotype. I conclude that mTORC1 is involved in pluripotency maintenance and differentiation through cross-talk with the Wnt signalling pathway. My screen has demonstrated the power of CRISPR-Cas9-mediated screen and provided further insights in biological pathways involved in regulating differentiation. It would be interesting to explore the remaining unstudied genes for better understanding of the mechanisms underlying mESC differentiation.

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases in zebrafish genome. On performing the genetic screen I identified 2 novel genes, hepatocytes growth factor like and prostasin involved in Factor VII activation.

Lipid droplet (LD) autophagy (lipophagy) is a recently discovered selective form of autophagy and is a pathway for LD catabolism through the lysosome or vacuole. Therefore, lipophagy has therapeutic potential in the treatment of a variety of lipid related diseases in which increased cellular LDs are associated with pathophysiologies, such as obesity or atherosclerosis. This ubiquitous process has been an ongoing area of research within the budding yeast, Saccharomyces cerevisiae. However, there remains a need to better understand the regulators of this process. I have developed and validated a lipophagy library in yeast for the assessment of novel genetic regulators of stationary phase induced lipophagy. Through the screening of my library for roles in lipophagy I have identified many genetic regulators of lipophagy which include CUE1, UBC7, LHS1, HSP31, PLN1, TFS1, LAM6, OSH3, OSH4 and OSH7, among others. My screen highlights the power of this library to identify lipophagy regulators in S. cerevisiae, which can be utilised in the future to further the understanding of lipophagy.

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. The mechanism by which these DA neurons die is still unclear and under investigation. Although mostly idiopathic, about 10% of PD cases have shown familial inheritance. Mutations in leucine-rich repeat kinase 2 (LRRK2), a large multi-domain protein with unknown physiological and pathological roles, have been linked to PD cases of autosomal dominant inheritance. A PD Drosophilamelanogaster model over expressing the human LRRK2(I2020T) kinase mutant using the GAL4/UAS system has shown a loss of DA neurons and locomotor deficiency. Additionally, ectopic overexpression of human LRRK2 in the eye caused a damaged eye phenotype characterized by roughness of the surface, loss of pigmentation and presence of black lesions (Venderova Ket. al., 2009). The presence of this identifiable eye phenotype has allowed us to perform a suppressor/enhancer screen to identify possible genetic interactors of LRRK2. The LRRK2(I2020T) transgenic flies were crossed with genomic deficiency lines and the eye phenotype screened for either suppression or enhancement. Twenty-two genes, which are implicated in a variety of biological processes, have been identified thus far. Fourteen of these 22 interacting genes were assessed in the DA neurons of the D.melanogaster model. This functional screen is a rapid method to provide us with potential genetic interactions between LRRK2 and other genes, which will in turn, aid in elucidating the functional role of LRRK2 in PD pathology.

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. The mechanism by which these DA neurons die is still unclear and under investigation. Although mostly idiopathic, about 10% of PD cases have shown familial inheritance. Mutations in leucine-rich repeat kinase 2 (LRRK2), a large multi-domain protein with unknown physiological and pathological roles, have been linked to PD cases of autosomal dominant inheritance. A PD Drosophilamelanogaster model over expressing the human LRRK2(I2020T) kinase mutant using the GAL4/UAS system has shown a loss of DA neurons and locomotor deficiency. Additionally, ectopic overexpression of human LRRK2 in the eye caused a damaged eye phenotype characterized by roughness of the surface, loss of pigmentation and presence of black lesions (Venderova Ket. al., 2009). The presence of this identifiable eye phenotype has allowed us to perform a suppressor/enhancer screen to identify possible genetic interactors of LRRK2. The LRRK2(I2020T) transgenic flies were crossed with genomic deficiency lines and the eye phenotype screened for either suppression or enhancement. Twenty-two genes, which are implicated in a variety of biological processes, have been identified thus far. Fourteen of these 22 interacting genes were assessed in the DA neurons of the D.melanogaster model. This functional screen is a rapid method to provide us with potential genetic interactions between LRRK2 and other genes, which will in turn, aid in elucidating the functional role of LRRK2 in PD pathology.

Thesis advisor: Marc A.T. Muskavitch / The Notch pathway is an evolutionarily conserved mechanism of intercellular signaling that plays a central role in the development of metazoans. Here I summarize two genetic screens that utilize a rough eye phenotype created by Delta overexpression in the Drosophila eye to identify modulators of Notch pathway signaling activity. Among the many "hits" obtained from both screens, I have mapped to the Not1 gene a single complementation group that exhibits strong genetic interactions with Notch pathway mutants. NOT1 is a component of the CCR4-NOT complex, a global regulator of gene expression that exerts its effects through a variety of mechanisms, including mRNA deadenylation and direct transcriptional repression. I have conducted a series of genetic and molecular experiments in an effort to obtain more insight into the relationship between the CCR4-NOT complex and the Notch pathway. Both Not1 EMS mutations and RNAi-mediated knockdown of NOT1 expression produce phenotypes that mimic those of Notch loss-of-function pathway mutants. Knockdown of NOT1 in the developing bristle organ disrupts Notch-mediated inhibition of neuronal specification, resulting in supernumerary neurons and aberrant sheath cell specification. Knockdown of NOT1 within the developing wing margin disrupts expression of the Notch target genes Cut and Wingless, as well as the Notch ligand Delta. Phenotypic rescue experiments imply that Not1 functions downstream of Notch signal activation and acts directly on Notch target gene expression. These results suggest that NOT1 is required for Notch signal transmission in certain developmental contexts and implicate the CCR4-NOT complex as a positive regulator of the Notch pathway. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Meiotic recombination promotes genetic variation by reciprocal exchange of genetic material producing novel allelic combinations that influence important agronomic traits in crop plants. Therefore, harnessing meiotic recombination has the potential to accelerate crop improvement via classical breeding. Numerous genes involved in crossover formation have been identified in model systems. For example, SPO11 mediates generation of meiotic DNA double-strand breaks (DSBs) across all eukaryotes, which may be repaired as crossovers. However, downstream regulators of recombination remain to be identified, including those with species-specific roles. To isolate crossover frequency modifiers I performed a high-throughput forward genetic screen using EMS mutagenesis of Arabidopsis carrying a fluorescent crossover reporter line called 420. The primary screen isolated nine mutants from ~3,000 scored individuals that showed significantly higher (high crossover rate, hcr) or lower (low crossover rate, lcr) crossover frequency, including a new fancm allele. Four mutants (hcr1, hcr2, hcr3 and lcr1) were mapped by sequencing and candidate genes identified. The hcr1 mutation was confirmed as being located within the PROTEIN PHOSPHATASE X-1 (PPX-1) gene, using isolation of an independent allele and complementation studies. Similarly, the lcr1 mutation was confirmed to be within the gene TBP-ASSOCIATED FACTOR 4B (TAF4B). Using immunocytological staining I observed that hcr1 did not show changes in DSB-associated foci (RAD51), but it did show a significant increase in crossover-associated MLH1 foci. The hcr1 mutation increases crossovers mainly in the sub-telomeric chromosome regions, which remain sensitive to crossover interference. Also the genetic interaction between the hcr1 and fancm mutations is additive. These results support a model where PPX- 1 acts to limit recombination via the Class I interfering CO pathway, downstream of DSB formation. In summary, this genetic screen has led to discovery of novel genes that regulate meiotic recombination and their functional characterization may find utility in crop breeding programs.

Inorganic phosphate \((P_i)\) is an essential nutrient whose intracellular levels are maintained by the PHO pathway in Saccharomyces cerevisiae. \(P_i\) limitation triggers upregulation of the PHO genes whose gene products primarily function to counterbalance the \(P_i\) deficiency. Despite a growing catalogue of genes that are involved in signaling of the PHO pathway, little is known about how cells actually sense \(P_i\) limitation. To better characterize the \(P_i\) sensing mechanism, I exploited two comprehensive and orthogonal approaches: 1) genome-wide genetic screening to identify novel genes involved in signaling \(P_i\) limitation through the PHO pathway and characterization of genetic interactions among these genes and 2) liquid chromatography /mass spectrometry (LC/MS)-based metabolic profiling to characterize the metabolomic response to changes in \(P_i\) availability. In genome-wide screening, I found that the aah1 mutant constitutively activated the PHO pathway and showed that AAH1 is involved in regulating PHO pathway activity. Moreover, I identified several novel genetic interactions of genes involved in inositol polyphosphate metabolism with those involved in purine metabolism and mitochondrial fatty acid biosynthesis.Through metabolomic profiling, I showed that all adenine nucleotides were downregulated in the constitutively induced ado1, adk1, and aah1 mutants in high \(P_i\) as well as in the wild type strain in low \(P_i\). These observations led to the hypothesis that downregulation of adenine nucleotides triggers activation of the PHO pathway. However, I find that decreases in adenine nucleotides appear to be the consequence of downregulation of glycolysis and of the pentose phosphate pathway rather than an activation signal for the PHO pathway.Among all the detected metabolites, S-adenosyl-L-homocysteine (SAH) responded the most quickly and significantly to changes in \(P_i\) concentration. It was known that SAH is an inhibitor of de novo synthesis of phosphatidylcholine (PC). I showed that overall PC levels were downregulated in low \(P_i\), suggesting that phospholipid metabolism is downregulated in low \(P_i\) conditions. Furthermore, I observed that exogenous SAH induces activation of the PHO pathway in high \(P_i\) implying a possible role of SAH as an initiating activation signal of the PHO pathway. / Chemistry and Chemical Biology