The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.

The Transforming Growth Factor beta (TGFb) superfamily of ligands are highly versatile, functioning throughout development and in adult organisms as diverse as worms and humans to regulate a myriad of biological activities. TGFb family members signal through their cognate serine/threonine kinase receptors to mediate the phosphorylation and activation of receptor-regulated Smads (R-Smads), that then complex with the common Smad (co-Smad/Smad4) to transduce TGFb signals from the membrane into the nucleus. This thesis recounts the first identification of a mammalian Smad-interacting transcription factor, Foxh1. Investigation of the Smad/Foxh1 DNA-binding complex, which mediates TGFb-dependent regulation of transcription from a Gsc enhancer, determined that both Smad and Foxh1 binding sites are required. These studies also defined the first known biological difference between the highly related R-Smads, Smad2 and Smad3. Specifically, it was shown that while both can similarly participate in Smad/Foxh1 DNA-binding complexes, Smad2 activates and Smad3 represses Foxh1-mediated TGFb-dependent transcription. A detailed analysis of the Gsc enhancer element subsequently defined the sequence req irements for a functional Smad/Foxh1 enhancer (SFE). This information was utilized to direct in silico, genome wide searches for genes harbouring evolutionarily conserved SFEs, which successfully expanded the repertoire of Smad/Foxh1 targets. Analysis of these targets revealed novel roles for Smad/Foxh1 signalling in forebrain development and retinoic acid production. Finally, the importance of Foxh1 to heart development was examined. The interaction between Foxh1 and the heart specific factor Nkx2-5 was characterized with respect to TGFb-dependent regulation of Mef2c expression via a compound Foxh/Nkx2-5 enhancer (FNE). Genome-wide searches for similar FNEs identified many potential Foxh1/Nkx2-5 targets, further analysis of which will provide greater insights into how Foxh1 functions in heart development. In summary, the work presented herein expands our understanding of the role of TGFb in development through the identification and characterization of Foxh1 and its genomic targets downstream of TGFb signalling.

Genetic and Molecular / Neurospora Duplications

My research project involved dissecting cis-acting promoter elements and attempting to identify binding proteins that regulate the expression and mediatc the DNA damage response of the yeast genes MAG1 and DDI1. MAG1 encodes a 3-methyladenine (3MeA) DNA glycosylase and protects cells against killing by MMS-induced DNA replication blocks (Chen et al., 1989 Proc. Natl. Acad. Sci. USA 86: 7961-7965). DDI1 was recently identified as a gene upstream of MAG1 and was inducible by DNA damaging agents (Xiao and Fontaine, unpublished). MAG1 and DDI1 are arranged in a head-to-head configuration and are transcribed divergently. These two genes are closely linked, with the fust ATG's of the two open reading frames being separated by 282 base pairs. The transcription of MAG1 is repressed by a URS (upstream repressing site) element and stimulated in response to DNA damage by a putative UAS (upstream activating site) (Xiao et al., 1993 Mol. Cell. Biol. 13: 7213-7221). The 46 bp sequence containing the putative UAS of MAG1 (UAS_MAG1) is located within the coding region of DDI1. The transcriptional and the translational starts of MAG1 and DDI1 were determined. My results showed that the two genes are indeed closely linked to each other. The UAS_MAG1 was identified within the protein coding region of DDI1. This is the first demonstration in yeast that a transcriptional regulatory element for one gene can be located within the protein coding region of another gene. Since MAG1 and DDI1 are co-induced by DNA damage in a similar manner, it was hypothesized that the two genes share one or more regulatory elements. A direct repeat sequence (DR) within the intergenic region between MACI andDDII was identified as a bi-directional transcriptional regulatory element for the expression of these two genes. Sequences similar to the direct repeat were also found in the promoters of several DNA repair, or DNA metabolism genes from S. cerevisiae. This is the first report of a situation where two DNA damage-inducible genes are co-ordinately regulated by physically sharing a regulatory element. MAG1 is one of the most extensively studied yeast DNA damage responsive genes. Previous studies (Xiao et al., 1993 Mol. Cell. Biol. 13: 7213-7221) have focused primarily on the mechanism of repression of MAG1 expression. The UAS_MAG1 element was not well defined and its role in the induction of MAG1 following DNA damage was not established. This work defined the UAS_MAG1 as a 24 bp sequence required for the expression of MAG1, but not DDI1. An UAS_MAG1-binding protein(s) was identified. The UAS_MAG1-binding protein(s) is probably a transcription activator that regulates the expression of MAG1. (Abstract shortened by UMI.)

<p>Chromatin is composed of DNA, histones, and other proteins and contributes to DNA packaging, controlling gene expression and DNA replication. This work focuses on the contributions of histones H3 and H4 to gene regulation in the yeast <italic>Saccharomyces cerevisiae</italic>. I identified a region of the nucleosome that is critical for three types of long-range transcriptional silencing but not for local repression mediated by some of the same proteins. </p><p>In <italic>S. cerevisiae</italic>, the Sir complex performs long range silencing of the mating type loci, while the promoter specific Sum1 complex represses mid-sporulation genes. Interestingly, the <italic>SUM1-1</italic> mutation changes the Sum1 repression complex into a silencing complex capable of long range spreading. Sum1-1 provides a good model to distinguish between properties of nucleosomes important for long-range silencing (common to Sum1-1 and Sir silencing), and specific interactions nucleosomes might make with the Sum1 complex (common to Sum1 and Sum1-1 complexes). Interactions between nucleosomes and silencing proteins are critical to Sir silencing, and the spreading ability of Sum1-1p suggests that a component of the Sum1-1 complex may also interact with nucleosomes. Since the Sum1-1 and Sum1 complex components are shared, histone contacts may also contribute to wild type Sum1 repression.</p><p>I investigated the contributions of histones H3 and H4 to Sum1-1 silencing and Sum1 repression using a genetic screen. Interestingly, I found histone mutations that disrupt Sum1-1 silencing and cluster in the LRS/H4 region of the nucleosome, which was previously identified to disrupt silencing at the mating type loci, telomeres, and rDNA. Therefore, this region of the nucleosome is important to silencing mediated by three distinct complexes- Sir, RENT, and Sum1-1. The Sir3p bromo-adjacent homology (BAH) domain binds this region of the nucleosome to facilitate Sir spreading and silencing, and I tested Orc1p, a paralog of Sir3p, to determine if it makes similar contributions to Sum1-1 silencing. Using reporter mating assays and chromatin immunoprecipitation, I found that mutations and deletion of the BAH domain of Orc1p disrupt Sum1-1 silencing. These results suggest that Orc1p may interact with this region of the nucleosome and contribute to Sum1-1 silencing outside of recruitment.</p><p>Surprisingly, Sum1 repression was not disrupted by histone mutations. I conducted <italic>in vitro</italic> binding assays to identify a region in Sum1p that may interact with histones and account for the spreading ability of Sum1-1p. Consistent with results that histones do not contribute to Sum1 repression, I did not find evidence of Sum1p binding to histone peptides. Therefore, interactions with histones H3 and H4 are important to Sir and Sum1-1 silencing and not Sum1 repression. These interactions with histones may facilitate the formation of higher order chromatin structures necessary for long range silencing complexes. </p><p>I also identified mutations in the H3 tail that disrupt Sum1-1 silencing. Surprisingly, these mutations did not disrupt the enrichment of Sum1-1p. Similar observations have been made for Sir proteins in the absence of the H3 tail, and the H3 tail may contribute to chromatin compaction and silencing after the assembly of silencing proteins. Therefore, the Sir and Sum1-1 complexes may share several features that facilitate silencing. The use of the LRS/H4 region of the nucleosome may be a common interaction surface with silencing proteins, and the H3 tail may assist in the formation of a specialized chromatin structure. These interactions may also be utilized in the formation of heterochromatin in higher eukaryotes.</p> / Dissertation

<p><p>Hematopoiesis is the process in which billions of blood cells are produced on a daily basis, and is vital for sustaining life. This process is tightly regulated by a dynamic balance between hematopoietic stem cell (HSC) self-renewal and differentiation, and maintenance of this balance is of critical importance as dysregulation of HSCs can lead to hematopoietic deficiencies or malignancies such as leukemogenesis. While the signaling mechanisms that regulate HSC homeostasis and function are not well understood, our previous studies have identified a calcium/calmodulin (CaM)-dependent protein kinase, CaMKIV, that is intrinsically required for regulating normal proliferation and survival in HSCs. These findings suggest not only the importance of calcium-initiated pathways including CaMKIV-dependent signaling in hematopoietic cells, but also the potential for other calcium/CaM-dependent effector proteins such as other CaM-kinases to be involved in regulating HSCs and hematopoiesis.</p> </p><p><p>The first major section of this dissertation work presented herein was based on the usage of RNA interference (RNAi) technology to specifically deplete HSCs of growth factor erv1-like (Gfer), a gene whose expression appeared to be absent in CaMKIV null HSCs based on comparative microarray analysis with wild-type HSCs, and seemed a potential target of CaMKIV. We showed that depletion of Gfer in HSCs compromised their <i>in vivo</i> engraftment potential and triggered a hyper-proliferative response that led to their exhaustion. We further assessed Gfer-depleted HSCs by using microscopy techniques and found that these cells possessed significantly reduced levels of the cyclin-dependent kinase inhibitor (CDKI) p27^kip1. In contrast, ectopic over-expression of Gfer in HSCs resulted in significantly elevated total and nuclear p27^kip1. We next performed immunoprecipitation-immunoblot analyses to determine whether alteration of Gfer levels would affect p27^kip1's binding with its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), that would subsequently lead to its ubiquitination, and determined that depletion of Gfer resulted in enhanced binding of p27^kip1 to Jab1. Conversely, over-expression of Gfer resulted in its enhanced binding to Jab1 and inhibition of the Jab1-p27^kip1 interaction. Furthermore, normalization of p27^kip1 in Gfer-KD HSCs rescued their <i>in vitro</i> proliferation deficits. These results provide evidence for a novel Gfer-Jab1-p27^kip1 pathway present in HSCs that functions to restrict abnormal proliferation.</p> </p><p><p>The second major section of this dissertation work describes our studies of a CaMKIV kinase, CaMKK2, and its role in HSCs and hematopoietic development. These studies were largely based on the usage of mice genetically ablated for the <i>Camkk2</i> gene in the germline. Herein, we identified a role for CaMKK2 in the restriction of granulocytic fate commitment and differentiation of myeloid progenitor cells. We performed bone marrow transplantation studies and discovered that engraftment by <i>Camkk2^-/-</i> donor cells resulted in the increased production of mature granulocytes in the bone marrow and peripheral blood. Similarly, we used fluorescence activated cell sorting (FACS) to determine that <i>Camkk2^-/-</i> mice possessed elevated numbers of common myeloid progenitor cells, and exhibited an accelerated granulopoietic phenotype in the bone marrow. Expression of ectopic CaMKK2 in <i>Camkk2^-/-</i> common myeloid progenitors was sufficient to rescue aberrant granulocyte differentiation, and when over-expressed in 32Dcl3 cells was also sufficient to impede granulocyte differentiation in a kinase activity-dependent manner. Collectively, our results reveal a novel role for CaMKK2 as an inhibitor of granulocytic fate commitment and differentiation in early myeloid progenitors.</p></p><p><p>While our original intent was to identify and link a downstream target and upstream kinase to CaMKIV in HSCs, our results ultimately did not suggest that Gfer or CaMKK2 function in the same pathway in HSCs as discussed in the following chapters. Nonetheless, our findings represent a considerable advance in identifying and characterizing the functions of two novel regulators, Gfer and CaMKK2, that are important for HSC proliferation and the commitment and early differentiation steps of granulopoiesis, respectively.</p></p> / Dissertation

Membrane proteins function as important communication channels of the cell and its environment that aid in regulating the overall homeostasis of organisms. Understanding the pathways by which these proteins adopt their three-dimensional structures can provide us with key insights into their functions. Failure of a membrane protein to fold into its native structure can lead to disruption of their functions and cause diseases. Through an understanding of the folding mechanisms of membrane proteins it may be possible to identify avenues for the treatment of such diseases. Towards these goals, this thesis describes the biophysical characterization of denatured states of rhodopsin, a model system selected to study helical membrane protein folding. The first contribution of this thesis was to establish approaches that can be used to identify suitable conditions for studying membrane protein folding in vitro. This required screening different denaturing conditions to obtain maximum unfolding without causing aggregation of rhodopsin. 30% SDS and 3% SDS + 8 M urea were found to be the most suitable denaturing conditions. Next, structural features of largely unfolded states of rhodopsin under optimized denaturing conditions were systematically characterized focussing on three levels of structural resolution: global, local and site-specific. Global tertiary structure changes upon SDS denaturation were observed to correlate with SDS micellar structure changes and also hinted at formation of compact intermediate states. Local structural dynamics, probed by NMR spectroscopy, showed that the cytoplasmic domain is more flexible than extracellular and transmembrane domains taken together in spite of an overall increase in flexibility with denaturation. Mobility studies probing site-specific changes by EPR spectroscopy, showed that specific extracellular residues retain more rigidity than cytoplasmic residues in denatured states. These results indicate that the former domain is involved in more stable interactions forming a possible folding core like structure, the location of which correlates with that described by the long-range interaction model of folding. Finally, the importance of dynamics in understanding folding mechanisms of rhodopsin led us to contribute to the development of two novel methodologies: terahertz spectroscopy to detect global motions and 19F NMR using new monofluoro labels to quantify residue specific motions.