Ribonucleoprotein complexes (RNPs) are involved in many essential cellular processes, of which the most prominent examples include the ribosome that functions in protein translation and the spliceosome which catalyzes pre-mRNA splicing. The biogenesis of RNPs often involves complex and elaborate pathways involving post-translational modifications, transit into specific cellular domains, and several auxiliary factors including assembly chaperones. One of the best-studied examples of such a chaperone is the survival motor neuron (SMN) protein, the disease gene in spinal muscular atrophy (SMA). SMN is part of a macromolecular protein complex and catalyzes the assembly of a heptameric core of Sm proteins onto small nuclear RNAs (snRNAs) to form spliceosomal snRNPs required for RNA splicing. The Sm and Sm-like (LSm) proteins are an evolutionarily conserved family of proteins that exhibit the propensity to form diverse heteromeric complexes with unique RNA-binding characteristics. The Sm/LSm proteins are thought to function as RNA chaperones whose association with their target RNAs plays a critical role in the maturation, transport, and stability of the resulting RNPs as well as modulation of RNA-RNA and RNA-protein interactions that are critical for RNP function. Sm/LSm containing RNPs have been shown to function in a variety of cellular pathways in addition to pre-mRNA splicing, including histone mRNA 3' end formation and mRNA decay. Interestingly, in addition to its direct binding to Sm proteins, SMN has been shown to associate in vitro with members of the LSm family as well as other RNA binding proteins, implicating the SMN complex in the biology of other cellular RNPs. The discovery of the full spectrum of RNPs that are dependent on SMN activity has important implications not only for our understanding of fundamental aspects of post-transcriptional gene regulation but also for SMA pathogenesis. To pursue this line of investigation, in this dissertation, I explore the hypothesis that SMN plays a general role in RNP assembly that extends to novel RNAs that function in diverse cellular pathways. First, I report the identification of a RNA polymerase III transcript of unknown function to be a novel cell type-specific RNA target of SMN function in ribonucleoprotein assembly. Second, I explore the role of SMN in the biology of the nuclear LSm2-8 complex active in splicing and the cytoplasmic LSm1-7 complex involved in mRNA decay. Finally, to facilitate the discovery of cellular pathways linked to SMN biology, I describe a novel cell-based model system for the phenotypic screening of genetic and pharmacological modifiers of SMN expression and function. Together, my studies significantly expand the repertoire of cellular RNAs that SMN is known to target and provide a unique platform for the identification of novel SMN-dependent cellular pathways, which have relevance for understanding RNA regulation and disease mechanisms and may help in the development of therapeutic approaches to SMA.

RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3' end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3' end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation. We further provide evidence that Thr 4 functions in histone mRNA 3' end formation (presented mostly in chapter 2 of this thesis). Chapter 3 mainly describes the studies regarding Ser 2, Ser 5, and Ser 7. Based on the DT40-Rpb1 cell line, I created stable cell lines expressing an Rpb1 carrying a CTD with Ser 2, Ser 5, or Ser 7 mutated to alanine, and investigated the phenotypes of these cells. We found that cells expressing an Rpb1 with S2A or S5A mutation were defective in transcription and RNA processing. Contrary to previous findings, we found Ser 7 was not involved in snRNA expression and 3' end processing. In fact, no phenotypes associated with Ser 7 mutation were detected by our measurements. Extending previous Thr 4 studies, we showed in vitro and in vivo that Fcp1 dephosphorylated Thr 4. Finally, Chapter 4 describes what we have found the functions of CTD Tyr 1. Using the DT40-Rpb1 cells, I created stable cell lines expressing an Rpb1 with all Tyr residues mutated to phenylalanine (Phe). We found these cells were inviable, and the mutant Rpb1-Y1F was degraded to a CTD-less protein. Interestingly, the instability of Rpb1-Y1F was restored by reintroduction of one Tyr residue at the last heptad repeat. Further analysis provided evidence showing the involvement of Tyr phosphorylation in preventing Rpb1 from degradation by the 20S proteasome. Next, using ChIP assay, we showed Tyr phosphorylation was detected mostly at promoters, indicating a function of Tyr phosphorylation in transcription initiation. Indeed, transcription initiation defects were uncovered by assessing the recruitment of general transcription factors in cells with Y1F mutation. Extending this, we found an accumulation of upstream antisense RNAs in about one hundred reference genes by RNA-Seq analysis.

Immediate Early Genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP Responsive Element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.

Recent advances in the field of axon guidance have revealed complex transcription factor codes that regulate neuronal subtype identity and their corresponding axon projections. Retinal axon divergence at the optic chiasm midline is key to the establishment of binocular vision in higher vertebrates. In the visual system of binocular animals, the ipsilaterally and contralaterally projecting retinal ganglion cells are distinguished by the laterality of their axonal projections. Specific axon guidance receptors and their ligands are expressed in retinal ganglion cells (RGCs) and at the chiasm, tightly regulating the development of the ipsilateral (uncrossed) and contralateral (crossed) retinal projections. Though many factors are known, their dysfunction leads to only partial misrouting of RGC axons. Moreover, the complex transcription factor codes that regulate RGC subtype identity are only beginning to be uncovered. Numerous gaps remain in our understanding of how these guidance molecules are transcriptionally regulated and how they are induced by the patterning genes that set up the different domains in which these RGC subtypes reside. An even more elusive question within the field is how the ipsilateral and contralateral RGC subpopulations acquire their different cell fates. In this thesis, I present my work on dissecting out the molecular signatures of the ipsilateral and contralateral RGC populations during embryonic development through gene profiling followed by the functional characterization of one candidate from this screen. In Chapter 2, I developed a cell purification method based on retrograde labeling of these two cell populations from their divergent axonal projections followed by cell sorting. This method can be used in studies requiring purified populations of embryonic RGCs. In Chapter 3, I conducted a microarray screen of purified ipsilateral and contralateral RGCs using the above method. Through subsequent validation of the in vivo expression patterns of select candidates, I identified a number of genes that are differentially expressed in ipsilateral and contralateral RGCs. Subsequent functional characterization of these genes has the potential to uncover novel mechanisms for regulating axon guidance, cell differentiation, fate specification, and other regulatory pathways in ipsilateral and contralateral RGC development and function. The results of this screen also revealed that ipsilateral and contralateral RGC may have distinct developmental origins and utilize different strategies for differentiation. In Chapter 4, I demonstrate a novel role for cyclin D2, one of the above candidates, in the production of ipsilateral RGCs. The G1-active cyclin D2 is highly expressed in the ventral peripheral retina preceding and coincident with the developmental window of ipsilateral RGC genesis. I further found that ipsilateral RGC production is disrupted in the cyclin D2 null mouse. The expression of cyclin D2 in a distinct proliferative zone that has evolutionary significance in ipsilateral RGC production and its subtype-specific requirement during retinal development suggest that cyclin D2 may mark a distinct progenitor pool for ipsilateral RGCs. Thus, these studies offer an important advance in our understanding of neuronal subtype diversification within the retina.

The ability to make transgenic animals has been a great tool for biologists to study living organisms. In C. elegans, the way transgenes are generated makes them problematic in many circumstances, and there is no single, simple, reliable approach that circumvents all of the problems with current methods of introducing transgenes into C. elegans. In Chapter 2, I discuss my attempt to develop a transgenic system in C. elegans using the bacteriophage phiC31 integrase system. I show evidence that phiC31 integrase is active in C. elegans somatic tissue. I have successfully integrated a transgene into the C. elegans genome in single-copy using phiC31 dependent recombination-mediated cassette exchange. However, attempts to repeat phiC31-mediated integration has been unsuccessful. In Chapter 3, I use genetic analysis to test many genes that were reported to be associated with the gamma-secretase complex in a mammalian tissue culture system. The gamma-secretase complex is an important component in the Notch signaling pathway. Not only is the gamma-secretase complex essential in the Notch pathway, it is also implicated in the pathology of familial Alzeheimer's disease (FAD). As gamma-secretase complex components show a Notch loss-of-function phenotype in C. elegans, a reverse genetic approach, using genes encoding proteins that associate with Presenilin was used to identify putative new Notch modulators. Several genes were identified that suppress a glp-1(gf) allele and one gene that suppress a gfp-1(lf) allele. These genes are unlikely to be core components of the Notch signaling pathway.

Cell motility is an essential process that depends on a coherent, cross-linked cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. In culture, a common feature of cells is the coherent movement of the endoplasmic reticulum and membranous organelles toward the periphery during substrate adhesion and spreading. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB-/- mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that leads to increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA-/- MEFs, but not FlnB-/- MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus I suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions. I also report that treatment with the calpain inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]- L-norleucine (ALLN) restores endoplasmic spreading and focal adhesion (FA) maturation in the absence of Fln. Further, expression of calpain-uncleavable talin, but not full-length talin, also rescues endoplasmic spreading in Fln-depleted cells and indicates a crucial role for stable, mature FAs in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, I also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a dominant-negative vimentin variant incapable of vIF polymerization exhibit deficient endoplasmic spreading as well as defects in FA maturation. ALLN treatment restores FA maturation despite the lack of vIFs, but does not restore endoplasmic spreading. Consistent with a role for vIFs in endoplasmic spreading, adhesive structures do not contain vIFs when the endoplasm does not spread. Fln-depleted cells also exhibit a microtubule-dependent mistargeting of vIFs. Thus, I propose a model in which cellular force generation and interaction of vIFs with mature FAs are required for endoplasmic spreading. Additionally, I discuss future lines of investigation concerning the role of FlnA in the endoplasmic spreading mechanism as well as mechanosensitive functions of FlnA. Finally, I speculate on a potential application of endoplasmic spreading deficiencies as hallmarks of metastatic breast cancer.

MYC (or c-myc) is a bona fide "cancer driver" oncogene that is deregulated in up to 70% of human tumors. In addition to its well-characterized role as a transcription factor that can directly promote tumorigenic growth and proliferation, MYC has transcription-independent functions in vital cellular processes including DNA replication and protein synthesis, contributing to its complex biology. MYC expression, activity, and stability are highly regulated through multiple mechanisms. MYC deregulation triggers genome instability and oncogene-induced DNA replication stress, which are thought to be critical in promoting cancer via mechanisms that are still unclear. Because regulated MYC activity is essential for normal cell viability and MYC is a difficult protein to target pharmacologically, targeting genes or pathways that are essential to survive MYC deregulation offer an attractive alternative as a means to combat tumor cells with MYC deregulation. To this end, we conducted a genome-wide synthetic lethal shRNA screen in MCF10A breast epithelial cells stably expressing an inducible MYCER transgene. We identified and validated FBXW7 as a high-confidence synthetic lethal (MYC-SL) candidate gene. FBXW7 is a component of an E3 ubiquitin ligase complex that degrades MYC. FBXW7 knockdown in MCF10A cells selectively induced cell death in MYC-deregulated cells compared to control. As expected, cellular MYC levels are stabilized when FBXW7 expression is attenuated. Notably, stabilization of MYC is more pronounced compared to other FBXW7 targets. FBXW7 knockdown with MYC deregulation results in cell cycle defects, as well as CDC45 accumulation on chromatin, suggesting DNA replication stress. Intriguingly, FBXW7 and MYC expression correlate most strongly in the luminal A-subtype of breast cancer associated with low to normal MYC expression. Together, our results suggest that knockdown of FBXW7 increases cellular MYC levels and promotes cell death possibly through accumulation of MYC-dependent genomic stress, and that FBXW7 inhibition may be selectively synthetic lethal with breast cancers that retain MYC-dependence. We also identified UVSSA and ERCC8, two genes involved in transcription-coupled repair (TCR), as MYC-SL candidates from our genome-wide screen. TCR is a DNA damage repair pathway associated with active RNA polymerase II-transcription complexes. We show that both UVSSA and ERCC8 knockdown confer increased lethality selectively in MYC-deregulated cells. This MYC-SL interaction is not exacerbated by exogenous UV irradiation, suggesting that TCR may be required for survival upon MYC deregulation independently of its role in UV damage repair. UVSSA knockdown with MYC deregulation results in cell cycle defects and CHK2 activation, suggesting genomic stress. Intriguingly, we observe that lethality associated with UVSSA down-regulation in cells expressing MYCER is alleviated by inhibiting transcription. This suggests that transcription-dependent aberrant genomic structures generated during MYC deregulation may require TCR for maintaining survival. Taken together, our results suggest that increased levels of transcription-dependent genomic stress may accumulate with MYC deregulation, and that TCR may have functions outside of repairing UV-induced damage in resolving these lesions or structures.

It is well established that the p53 tumor suppressor plays a crucial role in controlling cell proliferation and apoptosis upon various types of stress. There is increasing evidence showing that p53 is also critically involved in various non-canonical pathways, including metabolism, autophagy, senescence and aging. Through a ChIP-on-chip screen, we identified a novel p53 metabolic target, pantothenate kinase-1 (PANK1). PanK1 catalyzes the rate-limiting step for CoA synthesis, and therefore, controls intracellular CoA content; Pank1 knockout mice exhibit defect in β-oxidation and gluconeogenesis in the liver after starvation due to insufficient CoA levels. We demonstrated that PANK1 gene is a direct transcriptional target of p53. Although DNA damage-induced p53 upregulates PanK1 expression, depletion of PanK1 expression does not affect p53-dependent growth arrest or apoptosis. Interestingly, upon glucose starvation, PanK1 expression is significantly reduced in HCT116 p53 (-/-) but not in HCT116 p53 (+/+) cells, suggesting that p53 is required to maintain PanK1 expression under metabolic stress conditions. Moreover, by using p53-mutant mice, we observed that PanK activity and CoA levels are lower in livers of p53-null mice than that of wild-type mice upon starvation. Similar to the case in Pank1 knockout mice, β-oxidation and gluconeogenesis are impaired in p53-null mice. Together, our findings show that p53 is critical in regulating energy homeostasis through transcriptional control of PANK1. Our study on PANK1 led us to the question of how p53 can differentially regulate a diverse array of downstream targets in a context-dependent manner. Studies have shown that p53 acetylation at K120 and K164 lysine residues contribute to p53-mediated apoptosis and growth arrest functions, which was further supported by the 3KR mouse model (K117/161/162R) that mirrors the K120/164R mutations in human p53. These studies also suggest that a potentially large number of p53 targets can still be regulated by p53 in the absence of K120/164 acetylation (K117/161/162R in mouse). To investigate whether additional modifications of p53 can further contribute to promoter-specific transactivation, we conducted a screen using mass spectrometry and identified a novel acetylation site at K101. Our data demonstrated that K101 in human p53, as well as the homologous K98 lysine residue in mouse p53, can be acetylated by acetyltransferase CBP. Acetylation at this novel site does not contribute to p53 stability or DNA-binding capabilities. Ablation of K98 acetylation in mouse p53 alone does not affect the transcriptional activity of p53. However, simultaneous loss of K98 acetylation with the previously characterized K117/161/162 acetylations (4KR98 p53) significantly abrogates p53-mediated activation of TIGAR and MDM2 genes. The 3KR mouse model, although cannot elicit canonical p53-mediated apoptotic and cell cycle arrest responses, still retains the ability to suppress tumor formation. We, therefore, investigated whether other non-canonical targets of p53 could potentially mediate tumor suppression. By RNA-seq profiling of gene expression in cells expressing 3KR p53, we identified TNFRSF14 (tumor necrosis factor receptor superfamily, member 14) as a novel p53 target. The TNFRSF14 receptor has been shown to be frequently mutated in follicular lymphoma and diffuse large B cell lymphoma, and stimulation by its ligand LIGHT leads to cell death in many cancer cells. We report that TNFRSF14 is a novel p53 target that can be activated by 3KR p53. Interestingly, transactivation of TNFRSF14 is defective by 4KR98 p53. Furthermore, LIGHT ligand stimulates cell death in TNFRSF14-expressing cells and cells expressing 3KR p53, but not those expressing 4KR98 p53. Altogether, our findings in these studies underscore the extensive scope of p53 functions and provide new insights into the versatility of non-canonical pathways. Not only does p53 mediate tumor suppression through both canonical and non-canonical downstream effectors, p53 can also contribute to cellular homeostasis and energy balance.

The development of insulin resistance (IR) in the liver is one of the key pathophysiologic events in the development of type 2 diabetes mellitus, but most patients do not become uniformly resistant to the hepatic actions of insulin. Although insulin loses its ability to blunt glucose production, it largely retains its capacity to drive lipogenesis. This "selective IR" results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. In this thesis, we take two approaches to better understand the mechanisms underlying selective IR. First, the compensatory chronic hyperinsulinemia (CHI) of insulin resistance downregulates levels of the insulin receptor (InsR). We have therefore modeled CHI in primary hepatocytes to demonstrate that the reduction in InsR number results in insufficient signaling capacity to halt glucose production while still leaving enough residual signaling capacity to promote lipogenesis. That is, the two processes are inherently differentially sensitive to insulin. Second, we hypothesize that FoxO1, a key insulin-inhibited transcription factor, coordinately regulates both hepatic glucose and lipid homeostasis. We have developed a transgenic mouse model heterozygous for a knocked-in allele of DNA binding-deficient FoxO1 and have proceeded to dissect the mechanisms by which FoxO1 differentially regulates glucose and lipid handling. We found that while the former requires FoxO1 to bind to its consensus sequences in target-gene promoters, the latter proceeds via a co-regulatory action of FoxO1. Taken together, these findings reveal novel connections between the glucose and lipid "arms" of the insulin-signaling pathway and how they may go awry in the run-up to diabetes.

Prostate cancer is a common malignancy with nearly one million annual diagnoses worldwide. Among a subset of patients, primary disease eventually progresses to disseminated castration resistant prostate cancer (CRPC). In recent years, treatment modalities that improve survival in CRPC have emerged including taxane chemotherapy and second generation androgen signaling inhibitors, among others. Indeed, today the first line chemotherapeutic docetaxel as well as the second line agent cabazitaxel are mainstays of treatment. However, CRPC inexorably progresses to a chemotherapy resistant state that ultimately precedes lethality. Elucidating the molecular determinants of aggressiveness in chemotherapy resistant CRPC may therefore stimulate new therapeutic strategies that improve clinical outcomes. We used laboratory models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Whole genome expression profiling, clinical validation and genetic screening approaches revealed that GATA2 regulates a signature of cancer progression associated genes. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a significant mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this regulatory axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in chemotherapy resistant prostate cancer and identify a therapeutic opportunity in this challenging disease.