Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

BAYESIAN STATISTICAL METHODS IN GENE-ENVIRONMENT AND GENE-GENE INTERACTION STUDIES

Complex diseases such as cancer result from multiple genetic changes and environmental exposures. Due to the rapid development of genotyping and sequencing technologies, we are now able to more accurately assess causal effects of many genetic and environmental factors. Genome-wide association studies have been able to localize many causal genetic variants predisposing to certain diseases. However, these studies only explain a small portion of variations in the heritability of diseases. More advanced statistical models are urgently needed to identify and characterize some additional genetic and environmental factors and their interactions, which will enable us to better understand the causes of complex diseases. In the past decade, thanks to the increasing computational capabilities and novel statistical developments, Bayesian methods have been widely applied in the genetics/genomics researches and demonstrating superiority over some regular approaches in certain research areas. Gene-environment and gene-gene interaction studies are among the areas where Bayesian methods may fully exert its functionalities and advantages. This dissertation focuses on developing new Bayesian statistical methods for data analysis with complex gene-environment and gene-gene interactions, as well as extending some existing methods for gene-environment interactions to other related areas. It includes three sections: (1) Deriving the Bayesian variable selection framework for the hierarchical gene-environment and gene-gene interactions; (2) Developing the Bayesian Natural and Orthogonal Interaction (NOIA) models for gene-environment interactions; and (3) extending the applications of two Bayesian statistical methods which were developed for gene-environment interaction studies, to other related types of studies such as adaptive borrowing historical data. We propose a Bayesian hierarchical mixture model framework that allows us to investigate the genetic and environmental effects, gene by gene interactions (epistasis) and gene by environment interactions in the same model. It is well known that, in many practical situations, there exists a natural hierarchical structure between the main effects and interactions in the linear model. Here we propose a model that incorporates this hierarchical structure into the Bayesian mixture model, such that the irrelevant interaction effects can be removed more efficiently, resulting in more robust, parsimonious and powerful models. We evaluate both of the 'strong hierarchical' and 'weak hierarchical' models, which specify that both or one of the main effects between interacting factors must be present for the interactions to be included in the model. The extensive simulation results show that the proposed strong and weak hierarchical mixture models control the proportion of false positive discoveries and yield a powerful approach to identify the predisposing main effects and interactions in the studies with complex gene-environment and gene-gene interactions. We also compare these two models with the 'independent' model that does not impose this hierarchical constraint and observe their superior performances in most of the considered situations. The proposed models are implemented in the real data analysis of gene and environment interactions in the cases of lung cancer and cutaneous melanoma case-control studies. The Bayesian statistical models enjoy the properties of being allowed to incorporate useful prior information in the modeling process. Moreover, the Bayesian mixture model outperforms the multivariate logistic model in terms of the performances on the parameter estimation and variable selection in most cases. Our proposed models hold the hierarchical constraints, that further improve the Bayesian mixture model by reducing the proportion of false positive findings among the identified interactions and successfully identifying the reported associations. This is practically appealing for the study of investigating the causal factors from a moderate number of candidate genetic and environmental factors along with a relatively large number of interactions. The natural and orthogonal interaction (NOIA) models of genetic effects have previously been developed to provide an analysis framework, by which the estimates of effects for a quantitative trait are statistically orthogonal regardless of the existence of Hardy-Weinberg Equilibrium (HWE) within loci. Ma et al. (2012) recently developed a NOIA model for the gene-environment interaction studies and have shown the advantages of using the model for detecting the true main effects and interactions, compared with the usual functional model. In this project, we propose a novel Bayesian statistical model that combines the Bayesian hierarchical mixture model with the NOIA statistical model and the usual functional model. The proposed Bayesian NOIA model demonstrates more power at detecting the non-null effects with higher marginal posterior probabilities. Also, we review two Bayesian statistical models (Bayesian empirical shrinkage-type estimator and Bayesian model averaging), which were developed for the gene-environment interaction studies. Inspired by these Bayesian models, we develop two novel statistical methods that are able to handle the related problems such as borrowing data from historical studies. The proposed methods are analogous to the methods for the gene-environment interactions on behalf of the success on balancing the statistical efficiency and bias in a unified model. By extensive simulation studies, we compare the operating characteristics of the proposed models with the existing models including the hierarchical meta-analysis model. The results show that the proposed approaches adaptively borrow the historical data in a data-driven way. These novel models may have a broad range of statistical applications in both of genetic/genomic and clinical studies.

{\it cis\/} -acting elements and {\it trans\/} -acting factors that control serum amyloid A gene expression during inflammation

To understand how the serum amyloid A (SAA) genes are regulated, the cis-acting elements and trans-acting factors involved in the regulation of mouse SAA3 and rat SAA1 genes expression during inflammation were analyzed.^ To identify DNA sequences involved in the liver-specific expression of the mouse SAA3 gene, the 5$\sp\prime$ flanking region of this gene was analyzed by transient transfection studies. Results suggest that C/EBP, a liver-enriched transcription factor, plays an important role for the enhanced expression of the mouse SAA3 gene in hepatocytes.^ Transfection studies of the regulation of the expression of rat SAA1 gene indicated that a 322 bp fragment ($-$304 to +18) of the gene contains sufficient information for cytokine-induced expression of the reporter gene in a liver cell-specific manner. Further functional analysis of the 5$\sp\prime$ flanking region of the rat SAA1 gene demonstrated that a 65 bp DNA fragment ($-$138/$-$73) can confer cytokine-inducibility onto a heterologous promoter both in liver and nonliver cells. DNase I footprint and gel retardation assays identified five putative cis-regulatory elements within the 5$\sp\prime$ flanking region of the gene: one inducible element, a NF$\kappa$B binding site and four constitutive elements. Two constitutive elements, footprint regions I and III, were identified as C/EBP binding sites with region III having over a 10-fold higher affinity for C/EBP binding than region I. Functional analysis of the cis-elements indicated that C/EBP(I) and C/EBP(III) confer liver cell-specific activation onto a heterologous promoter, while sequences corresponding to the NF$\kappa$B element and C/EBP(I) impart cytokine responsiveness onto the heterologous promoter. These results suggest that C/EBP(I) possesses two functions: liver-specific activation and cytokine responsiveness. The identification of two cytokine responsive elements (NF$\kappa$B and C/EBP(I)), and two liver-specific elements (C/EBP(I) and C/EBP(III)) implies that multiple cis-acting elements are involved in the regulation of the expression of the rat SAA1 gene. The tissue-specific and cytokine-induced expression of rat SAA1 gene is likely the result of the interactions of these cis-acting elements with their cognate trans-acting factors as well as the interplay between the different cis-acting elements and their binding factors. (Abstract shortened with permission of author.) ^

Regulation of {\it neu\/} gene expression by the simian virus 40 large T antigen and tumor suppressors Rb and p53

The neu gene (also c-erbB-2 or HER2) encodes a 185 kilodalton protein that is frequently overexpressed in breast, ovarian and non-small cell lung cancers. Study of the regulation of neu indicates that neu gene expression can be modulated by c-myc or by the adenovirus 5 E1a gene product. This study demonstrates that the transforming protein, large T antigen, of the simian virus 40 represses neu promoter activity. Repression of neu by large T antigen is mediated through the region $-$172 to $-$79 (relative to first ATG) of the neu promoter--unlike through $-$312 to $-$172 for c-myc or E1a. This suggests a different pathway for repression of neu by large T antigen. The 10 amino acid region of large T required for binding the tumor suppressor, retinoblastoma gene product, Rb, is not necessary for repression of neu. Moreover, the tumor suppressors, Rb and p53 can independently inhibit neu promoter activity. Rb inhibits neu through a 10 base pair G-rich enhancer (GTG element) ($-$243 to $-$234) and also through regions close to transcription initiation sites ($-$172 to $-$79). Mutant Rb unable to complex large T is able to repress the region close to transcription initiation but not the GTG enhancer. Thus, Rb inhibits the two regulatory domains of the neu gene by different mechanisms. Both Rb and p53 can repress the transforming activity of activated neu in focus forming assays. These data provide evidence that tumor suppressors regulate expression of growth stimulatory genes such as neu. Therefore, one reason for the overexpression of neu that is frequently seen in breast cancer cells may be due to functional inactivation of Rb and p53 which is also a common occurrence in breast cancer cells. ^

Functional studies of {\it twist\/} during mouse embryogenesis

A fundamental task in developmental biology is to understand the molecular mechanisms governing early embryogenesis. The aim of this study was to understand the developmental role of a putative basic helix-loop-helix (b-HLH) transcription factor, twist, during mouse embryogenesis.^ twist was originally identified in Drosophila as one of the zygotic genes, including snail, that were required for dorsal-ventral patterning. In Drosophila embryogenesis, twist is expressed in the cells of the ventral midline destined to form mesoderm. In embryos lacking twist expression, their ventral cells fail to form a ventral furrow and subsequently no mesoderm is formed.^ During mouse embryogenesis, twist is expressed after initial mesoderm formation in both mesoderm and cranial neural crest cell derivatives. To study the role of twist in vivo, twist-null embryos were generated by gene targeting. Embryos homozygous for the twist mutation die at midgestation. The most prominent phenotype in the present study was a failure of the cranial neural tube to close (exencephaly). twist-null embryos also showed defects in head mesenchyme, branchial arches, somites, and limb buds.^ To understand whether twist functions cell-autonomously and to investigate how twist-null cells interact with wild-type cells in vivo, twist chimeras composed of both twist-null and wild-type cells marked by the expression of the lacZgene were generated. Chimeric analysis revealed a correlation between the incidence of exencephaly and the contribution of the underlying twist-null head mesenchyme, thus strongly suggesting that twist-expressing head mesenchyme is required for the closure of the cranial neural tube. These studies have identified twist as a critical regulator for the mesenchymal fate determination within the cranial neural crest lineage. Most strikingly, twist-null head mesenchyme cells were always segregated from wild-type cells, indicating that the twist mutation altered the adhesive specificity of these cells. Furthermore, these results also indicated that twist functions cell-autonomously in the head, arch, and limb mesenchyme but non-cell-autonomously in the somites. Taken together, these studies have established the essential role of twist during mouse embryogenesis. ^

Functional analysis of p53 phosphorylation and a p53 hot spot mutation

The tumor suppressor p53 is a phosphoprotein which functions as a transcriptional activator. By monitoring the transcriptional activity, we studied how p53 functions is regulated in relation to cell growth and contact inhibition. When cells were arrested at G1 phase of the cell cycle by contact inhibition, we found that p53 transactivation function was suppressed. When contact inhibition was overridden by cyclin E overexpression which stimulates cell cycle progression, p53 function was restored. This observation led to the development of a cell density assay to study the regulation of p53 function during cell cycle for the functional significance of p53 phosphorylation. The murine p53 is phosphorylated at serines 7, 9, 12, 18, 37, 312 and 389. To understand the role of p53 phosphorylation, we generated p53 constructs encoding serine-to-alanine or serine-to-glutamate mutations at these codons. The transcriptional activity were measured in cells capable of contact inhibition. In low-density cycling cells, no difference in transcriptional activity was found between wild type p53 and any of the mutants. In contact-inhibited cells, however, only mutations of p53 at serine 389 resulted in altered responses to cell cycle arrest and to cyclin E overexpression. The mutant with serine-to-glutamate substitution at codon 389 retained its function in contact inhibited cells. Cyclin E overexpression in these cells induced p53 phosphorylation at serine 389. Furthermore, we showed that phosphorylation at serine 389 regulates p53 DNA binding activity. Our findings implicate that phosphorylation is an important mechanism for p53 activation.^ p53 is the most frequently mutated gene in human tumors. To study the mechanism of p53 inactivation by mutations, we carried out detailed analysis of a murine p53 mutation with an arginine-to-tryptophane substitution at codon 245. The corresponding human p53 mutation at amino acid 248 is the most frequently mutated codon in tumors. We showed that this mutant is inactive in suppressing focus formation, binding to DNA and transactivation. Structural analysis revealed that this mutant assumes the wild type protein conformation. These findings define a novel class of p53 mutations and help to understand structure-function relationship of p53. ^

The structural and functional relationship of the p53 tumor suppressor protein

The tumor-suppressing function of p53 can be affected in a variety of manners. Here, we describe a novel mechanism of transformation by mutant p53. Previously, it had been believed that mutant p53 molecules transform cells by oligomerizing with wild-type p53 and inactivating it. However, we demonstrated that there exists an additional mechanism of inactivation of p53 available to p53 mutants. It involves sequestration of cofactors necessary to p53, and subsequent interruption of its transactivation and tumor suppression functions. The p53 amino or carboxyl termini, known to interact with a large number of cellular factors, can affect wild-type p53 in this manner. Although they are unable to oligomerize with wild-type p53, they transform cells containing p53, and inhibit its transactivation ability. In addition, they interrupt growth suppression by p53, but not RB, confirming that they specifically affect p53 function, rather than having a general growth-stimulatory phenomenon. Also, we have cloned a p53 tumor mutation which results in expression of the amino terminus of p53. This provides a means to study the factor-sequestration transforming mechanism in vivo. Additionally, we found that the published sequence of the mdm2 gene is in error. mdm2 is a gene intimately involved with p53, blocking its ability to transform cells. Finally, previous data had established the influence of cell-cycle status on p53 function. In growth-arrested cells, wild-type p53 expressed by a transgene cannot activate transcription, but if these cells are forced to cycle by addition of cyclin E, p53 once again becomes functional. In this study, we extend these findings by examining only those cells successfully transfected, using fluorescence-activated cell sorting. Our results support the previous data, that cyclin E pushes growth-arrested cells back into the cell cycle. In summary, we have demonstrated the potential importance of cofactor association and protein modification to the abilities of p53 to cause transcription activation and repression, inhibition of DNA replication and induction of DNA repair, and initiation of cell-cycle arrest and apoptosis. Further elucidation of these processes and their roles in tumor suppression will prove fascinating indeed. ^

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Identification and characterization of cellular genes differentially expressed in a subset of tumors with non -functional WT1

Wilms tumor (WT) or nephroblastoma is a genetically heterogeneous pediatric renal tumor that accounts for 6–7% of all childhood cancers in the U.S. WT1, located at 11p13, is the sole WT gene cloned to date. Additional genomic regions containing genes that play a role in the development of Wilms tumor include 11p15, 7p, 16q, 1p, 17q and 19q. This heterogeneity has made it extremely difficult to develop an understanding of the pathways involved in the development of WT, even in the 5–20% of tumors that show mutations at the WT1 locus. My research addresses this gap in our current comprehension of the development of WT. ^ I have used two complementary approaches to extend the current understanding of molecular changes involved in the development of WT. In order to minimize complexities due to genetic heterogeneity, I confined my analysis to the WT1 pathway by assessing those genetically defined tumors that carry WT1 mutations. WT1 encodes a zinc finger transcription factor, and in vitro studies have identified many genes that are potentially regulated in vivo by WT1. However, there is very little in vivo data that suggests that they are transcriptionally regulated endogenously by WT1. In one approach I assessed the role of WT1 in the in vivo regulation of PDGFA and IGF2, two genes that are strong contenders for endogenous regulation by WT1. Using primary tissue samples, I found no correlation between the level of RNA expression of WT1 with either PDGFA or IGF2, suggesting that WT1 does not play a critical role in their expression in either normal kidney or WT. ^ In a parallel strategy, using differential display analysis I compared global gene expression in a subset of tumors with known homozygous inactivating WT1 mutations (WT1-tumors) to the gene expression in a panel of appropriate control tissues (fetal kidney, normal kidney, rhabdoid tumor and pediatric renal cell carcinoma). Transcripts that are aberrantly expressed in this subset of Wilms tumors are candidates for endogenous transcriptional regulation by WT1 as well as for potentially functioning in the development of WT. By this approach I identified several differentially expressed transcripts. I further characterized two of these transcripts, identifying a candidate WT gene in the process. I then performed a detailed analysis of this WT candidate gene, which maps to 7p. Future studies will shed more light on the role of these differentially expressed genes in WT. ^

Functional activation of p53 induces apoptosis in the absence of MDM2

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The mdm2 oncogene abrogates p53 transactivation function. Amplification of mdm2 occurs in 36% of human sarcomas, which often retain p53 in wild type form, suggesting that overexpression of mdm2 in tumors results in p53 inactivation. Thus, the relationship of p53 to mdm2 is important in tumorigenesis. The deletion of mdm2 in the mouse results in embryonic lethality by 5.5 days post coitum. Embryonic lethality of the mdm2 null embryos was overcome by simultaneous loss of the p53 tumor suppressor, which substantiates the importance of the negative regulatory function of MDM2 on p53 function in vivo. These data suggest that the loss of MDM2 function allowed the constitutively active p53 protein to induce either a complete G1 arrest or the p53-dependent apoptotic pathway, resulting in the death of the mdm2−/− embryos.^ The present study examines the hypothesis that the absence of mdm2 induces apoptosis due to p53 activation. Viability of the p53−/−mdm2−/− mice has allowed establishment of mouse embryo fibroblasts (MEFs) and a detailed examination of the properties of these cells. To introduce p53 into this system, and essentially recreate a mdm2 null cell, a temperature sensitive p53 (tsp53) point mutant (A135V) was used, which exhibits a nonfunctional, mutant conformation at 39°C and wild type, functional conformation at 32°C. Infected pools of p53−/− and p53−/−mdm2−/− MEFs with the tsp53 gene were established and single-cell clonal populations expressing tsp53 were selected. Shifting the cells from 39°C to 32°C caused p53−/−mdm2 −/− lines expressing tsp53 to undergo up to 80% apoptosis, which did not occur in the p53−/− lines expressing tsp53 nor the parental lines lacking p53 expression. Furthermore, the amount of p53 present in the clonal population determined the extent of apoptosis. Tsp53 is transcriptionally active in this system, however, it discriminates among different target promoters and does not induce the apoptosis effector targets bax or Fas/Apo1. ^ In summary, this study indicates that the presence or absence of mdm2 is the determining factor for the ability of p53 to trigger apoptosis in this system. The loss of mdm2 promotes p53-dependent apoptosis in MEFs in a cell cycle and dose-dependent manner. p53 is differentially phosphorylated in the presence and absence of mdm2, but does not induce the apoptosis effectors, bax or Fas/ Apo1. ^

Functional studies of a LIM -homeodomain transcription factor lmx1b in murine mid -hindbrain and limb development

This thesis is centered on applying molecular genetics to study pattern formation during animal development. More specifically, this thesis describes the functional studies of a LIM-homeodomain gene called lmx1b during murine embryogenesis. Lmx1b expression is restricted to the mid-hindbrain junction as well as to the dorsal mesenchyme of the limb, suggesting important functions during mid-hindbrain and limb development. To test these possibilities, lmx1b homozygous mutant mice were generated and their limb and CNS phenotypes examined. Lmx1b homozygous mutant mice exhibit a large reduction of mid-hindbrain structures, and that their limbs are symmetrical along the dorsal-ventral axis as the result of a dorsal to ventral transformation. Taken together, these studies define essential functions for lmx1b in mid-hindbrain patteming and in dorsal limb cell fate determination. However, the molecular mechanisms which accounts for these phenotypes are unknown, and whether lmx1b has same or distinctive functions during the mid-hindbrain and limb development is also unclear. ^ Recently, insight into molecular mechanisms of mid-hindbrain patterning and limb development has resulted from the identification of several factors with restricted expression patterns within these regions. These include the secreted factors wnt-1, fgf-8, wnt-7a and the transcription factors pax-2, and en-1. Targeted disruption of any of these genes in mice suggests that these genes might be involved in similar regulatory pathways. Analysis of the expression of these genes in lmx1b mutants demonstrates that lmxlb is not required for the initiation, but is required to maintain their expression at the mid-hindbrain junction. Thus, lmxlb is not required for specifying mid-hindbrain cell fates, rather, it functions to ensure the establishment or maintenance of a proper organizing center at the mid-hindbrain junction. Interestingly, lmxlb functions cell non-autonomously in chimera analysis, which indicates that lmx1b might regulate the expression of secreted factors such as wnt-1 and/or fgf-8 in the organizing center. In contrast, lmx1b functions cell autonomously in the dorsal limb to govern dorsal ventral limb development and its expression is regulated by with wnt-7a and en-1. However, single and double mutant analysis suggest that all three genes have partially overlapping functions as well as independent functions. The results point toward a complicated network of cross-talks among all three limb axes. ^

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Isolation and characterization of a novel human Mix -like homeobox gene MIXL

Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1–4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from zebrafish (bon), chicken (CMIX) and mice (MmI/MIXL1), isolation of a human Mix-like gene has remained elusive. ^ We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and MmI/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of MmI/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted immature B and T lymphoid cells. Several acute leukemic cell lines of B, T and myeloid lineages express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the α-globin gene, suggesting a functional conservation of the homeodomain. ^ Biochemical analysis revealed that MIXL proteins are phosphorylated at multiple sites. Immunoprecipitation and immunoblotting confirmed that MIXL is tyrosine phosphorylated. Mutational analysis determined that Tyr20 appears to be the site for phosphorylation. However, deletion analysis preliminarily showed that the proline-rich domain appears not to be necessary for tyrosine phosphorylation. The novel finding will help us make a deeper understanding of the regulation on homeodomain proteins by rarely reported tyrosine phosphorylation. ^ Taken together, isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation. ^

Expression regulation of AP -2 gamma gene

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placenta development. In an effort to identify factors regulating AP-2γ gene expression we isolated and characterized the promoter and 5′ flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ RACE. Transient gene transfer studies showed that basal promoter activity resides within a highly conserved ∼200 by DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp and AP-2 binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and non-trophoblast cells, demonstrating the functional importance of these sites in AP-2γ gene expression. ^ Mutational analysis of the 5′-flanking region revealed a 117-bp positive regulatory region of the mouse AP-2γ gene located between −5700 and −5583 upstream of the transcription start site. This 117-bp positive regulatory element provided approximately 7-fold enhancement of reporter gene expression in cultured trophoblast cells. A C/EBP-Sp1 transcription factor-binding module is located in this DNA sequence. Electrophoretic mobility shift assays demonstrated that transcription factors Sp1, Sp3 and C/EBP bind to the enhancer element. Mutation of each protein-binding site reduced the enhanced expression significantly. Mutagenesis assays showed that two other protein-binding sites also contribute to the enhancer activity. In summary, we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter region and contribute to basal promoter activity. We have identified a 117-bp positive regulatory element of AP-2γ gene, and we have shown that Sp and C/EBP proteins bind to the cis -regulatory elements and contribute to the enhanced gene expression. ^