Xiaozhong Wang - US grants

The normal function of the brain relies on precise patterns of neuronal connections, and aberrant connectivity leads to human neurological and psychiatric disorders. The human brain consists of approximately100 billion neurons with trillions of synapses. Because of the enormous neuronal diversity and staggering synaptic complexity, very little is known about the molecular mechanisms that lead to the assembly of specific synapses in different neuronal types. Protocadherin (Pcdh) genes (14 Pcdh-a, 22 Pcdh-fi and 22 Pcdh-y in mouse) are attractive candidates for such a role because they can potentially generate a significant number of cell-surface "codes" through a combination of cell-specific promoter activation andcis- alternative splicing. It has been suggested that the distinct combinatorial Pcdh expression patterns might specify neuronal types and their connectivity. To evaluate their roles in neural development, we initiated functional analyses of these genes using genetically modified mice. Our analyses on Pcdh-j mutant mice provide the first in vivo evidence that protocadherins are essential for vertebrate CNS development and play an important role in establishing neuronal connectivity. However, Pcdh-y's function during synaptic development is not well defined and its molecular mechanisms of action are completely unknown. We plan to combine molecular and genetic approaches to further our understandingof Pcdh-y's functions.Specifically, we propose 1) to investigate the rules of expression for individual isoforms of Pcdh-y;2) to define the role of Pcdh-y's diversity;and 3) to identify and characterize the signaling components of Pcdh-y. The attainment of these goals will shed light on our understandingof the molecular basis for the precision and complexity of neuronal circuitry in the brain.

[unreadable] DESCRIPTION (provided by applicant): Embryonic stem (ES) cells are accessible for extensive genetic manipulations, therefore, providing a means to unveil functions of human genes. Our lab focuses on establishing new approaches for genetic analysis using ES cells. A major obstacle to performing recessive genetic screens in mammalian cells is the diploid nature of the genome. To overcome this barrier, we have been using an ES cell line that are Bloom- syndrome protein deficient (blm-/-). Bloom-deficient cells exhibit a much higher loss of heterozygosity than wild-type cells and can be used to generate homozygous recessive mutants required for screening. Taking advantage of this feature of Bloom deficient ES cells, we have devised a novel genetic screen to identify components of mammalian RNA interference (RNAi) pathway. RNAi is a recently discovered gene silencing phenomenon that occurs in a wide variety of organisms. RNAi has been implicated in a variety of biological and pathological processes, from normal embryonic developemnt to human cancers and neurodegenerative diseases. Because much of our understanding of RNAi pathway come from genetic and biochemical analysis in worms, plants and flies, a genetic screen in mouse ES cells will provide an exciting opportunity to examine the unique aspect of RNAi in a vertebrate system. In our design, RNAi competent blm-/- ES cells will be mutated using a retroviral gene trap that randomly integrates within the genome. The blm-/- cells create homozygous mutants for the retroviral integration events. We will then use drug selection to isolate putative RNAi mutants. To prove the principle of our design, we have successfully performed a small-scale screen and identified Argonaute 2, a known component of RNAi pathway and other putative RNAi defective mutant ES cell lines. These results have therefore validated our screen for novel RNAi components in mammalian cells. We propose to carry out a large scale screen for RNAi mutants using our established selection system in Bloom deficient ES cells. By completing a genome-wide recessive genetic screen in mammalian cells, we will not only have the unique opportunity to examine the RNAi pathway in the unexplored territory of the vertebrate genome, but also set an example for deciphering other genetic pathways using recessive genetic screens. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuronal death is not only essential in shaping the size and connectivity of the developing nervous system, but also contributes significantly to the pathogenesis of neurodegenerative diseases and stroke. Accumulating genetic evidence shows that clustered protocadherins (Pcdhs) play an important role in the regulation of neuronal survival. Our preliminary data show that PDCD10, also known as CCM3, a causative genetic defect for Cerebral Cavernous Malformations in human, acts downstream of Pcdh-?s to mediate this function. To better understand molecular pathways by which Pcdhs regulate neuronal survival, we will define the molecular pathways downstream of PDCD10 by evaluating the role of individual components in PDCD10 protein interaction network using genetically modified mouse models. PUBLIC HEALTH RELEVANCE: Developmental neuronal death ensures the appropriate size and connectivity of the nervous system and aberrant neuronal death in adulthood is commonly associated with chronic neurodegenerative diseases such as Alzheimer's disease, and acute cerebral ischaemia/stroke. The objective of this project is to elucidate how neurons signal through a large family of cell surface molecules-protocadherins to regulate neuronal survival during normal development and how misregulation of this pathway leads to neurodegeneration.

DESCRIPTION (provided by applicant): The function of the brain relies on the precise assembly of approximately 100 billion neurons with trillions of synaptic connections. The development of neural circuitry involves a complex interplay of cell-cell adhesion, inter-neuronal signaling and assembly of intracellular macromolecular protein complexes. The three tandem-arrayed protocadherin (Pcdh) gene clusters, namely Pcdh-¿, Pcdh-¿ and Pcdh-?, regulate neuronal survival and synaptic development by distinct mechanisms. In this project, we will develop a new mouse genetic model to define the overlapping function of clustered Pcdhs in circuit development, independently from their role in neuronal survival. This line of investigation will advance our understanding of molecular diversity underlying the precision of neural circuitry in the brain.

DESCRIPTION (provided by applicant): Accumulating evidence suggests that a variety of human cancers originate from the cancer stem cells (CSC) as a result of their ability to hijack self-renewal pathways that are employed by normal embryonic or adult stem cells. WNT, BMI1, Notch, Hedgehog and PTEN pathways are often deregulated in human cancer and these pathways are critical for the self-renewal of stem cells. More recently, microRNAs have also been shown to regulate many important aspects of stem cell biology and tumorigenesis including proliferation, differentiation, apoptosis, invasion and metastasis. However, it still remains poorly understood how these different self-renewal pathways interact with each other to coordinate self-renewal in normal stem cells and how the deregulation of one particular pathway in CSCs affects other pathways during tumorigenesis. Using a genetic screen, we identified a novel function for the tumor suppressor PTEN as a modifier of the microRNA silencing pathway in embryonic stem cells. Loss-of-PTEN mutations have been associated with a wide range of human cancers. Thus, the objectives of this project are to elucidate the molecular mechanisms by which PTEN modulates the efficacy of microRNA silencing machinery in stem cells and to determine the contribution of microRNAs to drive malignant transformation in loss-of-PTEN cancer cells. By characterizing the difference of microRNA silencing mechanisms between normal and PTEN negative stem cells, we hope to develop new therapeutic strategies that specifically target loss-of- PTEN cancer stem cells. PUBLIC HEALTH RELEVANCE: Understanding the similarities and differences between self-renewal mechanisms in normal stem cells and cancer stem cells offers great promises for better treatment of cancer. The tumor suppressor PTEN (phosphatase and tension homolog) pathway has been shown to control normal stem cell maintenance and self-renewal, whereas the loss of PTEN, frequently found in human cancers, promotes the propagation of cancer stem cells and tumor formation. In this study, we will define a novel modifier function of PTEN in the microRNA silencing pathway, and provide a new avenue to distinguish PTEN- negative cancer stem cells versus normal stem cells.

DESCRIPTION (provided by applicant): Multiple levels of epigenetic regulation are essential to maintain the pluripotent state of embryonic stem (ES) cells. Histone modification and DNA methylation have been shown to control the stemness of ES cells. Despite the importance of nucleosome positioning in epigenetic regulation, whether and how nucleosomes regulates stem cell functions remains poorly defined, in part due to technical obstacles to obtain high-resolution nucleosome maps in higher organisms. Recently we obtained a yeast nucleosome map at base-pair resolution by combining a novel chemical mapping approach and a Bayesian deconvolution algorithm. This current project seeks to extend this new approach to map nucleosomes for the mouse genome. Using ES cells expressing an engineered histone H4, our preliminary results have demonstrated the feasibility of constructing high-resolution nucleosome maps for higher organisms. The chemical mapping requires introducing a unique cysteine into histone H4 at position 47 to covalently attach a sulfhydryl-reactive copper-chelating label. This procedure is complicated by existence of multiple copies of histone H4 genes in the mouse genome. Thus our first aim is to develop a chemical mapping protocol, and carry out genome-wide chemical mapping in cultured mammalian cells. To define the center positions of nucleosomes based on chemical data requires deconvolution of cleavage signals from locally overlapping nucleosomes. The Bayesian algorithm we developed previously for yeast is computationally inefficient for the mouse data. Thus our second aim is to develop a more efficient computing algorithm and software tools. With above aims achieved, we will generate chemical maps of nucleosomes for both pluripotent ES cells and differentiated fibroblast cells, and quantify the gene expression in parallel by RNA-seq experiments. We will perform high-resolution analysis on global features of nucleosome positioning for the mouse genome, and investigate how nucleosomes regulate gene expression in coordination with other epi-regulators. Lastly, using chemical nucleosome maps we aim to determine the impact of the repressive chromatin mark H3K27me3 on nucleosome positioning throughout the genome. Taken together, the proposed work will delineate the nucleosome landscape of embryonic stem cells in unprecedented details and accuracy, providing insight into a new aspect of epigenetic regulation of the pluripotent cellular state.

Project Summary The unfolded protein response (UPR), a well-known adaptive mechanism for cells to maintain ER homeostasis, has been implicated in the pathogenesis of a variety of human diseases. Genetic deletion of the UPR signaling components can significantly influence the disease progression in mouse models for diabetes, hepatic steatosis, IBD, ALS and other diseases. We have recently performed a synthetic biology screen to interrogate the mammalian UPR pathway. From this screen, we have identified RtcB as a long sought RNA ligase that catalyzes unconventional XBP-1 splicing during ER stress. RtcB is a multifunctional RNA ligase that also acts in tRNA splicing. Genetic rescue of RtcB knockout cells suggests that RtcB-mediated ligation of XBP1 and tRNA may occur at different subcellular compartments. We will therefore determine subcellular compartment-specific RtcB function. By generating Trpt1 and RtcB double knockout cells and performing genetic rescue, we further demonstrate that a second biochemically distinct RNA ligation pathway cooperates with RtcB in mammalian cells to regulate XBP1 and tRNA splicing. Therefore, we will examine the role of other putative components in the RNA ligation pathway. Lastly we will identify unknown RNA ligases of the new ligation pathway by a genetic suppressor screen using RtcB knockout cells. By revealing the complete molecular repertoire of RNA ligation pathways, we will hopefully gain new insights and uncover new therapeutic targets to modulate the mammalian UPR.

ABSTRACT Vibrio vulnificus is a Gram-negative bacterial pathogen that causes severe life-threatening infections in humans after eating shellfish or swimming in warm seawater. In the US, there are about 200 cases annually with high rates of mortality and morbidity. While the number of cases is low compared to other food-borne pathogens, the high rates of death among patients exerts a significant burden on human life and the economy. This is expected to increase as incidence of V. vulnificus infections is rising due to the climate crisis. The most prominent virulence factor of V. vulnificus is a 5208 aa MARTX family toxin that delivers cytotoxic effectors to cells and these effectors are essential for virulence in a mouse model. While extensive information is known about the mechanisms of the effectors, very little is known about the remaining over 2800 aa of the protein toxin. These regions are known to be both necessary and sufficient for secretion of the toxin from the bacteria, interaction of the toxin with the eukaryotic cell surface, and translocation of the effectors across the plasma membrane. The project proposes to identify host cell receptors and host factors essential for intoxication using a genetic screen and selection for mutant cells that survive the cytolytic action of the toxin. We will also use a structure-function based approach to identify regions of the MARTX toxin essential for Type I secretion from the bacterium, binding of the toxin to a putative surface receptor, formation of a pore in the plasma membrane, and translocation of effectors to the eukaryotic cell cytosol. This study will advance our understanding of the function of the MARTX toxin repeat regions in detail and is expected to impact our understanding of pathophysiology during V. vulnificus infection.