Craig Cameron Mello, Ph.D. - US grants

Initial anterior-posterior (a-p) polarity in C. elegans is established at fertilization. A set of asymmetric cell divisions follow during which cell fate determinants become localized differentially in anterior and posterior cells. As development proceeds a Wnt signal, MOM-2, functions to maintain and to propagate a common a-p cellular polarity. In addition, cells receive a parallel but genetically distinct polarity input from apr-1, a gene related to the human Adenomatous polyposis coli gene (APC). These pathways share several common components including WRM-1 (a beta-catenin related gene), LIT-1 (a Ser/Thr Kinase) and POP-1 an HMG-domain transcription factor. The long-term goal of the proposed research is to understand the genetic and molecular relationships between these factors and hence to understand their role in controlling cellular polarity in the C. elegans embryo. The proposed studies will include in vitro and in vivo biochemical studies of WRM-1, LIT-1 and POP-1 protein interactions. These studies will focus on the mechanism through which WRM-1 and LIT-1 down-regulate POP-1 activity in response to polarity signaling. Yeast two hybrid screens, reverse genetic screens and conventional forward genetic screens will identify additional interacting factors that function along with WRM-1 and LIT-1. Further genetic and reverse genetic screens will identify new factors involved in these pathways. The proper control of cellular polarity is essential for the development and homeostasis of tissues in humans, and defects in this process are implicated in numerous forms of cancer. The significance of this work lies in the opportunity it provides to study important regulators of cellular polarity within a relatively simple and well characterized genetic model system.

The targeted inhibition of gene expression is an essential tool for basic medical research in numerous fields and has many potential clinical applications. However, improved tools are needed for probing the functions of cloned genes, especially in light of the comprehensive genome sequencing projects now underway for many organisms. The present proposal is directed at understanding and improving one such tool known as "RNAi" for RNA mediated genetic interference. RNAI was discovered in the course of attempts to use "antisense" methodology to interfere with gene expression in C. elegans. These studies led to the surprising observation that preparations of either "sense" or "antisense" RNA strands can induce potent and specific genetic interference upon microinjection into this organism. Findings described in this proposal suggest that a cellular and organismal response may underlie this genetic interference. Remarkably, interfering effects are observed not only throughout the tissues of the injected animal but are inherited by 100% of the injected animal's progeny. In subsequent generations genetic interference can be transmitted in the sperm or oocytes as a dominant extragenic factor. Microinjection of RNA (but not DNA) can induce interference. Surprisingly, double stranded RNA is much more effective at inducing interference than is either single strand. This proposal outlines a systematic approach that will dissect the molecular and genetic mechanisms that underlie RNAi. Aims will include: properties associated with interference. Improvements in the methodology. And identification of the C. elegans genes whose projects mediate genetic interference. Insights from these studies may lead directly to the development of related RNA interference technologies for probing gene function in other organisms including humans.

DESCRIPTION (provided by applicant): Project Summary/Abstract RNA interference (RNAi) was originally described as a gene silencing mechanism triggered by the experimental introduction of double stranded (ds)RNA into the nematode C. elegans (Fire et al., 1998). The term RNAi is now used to refer to a diverse set of gene regulatory mechanisms that share common features including the involvement of a short 21-30 nucleotide (nt) long RNA and a protein cofactor of the Argonaute (RNase H-related) protein family. As an experimental tool, RNAi is of broad relevance to basic medical research in numerous fields, and RNAi therapeutics are now under development for several clinical applications. Furthermore, RNAi-related mechanisms function in conserved gene-regulatory pathways that are of basic and fundamental importance to human cellular and developmental biology. The proposed genetic and biochemical studies will advance our understanding of RNAi and related pathways. The ability to combine classical genetics with the newer disciplines of deep-sequencing, functional genomics and proteomics, make C. elegans an ideal system for these studies. In all animals studied to date, multiple RNAi-related pathways co-exist within cells. In C. elegans, three AGO pathways have the potential to mediate genome-wide or transcriptome-wide surveillance. These pathways are: (i) the WAGO pathway, which targets transposons, pseudogenes and other cryptic loci, as well as some protein encoding genes; (ii) the CSR-1 pathway, which targets most, if not all, protein-encoding mRNAs expressed in the germ line; and (iii) the PRG-1 pathway, which targets at least one transposon family but has more than fifteen thousand additional genomically-encoded small RNA cofactors whose targets are not known. An important goal of this work is to understand how these distinct pathways identify targets and mediate specific regulatory outcomes. The proposed studies will investigate the function and interrelationship of these pathways, using an array of biochemical, molecular and genetic approaches. The mechanisms and protein families that mediate RNAi are highly conserved in animals, therefore, insights from the proposed studies will be directly relevant to human biology and disease. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page

DESCRIPTION (provided by applicant): Initial anterior-posterior (a-p) polarity in C. elegans is established at fertilization. A set of asymmetric cell divisions follow during which cell-fate determinants become localized differentially in anterior and posterior cells. As development proceeds a Wnt signal, MOM-2, functions to maintain and to propagate a common ap cellular polarity. At the 4-cell stage, Wnt signaling acts in parallel with signaling mediated by the tyrosine kinase pp60 Src to control both cell fate and cell division orientation in the endoderm precursor cell named EMS. The Wnt and Src pathways converge on several common components including WRM-1 (a beta-catenin related protein), LIT-1(a Serfrhr Kinase), and POP-1 an HMG-domain transcription factor. Recent genetic studies indicate that coordination of the cell-division axis with the polarized axis of the EMS cell depends on signaling mediated by the major cell-cycle regulator CDK-1 and its 13 kd binding partner CKS-1. Our findings suggest a model in which CDK-1 phosphorylates WRM-1 during mitosis to unmask cues that direct the rotation of the EMS spindle onto the a-p axis of the cell. The activated WRM-1 is then free to transduce to the nucleus of the posterior EMS daughter, called E, where it functions to down regulate and/or to promote nuclear export of the endoderm repressor, POP-1. The proposed studies will use cell-biological as well as biochemical methods to test the above model for endoderm induction. These studies will focus on the mechanisms of WRM-1 regulation by the Wnt, Src, and CDK-1 pathways. Protein interaction screens, suppressor-genetic screens, and conditional forward genetic screens will identify additional components of each signaling pathway. The localization of WRM-1 protein will be examined both in wild-type strains and in strains with defects in each signaling pathway, including strains that express specific mutant forms of WRM-1 itself. Proper control of cellular polarity is essential for the development and homeostasis of tissues in humans, and defects in this process are implicated in numerous forms of cancer. The significance of this work lies in the opportunity it provides to study important regulators of cellular polarity within a relatively simple and well characterized genetic model system.

Project I. piRNA function in epigenetic inheritance. Craig Mello, P.I. Project summary Darwin recognized that evolution by natural selection required two self-evident but entirely mysterious biological mechanisms: (i) a property of inheritance such that offspring share features of parents, and (ii) a source of variation so that novel inherited features, adaptations, can arise. The discovery of Mendelian genetics and of the structure and properties of DNA seemed to solve these mysteries, creating the widely held DNA-centric view of inheritance. However, beginning with Barbara McClintock's classic work on variegation in maize, a growing field of epigenetics has begun to reveal how RNA and chromatin can contribute to inheritance. Recently, we have shown that an RNAi-related small-RNA pathway called the Piwi pathway can initiate a very stable transgenerational mode of epigenetic silencing in the C. elegans germline. Remarkably, this pathway involves piRNA signals that constitute not only a memory (and enforcer) of gene silencing but also involves an epigenetic memory (and protector) of expressed germline genes. PIWI Argonautes and their small RNA cofactors (piRNAs) are conserved regulators of germline development in animals. piRNA-pathway mutants exhibit embryonic lethality and partial or complete deficits in fertility, characterized by under- proliferated germlines and activation of transposable elements. While the role of piRNAs in transposon silencing is well established, how piRNAs promote germ cell development and function is not understood. Here we propose a set of genetic, biochemical and bioinformatic studies to further characterize and understand: 1) the molecular mechanisms and outcomes of targeting by PIWI/piRNA complexes, 2) the specificity of individual PIWIs, and 3) the epigenetic functions of piRNA pathways. Insights from parallel projects in the laboratories of Drs. Theurkauf and Zamore, with informatics-based model building by the Weng lab, will inform this analysis and provide new tools and methods for probing piRNA activities and downstream functions. Relevance These studies will advance our basic understanding of piRNA function in fertility and epigenetic inheritance, and should guide our understanding of how related pathways protect the human genome, maintain somatic cells, and promote the pluripotency and immortality of germ cells.

Project I. piRNA function in epigenetic inheritance. Craig Mello, P.I. Project summary Darwin recognized that evolution by natural selection required two self-evident but entirely mysterious biological mechanisms: (i) a property of inheritance such that offspring share features of parents, and (ii) a source of variation so that novel inherited features, adaptations, can arise. The discovery of Mendelian genetics and of the structure and properties of DNA seemed to solve these mysteries, creating the widely held DNA-centric view of inheritance. However, beginning with Barbara McClintock's classic work on variegation in maize, a growing field of epigenetics has begun to reveal how RNA and chromatin can contribute to inheritance. Recently, we have shown that an RNAi-related small-RNA pathway called the Piwi pathway can initiate a very stable transgenerational mode of epigenetic silencing in the C. elegans germline. Remarkably, this pathway involves piRNA signals that constitute not only a memory (and enforcer) of gene silencing but also involves an epigenetic memory (and protector) of expressed germline genes. PIWI Argonautes and their small RNA cofactors (piRNAs) are conserved regulators of germline development in animals. piRNA-pathway mutants exhibit embryonic lethality and partial or complete deficits in fertility, characterized by under- proliferated germlines and activation of transposable elements. While the role of piRNAs in transposon silencing is well established, how piRNAs promote germ cell development and function is not understood. Here we propose a set of genetic, biochemical and bioinformatic studies to further characterize and understand: 1) the molecular mechanisms and outcomes of targeting by PIWI/piRNA complexes, 2) the specificity of individual PIWIs, and 3) the epigenetic functions of piRNA pathways. Insights from parallel projects in the laboratories of Drs. Theurkauf and Zamore, with informatics-based model building by the Weng lab, will inform this analysis and provide new tools and methods for probing piRNA activities and downstream functions. Relevance These studies will advance our basic understanding of piRNA function in fertility and epigenetic inheritance, and should guide our understanding of how related pathways protect the human genome, maintain somatic cells, and promote the pluripotency and immortality of germ cells.

Project I. piRNA function in epigenetic inheritance. Craig Mello, P.I. Project summary Darwin recognized that evolution by natural selection required two self-evident but entirely mysterious biological mechanisms: (i) a property of inheritance such that offspring share features of parents, and (ii) a source of variation so that novel inherited features, adaptations, can arise. The discovery of Mendelian genetics and of the structure and properties of DNA seemed to solve these mysteries, creating the widely held DNA-centric view of inheritance. However, beginning with Barbara McClintock's classic work on variegation in maize, a growing field of epigenetics has begun to reveal how RNA and chromatin can contribute to inheritance. Recently, we have shown that an RNAi-related small-RNA pathway called the Piwi pathway can initiate a very stable transgenerational mode of epigenetic silencing in the C. elegans germline. Remarkably, this pathway involves piRNA signals that constitute not only a memory (and enforcer) of gene silencing but also involves an epigenetic memory (and protector) of expressed germline genes. PIWI Argonautes and their small RNA cofactors (piRNAs) are conserved regulators of germline development in animals. piRNA-pathway mutants exhibit embryonic lethality and partial or complete deficits in fertility, characterized by under- proliferated germlines and activation of transposable elements. While the role of piRNAs in transposon silencing is well established, how piRNAs promote germ cell development and function is not understood. Here we propose a set of genetic, biochemical and bioinformatic studies to further characterize and understand: 1) the molecular mechanisms and outcomes of targeting by PIWI/piRNA complexes, 2) the specificity of individual PIWIs, and 3) the epigenetic functions of piRNA pathways. Insights from parallel projects in the laboratories of Drs. Theurkauf and Zamore, with informatics-based model building by the Weng lab, will inform this analysis and provide new tools and methods for probing piRNA activities and downstream functions. Relevance These studies will advance our basic understanding of piRNA function in fertility and epigenetic inheritance, and should guide our understanding of how related pathways protect the human genome, maintain somatic cells, and promote the pluripotency and immortality of germ cells.