The specific aim of this proposed research is to understand the mechanism by which Polycomb group proteins repress gene expression. This goal will be achieved by using combination of structural and biochemical methods. My research will focus on PcG proteins of PRC1. I will study the structures and functions of the PcG proteins of PRC1 and its functional complexes with nucleosome(s) and the transcription machinery. X-ray crystallography will be used to determine the three dimensional structures of individual PcG proteins of PRC1. In addition, the core PRC1 complex, also in its functional state with nucleosome(s), will be investigated with hybrid methods by x-ray crystallography and electron microscopy. Biochemical studies based on the structural information will be performed, to elucidate the mechanism of PRC1 in chromatin remodeling. I will also try to determine the interactions and structures of PRC1 and the components of general transcription machinery. Biochemical methods will be used to map the interactions between the components of the general transcription machinery with the PRC1. I will determine the structure(s) of these assemblies to understand the mechanism of PRC1 silencing by direct interaction with the basal transcription machinery. This combined structural and biochemical approach is critical to understand the way PRC1 functions in gene regulation and how the deregulation of PcG expression causes cancer.

Most physiological and behavioural processes in mammals are governed by a complex intrinsic circadian clock. The mammalian circadian clock is composed of a central pacemaker in the brain that coordinates subsidiary oscillators in most peripheral tissues. The intracellular clock mechanism involves interacting positive and negative transcriptional feedback loops that drive rhythmic expression of key clock components. Experimental evidence suggests a strong link between metabolism and circadian rhythms. Circadian clocks respond to feeding cycles and control the level of many cellular metabolic enzymes and nutrients. These observations point out a feed back-loop between the circadian clock and the cellular metabolic status. However, the molecular mechanisms that govern the interplay between metabolism and circadian oscillators are not yet clear. Recently it was demonstrated that, at least in vitro, the cellular redox state and NAD(P)+/NAD(P)H levels regulate the activity of the clock components. Here we propose to further study the interplay between cellular redox state and circadian clocks in living cells. We intend to develop a novel method that follows NAD(P)+/NAD(P)H levels in living cells and animals. Such a method will allow us to study the cross talk between cellular NAD(P)+/NAD(P)H levels and circadian oscillators. This project is aimed at understanding fundamental aspects of the molecular mechanisms within the mammalian circadian clock in connection with cellular metabolic state, in particular the involvement of cellular NAD(P)+/NAD(P)H levels in the phase entrainment of peripheral circadian oscillators.

Polyadenylation is a fundamental step in the formation of almost every eukaryotic mRNA. The 3’ poly(A) tail plays key roles in the mRNA life cycle, including nuclear export, turnover, and translational activity. Despite more than 40 years of studies on mRNA polyadenylation, the mechansism of poly(A) tail length control is still poorly understood. We have recently characterized an evolutionarily conserved nuclear poly(A) binding protein (PAB2) in the fission yeast Schizosaccharomyces pombe. Significantly, mRNAs from pab2-null are hyperadenylated, indicating a key role for PAB2 in polyadenylation control in vivo. Our objective is therefore to understand how PAB2 determines and regulates poly(A) tail length control. To achieve this objective, the proposed research is designed to test the hypothesis in which PAB2 is recruited by the RNA polymerase II transcriptional machinery and transferred to 3’ ends of pre-mRNAs to regulate polyadenylation. We also intend to determine the functional role of a posttranslational modification where specific arginine residues of PAB2 are methylated. Given the critical role of fission yeast PAB2 in poly(A) tail length regulation, our rationale is that a detailed understanding of PAB2 function will provide novel and significant insights into the mechanism of polyadenylation control in eukaryotes. The HFSP long-term fellowship was instrumental in the establishment of my current research program and my return to Canada yet, I now need further support to continue this important work. An HFSP CDA award would give me the financial and scientific independence, support, and tools that I need to make this work possible.

The interaction between several cell types inside a neuronal circuit enables an organism to perceive information from its surroundings and produce an appropriate behaviour. To understand this processing of sensory information, the contribution of each cell type to the logic of neuronal circuitry must be defined. The Drosophila visual system, with its relatively simple anatomy and its amenability to genetic manipulation of neuronal activity is ideal for such studies. The aim of the proposed work is to decipher how the neurons of the fly medulla, the neuropil of the optic lobe specialised in colour vision, process colour information coming from the sensory end of the circuit, the photoreceptors. Using two-photon imaging of genetically encoded sensors of neuronal activity, I propose to develop a system in which the activity of the different subtypes of medulla neurons is precisely monitored when light of a specific wavelength is applied. In parallel, I will validate techniques to artificially depolarise or hyperpolarise genetically targeted neurons that will serve to map the next level of connectivity in this circuit. This work will enable me to build a functional map of the colour vision system and will lead to an understanding of how colour is represented in the brain.

I propose to study olfactory signaling for the fellowship period. My primary goal will be to combine physiology and molecular biology to study the recognition of odorants by receptor proteins, ad to study the downstream signaling steps. Because the standard approach of expressing and studying cloned odorant receptors in heterologous expression systems has so far met with limited success, I propose to approach the problem in the opposite direction, namely, studying these receptor proteins in primary olfactory receptor neurons with a panel of odorants by using electrophysiology (suction-pipette recording) and then characterizing the molecular structures of the same receptors by using molecular biology (single-cell polymerase chain reaction). Although an analogous approach has been undertaken previously by others using Ca-imaging to probe receptor responses to odorants, this assay is not as detailed in characterizing receptor-odorant interactions as electrophysiological recording, especially the suction-pipette recording method that I propose to use, which has a resolution capable of examining the response of a receptor cell to the binding of a single odorant molecule. A correlation between the properties of the odorant receptor proteins and their structures will be an important step toward understanding the olfactory code. In addition, I propose to study two questions not previously addressed by others. One is constitutive activity of the odorant receptors, and the other is adaptation mechanisms of the olfactory receptor cell.

The integrity of stratified epithelial sheets in skin is dependent on intercellular adhesion, which is in part mediated by adherens junctions. These junctions are dynamic, allowing for tissue remodeling during morphogenesis and tissue repair following injury. Wound healing is an orchestrated process that involves movement of epidermal cells to close the gap, inflammatory responses to prevent infection at the injured site, and hyperproliferation to replenish lost cells. Targeted loss of the adherens junction protein alpha-catenin in skin results in enhanced inflammatory, hyperproliferative, and migratory behaviors, changes in epithelial cell morphology, and development of squamous cell carcinoma in situ. The molecular roots of these phenotypes appear to be intrinsic to the epidermal keratinocyte. I have developed an approach to dissect the mechanisms underlying the complex phenotypes arising from alpha-catenin loss in skin. I devised a series of RNAi screens in wild type and alpha-catenin mutant keratinocytes to assay how the genes that are differentially expressed in these cells contribute to each phenotype. I then propose functional analyses of my identified factors to address the phenotypic consequence of their loss in skin, the nature of their interaction with alpha-catenin, and their function in wound repair and squamous cell carcinoma tumor development and progression.

During development, a few neural stem (NS) cells give rise to all the different cell types of the mammalian central nervous system. To do so, they can divide asymmetrically into another self-renewing NS cell and a lineage-committed sibling. Defective cell fate choices can cause aberrant neurogenesis, including microcephaly or tumor formation. The mechanisms regulating asymmetric cell division and lineage choice in NS cells are, however, only poorly understood. Here I propose the use of homogeneous NS cell cultures to explore cellular and molecular mechanisms regulating NS cell differentiation. I plan to establish an assay for asymmetric divisions of NS cells that will be used to genome-wide RNAi screening for genes regulating cell fate decisions. I furthermore plan to screen under selective culture conditions to identify genes that are required for differentiation of NS cells into neurons and astrocytes. Candidate genes will be further characterized and their in vivo function analyzed in cortical slice cultures. The principles gained from this system will provide insight into vertebrate neurogenesis and might reveal general strategies of somatic stem cell divisions.

Post-transcriptional regulation of gene expression in metazoa involves a complex cellular network. Many trans-acting factors, including microRNAs, influence mRNAs stability, translatability, or mRNA subcellular localization by binding to specific cis-acting sequences or structures at the 3’ and 5’ untranslated regions of a mRNA. These interactions are decisive for translation of mRNA into protein and are influenced by the cellular context in time and space. Deregulation of cellular mRNA homeostasis is associated with many human diseases. The cellular microenvironment is composed of an intricate blend of extracellular matrix components and numerous neighboring cell types, and influences cellular behavior and gene expression profile in vivo. However, the effect of cellular microenvironment on post-transcriptional regulation of gene expression has never been addressed in detail in metazoa. I will study the effects of cellular microenvironment on post-transcriptional gene regulatory networks in mammals. Multidimensional cell culture systems and arrays of microenvironments will be used for identification of extracellular factors regulating miRNA-dependent and miRNA-independent post-transcriptional gene regulation machineries in mammalian cells. Experiments will also be designed to study the microenvironmental effects on miRNA mediated suppression of genes in a mouse model. Understanding how multiple extra- and intracellular signals are integrated to determine the differential fate of a mRNA in human cells, will pave the path for future studies on the importance of altered stability and translatability of mRNAs during tumorogenesis or organogenesis.

The loss of axons and dendrites usually precedes the death of the cell bodies in many neurodegenerative diseases. Since the molecular pathways involved in the death of cell bodies are pretty well understood, those underlying the degeneration of the axons and dendrites remain to date largely elusive. In a work from the host lab currently under review, Galectin-1 is identified as a major effector of the neuronal processes degeneration following injury of the adult nervous system. The receptor p75NTR is expressed early during neurons development and is also known to be up-regulated following nervous system’s injury. Using neurons generated from embryonic stem cells, the authors compare the proteome of wild type cells with these of p75NTR over-expressing neurons, and observe a three-fold over-expression of Galectin-1. Its transcriptional regulation is under the control of p75NTR through c-Jun N-terminal kinase and RhoA signaling, but do not seem to depend on them for the induction of the degeneration mechanisms observed. The research project I propose will first focus on the identification of the receptor(s) of Galectin-1. Many candidates exist, and we propose to analyze their profile of expression, as well as to use a biochemical approach to identify cell surface receptors interacting with Galectin-1. Once identified we plan to deplete, using RNA interference tool, the transcript(s) of Galectin-1 receptor(s) in order to test its/their involvement in the degeneration process. A second aim of this project will be to study the precise cellular and molecular mechanisms involved in the Galectin-1 signaling, causing the degeneration of neuronal processes.

Since their discovery over 100 years ago, enzymes are a topic of intensive research. Recently, it was shown that enzymes are dynamic entities existing in different conformations. These conformations, which are represented by different wells in the energy landscape, exhibit different rate constants for the catalytic process. It is likely that, during enzyme evolution, the original activity and the newly evolving activity are linked with different conformations. The proposed project aims to analyze the presence and evolution of different conformations when an enzyme is subjected to directed in vitro evolution. It will combine two new and innovative approaches for the evolution of enzymes and for the characterization of the dynamics of single enzymes. The evolution of enzymes will be carried out in a process combining in vitro compartmentalization with microfluidics, allowing the systematic control of all evolutionary steps (mutation, recombination and selection). A well-known model protein for the analysis of the evolution of enzyme activity (evolved beta-galactosidase from E. coli) will either be evolved to exhibit a higher activity or a different substrate specificity. In either case, single molecule experiments will be performed to determine the distribution of the rate constants. The results provide information about the existence and distribution of different enzyme conformations and will allow testing to what extent a stabilisation of different conformations with different activities contributes to the evolution of enzymes with higher activity or new functions.

This proposal concerns the role of clathrin and actin in the invasion of host cells by the intracellular bacterium Listeria monocytogenes. Listeria uptake proceeds by a clathrin-mediated route distinct from the canonical clathrin coated-pit/coated vesicle pathway, and it thus provides a way to probe molecular mechanisms in the non-canonical mode of uptake. The live cell imaging methods developed in Dr. Kirchhausen’s laboratory are a powerful way to decipher mechanisms underlying clathrin-mediated traffic. (1) I will use these methods to study the dynamics of clathrin and actin recruitment during Listeria entry and hence to probe their relative functions. Subsequently, the pattern of recruitment of proteins involved in generation of membrane curvature and vesicle pinching from the donor membrane will be investigated. (2) I will dissect which properties of a cargo, its size or its association with a particular receptor, will determine its route of entry. For this purpose beads of varying sizes will be modified with (a) the Listeria surface proteins InlA and InlB necessary for invasion and (b) with transferrin and other ligands that rely on the canonical pathway mediated by coated pits and coated vesicles to serve as model cargoes for live cell imaging studies. Hereby I will capitalize on my background in surface chemistry to conjugate the proteins in a well-defined density and orientation to the beads. The proposed studies will allow me to investigate the general principles governing the uptake of cargo via canonical and non-canonical clathrin-mediated endocytosis.