Nonribosomal peptide synthetases (NRPS) are huge macromolecular machines that produce a vast variety of small molecules with important and diverse biological activities, including anti-virals, immunosuppressants and antibiotics, such as penicillin and cyclosporin. Unlike ribosomal peptides, NRPS peptides can include non-standard amino acids and are often cyclic. NRPSs are organized into modules of ~1000 residues, with each module responsible for the addition of one specific amino acid to the peptide, in an assembly line fashion.
NRPS are intrinsically interesting because they are among the largest and most complicated molecular machines in nature. Additionally, the wide-ranging activities of their small peptide products, and the potential to engineer NRPSs for production of new bio-active molecules, make NRPS systems an ideal source of novel therapeutics and chemicals.
I propose to expand our structural knowledge of non-ribosomal peptide synthetases and the mechanisms they employ to synthesize bio-active molecules. X-ray crystallography and mutational analysis of the peptide bond forming “condensation” domain will clarify the catalytic mechanism and elucidate the determinants of substrate specificity. I propose to determine the structure the penicillin producing NRPS, ACV synthase, by X-ray crystallography. I will also study NRPSs by electron microscopy (EM), using ACV synthase and cyclosporin synthase, a gigantic 1.7 MDa NRPS. These EM studies, in combination with X-ray crystallography, will reveal the architecture of large NRPSs and visualize the movements required for NRPS function.

In order to equalize the gene dosage between the sexes, one X-chromosome is randomly chosen for inactivation during early embryonic female development. Initiation of this X-chromosome inactivation (XCI) process involves ‘sensing’ the presence of more than one X in the cell, ‘counting’ the number of X’s and choosing the X to be inactivated. The outcome of these events is the monoallelic upregulation of the key molecule that ensures silencing of one X chromosome, the Xist non-coding RNA. However, the molecular mechanisms as well as the interplay of cis-acting elements and trans-acting factors that mediate sensing, counting and choice remain elusive.
In the proposed project, I will combine quantitative experiments and mathematical modeling to address this problem. I will follow several processes involved in XCI during differentiation of female ES cells, such as the expression of Xist and down-regulation of its repressive antisense transcript, Tsix; transient co-localization of the X chromosomes during activation and monoallelic regulation of Xist expression; dynamics of developmentally regulated (e.g. Oct4) and constitutively active (e.g. CTCF) trans-acting factors. Perturbation of these processes using mutant cell lines, over-expression and knock-down techniques will be linked to the kinetics and to the outcome of XCI. Based on this integrated data set I will develop mathematical models for the molecular processes governing sensing, counting and choice. Comparison of different simulations with experimental measurements will allow me to distinguish alternative models that differ in the temporal and causal order of molecular events and to predict missing steps and regulators.

MicroRNAs (miRNAs) guide the repression of mRNAs to which they are complementary. Over the last 6 years, a dogma has emerged: miRNAs regulate a large fraction of genes in animals. Tens or hundreds of mRNAs have conserved binding sites to each miRNA – and there are tens or hundreds of miRNAs per species.
This dogma is facing three paradoxes:
1.In a few well-studied cases, miRNAs seem to regulate a very limited number of targets.
2.When quantified, miRNA-mediated repression is lower than the usual noise in gene expression.
3.While miRNAs can be extremely well conserved across animals, their interaction with most of their predicted targets is not.
I recently proposed another hypothesis: most predicted miRNA “targets” are in fact competitive inhibitors (they are too modestly repressed by miRNAs to benefit from that regulation; but titrating miRNAs in a cell type-specific manner, they would regulate them: that would be the reason for the conservation of their miRNA binding sites). I am now proposing to test this hypothesis, assessing four of its predictions:
1.mRNAs compete for their interaction with miRNAs.
2.Poorly abundant pseudo-targets have a weaker regulatory effect than the abundant ones.
3.Pseudo-targets do not need to be coding.
4.MiRNA binding sites on pseudo-targets are antagonists of the miRNA.
Whether this hypothesis turns out to be supported or not, it needs to be put to the test. The outcome of this project could be a re-definition of miRNA targets (that would profoundly affect any research involving miRNAs); if the hypothesis turns out to be wrong, it will raise a new question in systems biology: how can miRNA targets be so sensitive to gene expression level?

Epigenetic marks, especially histone modifications, play pivotal roles in regulating gene expression in eukaryotes. Several studies suggest that the mutually exclusive expression of var genes in Plasmodium falciparum is under epigenetic control, but it is not known what initiates histone mark changes at active and silent var genes. Studies in several organisms indicate that non-protein-coding RNAs (ncRNAs) may play a role in recruiting histone-modifying enzymes to specific genomic loci.
In P. falciparum, ncRNAs transcribed from var-gene introns associate with chromatin close to var genes, indicating that ncRNA may play a role in regulating var gene expression but, to date, no mechanistic data exist to support this hypothesis.
I propose to test the hypothesis that ncRNAs play important roles in transcriptional regulation in P. falciparum by following three independent but complementary approaches. First, using high-throughput RNA-sequencing, I will determine genome-wide levels of ncRNAs. Second, to establish a mechanistic link between ncRNA and nuclear RNA-processing enzymes, I will determine the RNA substrates of two subtelomeric RNases recently identified in the Scherf laboratory. Third, to identify ncRNA associated with active or silent var genes, I will perform RNA-ChIP-sequencing with antibodies specific for histone modifications found at those loci.
This work will represent a significant contribution by formulating strand-specific RNA-sequencing and ChIP-sequencing for P. falciparum and using these techniques to gain a better understanding of the role of ncRNA in transcriptional regulation in general and regulation of var-gene expression in particular.

The ability to precisely determine the proteins content of a cell at a given time point is fundamental for the understanding of basic cell biology principals. I plan to employ a unique method (called ribosome profiling) to determine protein translation, in which deep sequencing technology is used to sequence the mRNA fragments that are protected by ribosomes. By using viral microRNAs (miRNAs) expressed in infected cells and the ribosome profiling technology I will try to reveal the mechanism(s) by which miRNAs repress translation. Furthermore, this unique large scale analysis will enable us to determine the identity and the relative levels of each of the viral and host proteins that are being translated during the course of infection and would therefore provide valuable information regarding translational control strategies used by viruses to reprogram the cellular host translation machinery. My previous experience concerning viral miRNAs biology together with the high through-put technology of ribosome profiling and the extensive experience Jonathan Weissman's lab has gained regarding the control of protein translation will allow me not only to successfully accomplish the goals of this research and concomitantly to reveal basic translation control processes, but also to mature as a scientist.

The histopathological hallmarks of multiple sclerosis (MS) are focal inflammation, demyelination, loss of oligodendrocytes, reactive astrogliosis, and axonal pathology. The precise pathogenesis of this disease is a subject of ongoing research. The exact interplay between inflammation, astrogliosis, neurodegeneration, demyelination and axonal damage in-vivo remains to be clarified, and any satisfactory understanding of the biology of MS must explain the temporal relationship between these different phases of MS.

An important piece of the puzzle can be uncovered by probing the disease's metabolism. In-vivo, the ideal tool for doing so is magnetic resonance spectroscopy. The spinal cord has barely been targeted in research due to the technical difficulties it presents; yet, the different phenotypes of MS suggest its manifestation in the cord might differ on a fundamental level from its manifestation in the brain, and therefore warrants a closer look.

The present proposal aims to carry out magnetic resonance spectroscopy in humans at high fields to investigate the metabolic pathways of MS in the spinal cord and optic nerve in order to clarify the pathogenesis of the disease. To this extent, new spectroscopy methodologies will be developed and consequently applied to human patients at varying stages of the disease. The high fields, coupled with the new methods for examining the spinal cord and optic nerve, are expected to yield insights regarding the disease's pathogenesis via the spectra obtained.

A central concept of biology is adaptation. Cells adapt to their environment by selectively remodeling the lipid and protein composition of their surface. To accomplish adaptation, the downregulation of activated cell surface receptors is essential. Activated receptors are ubiquitinated, removed from the surface and subsequently delivered into lysosomes where they are finally degraded. An essential sorting step occurs on endosomes. There, the ‘endosomal sorting complexes required for transport’ (ESCRTs) bind to and package the ubiquitinated membrane proteins into small vesicles that bud into the lumen of endosomes. This generates multivesicular bodies (MVB). When MVB fuse with lysosomes, the luminal vesicles that carry the membrane proteins are delivered into lysosomes, where they are degraded. Hence, loss of ESCRT function disrupts the degradation of most if not all membrane proteins. Probably all components of the core ESCRT machinery have been identified. Their function is reasonably well understood. Yet, the molecular mechanism underlying the regulation of ESCRT activity and the consequences of its dysfunction, raise important questions. Among the most important are:
1. How is the activity of the ESCRT machinery regulated?
2. How do cells react/adapt to the loss of ESCRT function?
The goal is to dissect the molecular mechanisms that regulate and survey the activity of the ESCRT machinery. The proposed research could identify a ‘quality control mechanism’ for the ESCRT machinery that monitors the cellular membrane protein repertoire and hence controls cell surface remodeling.

The contribution of oncogenic and tumor-suppressive perturbations to global protein stability in cancer cells will be studied to discover post-translational changes in protein levels that can contribute to cell transformation, cell proliferation,anti-apoptotic properties of cancer cells.and finally to cancer initiation and progression. Technically, a screen will be performed with the utilization of a recently published multiplexing strategy to monitor protein turnover of individual proteins on a global scale. This method will be applied to study the effect of oncogenic Ras expression and of shRNA-mediated knock-down of the tumor-suppressor PTEN in isogenic cell lines. The goal is to discover candidates and pathways whose changes in protein stability are critical for transformation, proliferation and anti-apoptotic effects conceptually revealing oncogene, non-oncogene or pathway addictions of the transformed cancer cell. As a follow-up, the consequences of these protein stability changes will be examined in various assays to reveal their specific function in cancer-associated phenotypes. In addition the underlying regulatory mechanism of selected key factors that show changes in stability will be studied with an RNAi approach and a transcriptome analysis. I anticipate these studies will contribute to a better understanding of molecular changes in transformation and to the discovery of novel candidates for cancer cell elimination or prevention of transformation.

The shape of bacteria is determined by the peptidoglycan cell wall, a single huge molecule that counteracts the turgor pressure from inside the cell. Recently, the structure of the peptidoglycan cell wall of the rodlike bacterium Escherichia coli was revealed in new detail by cryotomography. However, the dynamical mechanism by which the cell achieves and maintains its shape during growth is not understood - in particular how does the cell cut existing peptidoglycan bonds to insert new material without risking lysis? We aim to pursue both experimental and theoretical approaches to tackle this problem. We will use state-of-the-art fluorescence microscopy experiments to track the trajectories of single molecules of both cell wall assembly enzymes and related cytoskeletal proteins. Once we can track these proteins' dynamics during growth, we will examine how their trajectories are regulated using genetic, chemical, and physical perturbations to the cell wall and the cytoskeleton. In parallel, we will complement our experiments with computational modeling of the process of cell wall growth. These theoretical studies will extend, with growth dynamics, a recently developed static biophysical model to both help understand the results of the proposed experiments and generate testable hypotheses regarding how new peptidoglycan is inserted during cell growth.

Embryonic development is a highly controlled process where biochemical networks are constantly challenged by environmental cues. In a growing embryo, cells grow, divide, migrate, differentiate or arrange to form structures in a mechanically active environment where they can physically alter their environment and react to these changes. Although there has been much progress in identifying the pathways leading to sensing physical environmental changes in vitro, very little is known about what happens in vivo. Furthermore, it is not clear how do biological systems sense their physical environment in a whole embryo or how positional information is linked with mechanical and geometrical constraints of a growing system. To date, the experimental data addressing these questions are few mainly due to the technical challenges of advanced and fast in vivo imaging required to analyze these physical changes.
The goal of this work is to isolate and describe new signaling pathways transducing information from environment to gene response during cardiovascular development. To do so, we have a descriptive approach to characterize the forces involved and to uncover the physical interaction between cells and their environment based on advanced imaging techniques. In parallel, we use conventional molecular biology techniques to isolate and characterize the genes involved in the pathways involved in the mechano transduction.

One of the central unanswered questions in neuroscience is how the brain encodes behaviour. Many behaviours, particularly those that are innate, are likely to be specified by genetic factors that establish the potential for a behavioural program to be carried out. A striking example of the genetic specification of behaviour is provided by the intimate link between the fruitless (fru) gene and male courtship behaviour in Drosophila. Male-specific transcripts of the fru gene are both necessary and sufficient to program the potential for male courtship behaviour into the fly’s nervous system. It is thought that fru acts by specifying sex-specific neural circuits within the CNS, which allow a particular stereotyped behavioural response. The underlying molecular mechanisms that mediate this circuit formation however, remain unknown. This project will explore, using fru as a model, how a single gene can direct the specification of neural circuitry underlying an innate behaviour, Drosophila courtship. This project will incorporate cutting edge molecular genetic and neuroscience techniques to investigate the gene networks that act downstream of fru and to understand how these regulatory cascades contribute to the properties of individual cells and their assembly and function in neural circuits. Understanding the molecular cascades directed by fru will not only give insight into how genes control courtship behaviour, but also more broadly suggest mechanisms by which the construction and coordination of neural circuits can be specified by genetic factors.

Organ morphogenesis is a complex process involving multiple steps from the cellular to tissue scale. A key question is how tissue polarity and growth are coordinated. Recent studies in the host laboratory, using a combination of molecular genetics, clonal analysis and computational modelling, have provided a working hypothesis of how seemingly complex shapes, such as that of the Antirrhinum flower, can be generated through an interplay between polarities and identities. In particular, polarities are established through a small number of organisers and these polarities then interact with region-specific transcription factors to determine the rates of growth along particular orientations. However, the nature of the polarity organisers and the way they influence the pattern of anisotropic growth are still unknown. To address this issue, I propose to study how identity factors control polarity organisers and signalling molecules during petal morphogenesis. I will use sector analysis to determine how developmental genes can influence the orientation of growth and to establish if CUP (a CUC homolog) is a key polarity organiser, following its local effects on the control of tissue polarity. In parallel, I plan to determine the relationship between variation in CUP expression, shifts in regional polarity and effects on growth patterns. Additionally, I intend to identify new polarity organisers by looking at available Antirrhinum mutants compromised in the control of local polarity. Finally, I will integrate my findings within a computer model of floral morphogenesis to gain a deeper understanding of how genes interact to control the evolution and development of biological form.

The centrosome organizes the microtubule network throughout the cell cycle. Centrosomes are composed of two centrioles which are duplicated once per cell cycle to allow the formation of the two centrosomes needed for bipolar spindle formation during mitosis. The polo-like kinase protein family member 4 (Plk4) is intimately involved in promoting centriole duplication and it has been shown that Plk4 overexpression induces the formation of extra centrosomes (centrosome amplification). Many studies have suggested a link between centrosome amplification, chromosome missegregation and tumor formation. Nevertheless, at present, evidence for this link remain largely correlative. I propose to develop a mouse model in which Plk4 can be conditionally overexpressed globally or in a tissue/cell type specific manner. Using this model, I will decipher the link between centrosome amplification, chromosome missegregation and tumorigenesis. Mice overexpressing Plk4 will be examined for spontaneous tumor formation and crossed with mice already predisposed to tumor formation. Using mouse embryonic fibroblasts from mice overexpressing Plk4, I will characterize the link between centrosome amplification and aneuploidy in cells. Together, these studies will increase our understanding of the role of centrosome amplification in tumorigenesis.

Mitochondrial AAA proteases conduct protein quality control and exert regulatory functions paramount to the maintenance of organellar integrity. Defects in the m-AAA (matrix) protease cause a recessive form of hereditary spastic paraplegia(Rugarli and Langer 2006) but the i-AAA (intermembrane space) protease has yet to be implicated in disease. The i-AAA protease is encoded by a single gene in yeast, YME1(Graef, Seewald et al. 2007), whose mammalian homologue is YME1L. To investigate the physiological role of the i-AAA protease we will create a conditional knockout mouse model for YME1L to determine why some cell types, in particular neurons, are more susceptible than others to defects in mitochondrial quality control. To study the protective effects of mitochondrial quality control and to identify novel substrates of the i-AAA protease, we will generate transgenic mice expressing: 1) a wild-type YME1L and 2) a dominant-negative version of YME1L (E381Q). We will examine whether increased expression of YME1L can augment cellular and cognitive function or increase lifespan. If the results indicate a cyto-protective or anti-apoptotic function of Yme1L we will then determine whether mitochondrial quality control by the i-AAA protease can influence cellular competition and proliferation in vivo by tracking the proliferation of two different cell populations in vivo that differ according to their expression of either a functional or proteolytically inactive (E381Q) version of YME1L.

Spinal cord injury (SCI) due to blunt trauma is the most common cause of paralysis. In such contusion injuries, some axons are transected immediately, while others initially only sustain functional damage. Despite the irreversibility of the transections, significant functional improvement occurs after SCI. This improvement could be due to spontaneous recovery of those axons that are incapacitated but remain contiguous. The dynamic events that determine the fate of such axons are unknown but can now be revealed by imaging the spinal cord in vivo. Using this technique, I will address the following questions: (1) What dynamic behaviors are displayed by non-transected axons after contusion SCI, and which changes predict recovery or degeneration? (2) Which subcellular changes underlie these dynamic behaviors and determine outcome?

I will use newly developed imaging tools to follow single spinal axons over minutes to months. I will induce contusion injury of graded severity in transgenic mice expressing fluorescent proteins in superficial spinal axons, and follow the fates of contused but non-transected axons to determine which changes predict outcome. Subsequently I will analyze the subcellular events that precede these predictive axon changes in novel transgenic mice in which axonal transport, mitochondrial health, and cytoskeletal integrity can be assayed.

My study will provide the first dynamic picture of single-axon behavior after contusion SCI. It will inform about how and when therapeutic interventions should best be applied and allow examining other critical post-traumatic changes such as inflammation, demyelination and altered axonal excitability.

Cell surfaces carry a wide variety of molecular structures to enable biological functionality such as transmembrane transport or extracellular communications like cell-cell and cell-virus interactions. Some of these structures show altered patterns in terms of abundance and/or structure in various diseases. Detecting such changes in living organism would lead to advances in our understanding of systems biology and deliver new tools for the diagnosis of disease. Several types of cancer are related to over expression of the transferrin receptor and show a modified structure of glycans that can also be observed at the onset of inflammation. The glycolysation and receptor profile is therefore often conceived as a code for cellular physiology and understanding of this code is an emerging field of research to decipher the biology of such diseases at a molecular level. We therefore propose a new approach that enables comprehensive cell profiling by means of addressing multiple parameters in a single setup that will ultimately be transferable to in vivo applications. Using xenon biosensors and the Hyper-CEST technique results in an overall 10 million-fold sensitivity enhancement compared to conventional NMR detection. We will develop an NMR bioreactor and remote detection techniques to utilise the high sensitivity and specificity of the xenon biosensors to demonstrate multiplexed cell tracking in cell culture and compare this approach to other NMR sensing techniques. These biosensors will also be used to detect the transcytosis of transferrin across the blood-brain barrier using in vitro models.

In most animals, the mother deposits mRNAs and proteins in the egg so that the embryo can undergo development until the activation of its own genome occurs [20]. How the zygote integrates signals that control maternal mRNA transcripts with those that activate the zygotic genome remains poorly understood. I propose to study the maternal-to-zygotic transition (MZT) in zebrafish during embryogenesis. First, using different experimental conditions, I will determine the different mechanisms of decay for maternally provided mRNAs (dependent on egg activation, fertilization and zygotic transcription). This approach will establish which mRNAs are controlled by maternal products, zygotic genes, and microRNAs. Second, using motif prediction software and statistical tools, I aim to identify the motifs responsible for each degradation mechanism. By combining yeast three hybrid assays, mRNA deletion constructs and in vivo mRNA injection, I propose to identify the core proteins, the cis-regulatory elements responsible for the post-transcriptional regulation of the maternal program and investigate their function during vertebrate development. Finally, I will use high-throughput sequencing to analyze whether alternative polyadenylation usage and splicing allow the differential regulation of maternal vs. zygotic mRNAs.
These experiments will define the RNA motifs and the recognition factors responsible for the regulation of maternal mRNAs. These results will uncover the mechanisms of mRNA turnover during embryonic development at the maternal-to-zygotic transition and are likely to shed light into the general mechanisms of RNA regulation in other biological contexts.

How do gene mutations lead to cancer? Myeloid leukemia is one of the best model systems to address this question, because hematopoiesis and myeloid differentiation are well understood and many mutations of genes implicated in myeloid leukemia evolution have been reported. Among them, the TET oncogene family member 2 (TET2) mis-sense mutations have been identified in several independent cohorts of patients with massive myeloproliferative disorders. However, the mechanism by which TET2 mutations cause these diseases is completely unknown. Importantly, TET1 catalyzes the conversion of 5-methylcytosine in DNA to 5-hydroxymethylcytosine, which hints that epigenetic regulation plays critical roles for leukemia development caused by TET2 mutations. The major goal of this project is to reveal how the TET2 mutations are involved in the pathogenesis of myeloid malignancies. To do this, an interdisciplinary strategy combining genomics, proteomics molecular biology and cell biology will be employed. The genome-wide approaches will be employed to profile the target genes of TET2 in hematopoietic stem cells (HSCs), with subsequent validation by traditional methods. TET2 protein complexes will be purified using proteomics approach to identify the proteins involved in TET function and regulation. The importance of TET2, its regulator proteins and their target genes on HSCs differentiation will be confirmed by cell biological analysis and murine model. This project will shed light on epigenetic regulation of TET2 proteins in hematopoiesis and contribute to our understanding of how myeloid cells acquire the malignancy through TET2 mutations.

In neurons, microtubules in axons and dendrites are organized differently. The microtubule bundles in the axon are uniformly polarized, with their plus ends distal from the soma; while in dendrites, microtubules have mixed orientations. Microtubule (MT) polarity in neurons is essential for the establishment and maintenance of the structure and function of axons and dendrites. However, how MT polarity is initiated and maintained remains largely unknown. The aim of this proposal is to develop and apply different genetics and cell biological tools to gain insight into the mechanisms that govern neuronal MT polarity. A forward genetics screen in the nematode C. elegans will be used to identify molecules crucial for neuronal MT polarity in DA9 neurons. To visualize MT organization in C. elegans neurons, two markers will be constructed. One of the markers specifically localizes in dendrites and soma, while the other accumulates at axonal tips. The localization of both of these markers is dependent on the polarized MTs. Worms with mutations, which will be chemically induced by EMS, will be screened according to the fluorescence and localization behavior of the markers.

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease share the common features of dendritic atrophy such as dendrite branch reduction and spine loss. Down syndrome has a progressive postnatal reduction in dendritic arbor of cortical neurons. Although dendrite maintenance is critical for the function of mature neurons and implicated in many neurological diseases, little is known about the mechanism underlying this especially in mammal. The focus of my research is to elucidate the mechanism of dendrite maintenance in mammalian brain. Specifically, I will address the role of Polycomb group (PcG) genes in dendrite maintenance in vivo in mouse brain. Considered as repressive chromatin remodeling factors, the PcG complexes play an important role in orchestrating DNA-based transcription and the maintenance of cellular memory. Recently, a study showed that PcG genes are necessary for dendritic maintenance in Drosophila. Many PcG genes are also expressed in adult rodent brain, but whether they are critical for dendrite maintenance in mammal is unknown. In vivo delivered short hairpin RNA by lentivirus will be used to knockdown PcG gene expression in mature neurons in adult mouse. Further, I will apply MADM method and Cre-Loxp system to conditionally knockout PcG genes in small subset of neurons. Finally, I will explore how dendrite maintenance deficit induced by PcG genes mutation affect neuronal function. This project will expose me in many new approaches like lentivirus packaging and state of the art mouse genetics. As a leading group in developmental neurobiology, the Jan lab will provide excellent environment for my training.