The activation of leukocytes plays an important role in immunity to control pathogen invasion. For instance, in macrophages, Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and induce macrophage activation and functioning, whereas in T-cells, the T-cell receptor (TCR) recognizes foreign antigens and initiates T-cell activation. However, beyond TLR and TCR signaling, relatively little is known how macrophages or T-cells are activated. Recent studies have now indicated the involvement of G-protein signaling in this process.
In the proposed study, I will analyze how G-protein coupled receptor (GPCR) signaling is involved in the activation of leukocytes. Using macrophages and T-cells, I will analyze which G-protein pathways are stimulated upon leukocyte activation. After determining the involved pathway(s), I will molecularly dissect these pathways using specific inhibitors and reporter systems. Finally, I will screen for the involved GPCRs in non-leukocyte cells expressing the complement C3 receptor and different GPCRs.
Together, to our knowledge, this study aims for the first time to systematically analyze the involvement of GPCRs in leukocyte activation. Thereby, this work will not only advance our understanding of leukocyte functioning and activation, but may also lead to novel drug targets against infectious and autoimmune diseases.

Protein degradation has two basic functions: (i) to eliminate abnormal proteins and (ii) to limit the lifetime of specific proteins and thus make them sensitive to regulation that can rapidly alter their activity levels. The latter function is often utilized during development. While previous studies have shown that the ubiquitin-proteasome system (UPS) is required for proper development, little is known about mechanism that controls UPS in response to developmental cues. To understand how these machineries are assembled and recognize specific substrates during development, I will utilize C. elegans oocyte-to-embryo transition system, in which the degradation of meiosis specific protein MEI-1 occurs in response to oocyte maturation. E3 ubiquitin ligase (E3) and posttranslational modification of Cullin regulates MEI-1 degradation. Interestingly, some of these components have both nuclear localization and export signals (NLS and NES), suggesting that they shuttle between the nucleus and cytoplasm. Moreover, the subcellular localization of E3 and MEI-1 are distinct, suggesting that the compartmentalization of these components is developmentally regulated. To analyze E3 assembly and substrate recognition, I will visualize these complexes and modified Cullins using Proximity Ligation Assay (PLA) to obtain spatial and temporal information that is not obtainable with methods used in previous studies. Concurrently, I will analyze the effects of NLS and/or NES depleted subunits on E3 assembly and function.

Analyzing the molecular mechanism of timely regulated E3 assembly and target recognition will contribute to a broader understanding of the developmental control of UPS.

Mental illnesses have been associated with immune responses. One well-documented example of this is the anxiety and depression found in Systemic Lupus Erythematosus (SLE) patients, referred to as autoimmunity associated behavioral syndrome (AABS) in the MRL/lpr and NZB/W mouse models of SLE. Although AABS has been characterized in these mouse models, underlying molecular mechanisms remain unknown due to the burden of backcrossing genetic deficiencies, from the C57BL/6 (B6) strain, onto these complex strains. The study of AABS on a B6 derived model of SLE would enable efficient study of required factors. We propose to study AABS in sanroque mice, a T cell driven B6 derived model of SLE. We will use the sucrose test, novelty-suppressed feeding test, marble-burying test, open field test, elevated plus maze test, and rotarod test. We will track elevation of anti-nuclear antibodies and cytokines in the serum of mice as this correlates with induction of AABS. We will then use flow cytometry and histology to characterize in more detail the lymphocytes infiltrating the CNS. Work by others suggests we should expect to find T cells producing interferon gamma (IFNg). Subsequently, we will test if IFNg is required for AABS during SLE using blocking antibodies. Finally, with extensive backcrossing or the availability of sanroque mice as a model, we will test genetically deficient mice, and use them to make standard and mixed bone marrow chimeras, to determine if and how AABS requires IFNg. This work will advance the field toward determining the precise molecular mechanisms of AABS and toward targeted therapies for SLE patients.

X-ray crystallography is one of the most powerful methods to reveal the structure and mechanism of biomacromolecules. However, some molecules, in particular many RNAs and ribonucleoproteins have properties unsuitable for crystallization: multiple conformation, structural flexibility as well as vulnerability to thermal or chemical denaturation. Engineering them for higher stability can improve their chances to be crystallized, but the engineering process is time-consuming, because it requires iterative expression and analysis of individual clones. We propose a synthetic biological approach to build a quick screen for crystallizable mutants. In this approach, the genotype and phenotype of a variant will be linked by co-segregation of its gene, encoded enzyme, and reaction system in a water-in-oil emulsion. This method would allow quick and efficient selection of variants with desired, high stability phenotype, which would then be iteratively screened for crystallization using high-throughput methods.

We will apply this approach to targets which contain RNA and have proven largely resistant to crystallization, 1) the RNA polymerase ribozyme, and 2) a ribonucleoprotein, telomerase. An artificial RNA polymerase ribozyme has been engineered by synthetic biological approach and is thought as a modern equivalent to the central ribozyme in the RNA world. Structural information of this ribozyme would greatly help to engineer it further, and to understand its function in mechanistic detail. Telomerase is a key enzyme of genome maintenance. A crystal structure of full-length human telomerase would serve as a basis for functional understanding of telomere synthesis and drug discovery

In the mammalian brain, dopamine (DA) transmission in the prefrontal cortex (PFC) is known to play an essential role in mediating executive functions such as decision making and working memory. How the DA signal exerts its influence differentially on distinct executive functions in the PFC, however, remains to be understood. In this project, I will tackle this question by applying optogenetic perturbation methods to mice performing a delayed response task that engages executive functions in the PFC. Although numerous studies have shed light on DA’s role in executive function in the PFC, the precise mechanisms by which DA modulates these functions remains poorly understood. By exploiting the high cell-type specificity and temporal/spatial precision of optogenetic methods, I will be able to avoid the issues such as poor cell-type specificity and/or the decreased temporal and spatial control of perturbations encountered in previous studies. In this project, I will first exploit the mouce’s superb capacity for olfaction by using a delayed-response task with olfactory cues. Second, I will be able to control neurons in the PFC and the ventral tegmental area (VTA) using channelrhodopsin-2 and halorhodopsin. Third, I will record and analyze the neuronal responses in the PFC during specific periods of the task, thus enabling direct comparison of the effect of DA signals on neuronal activity and on animal behavior under specific cognitive demands. By integrating these strategies, I will elucidate the differential effects of dopaminergic modulation on prefrontal executive functions such as working memory and decision making.

The emergence of structure and function from the collective interactions of individual components such as biopolymers, cells and even whole organisms is an often seen but very poorly understood facet of the living world. We would like to use a bacterial system as a model to investigate collective behavior emerging in a predator-prey system. Such behavior gives rise to the rich socio-biology and evolution of co-operation in microbes. In particular, we want to study collective Myxobacterial behavior that manifests as a co-operative predation phenomenon of Myxococcus Xanthus, known as the predatory rippling motility, when they invade colonies of E. Coli. Using a combination of techniques such as microfluidics, genetic engineering and 3-D fluorescence microscopy, we would like to develop a picture of the events, at the level of individual cell interactions that lead to the large scale collective rippling motion of the Myxobacteria. The experiments would be performed with a view to isolate the purely physical mechanisms that lead to the collective behavior from those involving biochemical signaling. We will then use the knowledge of the individual interactions and apply physical and statistical mechanical theories on self-organization, to extract general principles that would be pertinent to a range of biological phenomena. We anticipate that such an approach will have the potential to generate a huge leap forward in our understanding, not just of M. xanthus predation, but also of how individual cells coordinate their activities to form large-scale structures in general, opening potentially new ways of thinking about biological organization.

Cancer progression often involves mutations in well-known oncogenes and tumor suppressors, but also depends on aberrations in other genes whose roles are only partially understood. These additional genes may play a major role in determining the unique features of specific cancer cells, and are thus of tremendous importance. In particular, chromatin factors have recently emerged as a specific class of recurrently-mutated genes in cancer, but their normal and aberrant functions are poorly understood. To decipher their role in cancer progression, I will study the effect of recurrent mutations in selected chromatin regulators on cancer gene expression and on genome-wide knock down effects. First, I will use available molecular and phenotypic profiles of a large set of cancer cell lines to develop computational models that predict which genes are essential for each cell-line. I will identify chromatin regulators with recurrent mutations and a predicted role in cancer and further explore their impact by deeply characterizing the epigenetic states of cell lines with and without such mutations. Finally, I will use computational modeling to generate specific hypotheses regarding the function of chromatin regulators, which will be further refined and tested by knocking down these regulators.

Among vertebrates, urodele salamanders show the highest potential for regenerative responses to injury-inflicted tissue loss. The neotenic salamander species Axolotl has proven particularly informative to study the process of limb regeneration after amputation, a multi-step process involving wound healing, blastema formation, re-growth and -patterning. Classical approaches such as grafting experiments have yielded key insights into these processes; yet the lack of a controllable genetic system has hampered their understanding at a more molecular level.
Here, I propose a novel combination of two different transgene delivery methods to circumvent these caveats. Their use will make the Axolotl limb regeneration system amenable to rapid and targeted interferences of gene function. Specifically, I will establish transgenic Axolotls expressing the avian retroviral receptor TVA in a tissue-specific manner, thereby allowing for viral-mediated mis-expression or knockdown of target genes. Delivery of TVA-expression constructs will be achieved through Tol2 transposon-mediated transgenesis. I plan to enhance the degree of transgene mosaicism in founder animals, by using recombinant transposase protein. This will by-pass the long generation time of the Axolotl system, (approx. 1 year to sexual maturity), and enable me to perform regeneration experiments straight in F0 animals.
As a proof of principle, I will apply the proposed transgenic toolkit to address the question of joint regeneration, as a model for the re-establishment of signaling centers and potential cellular re-specifications during vertebrate limb regeneration.

Metabolism-first theories on the origin of life state that there may have been prebiotic systems capable of propagation and Darwinian evolution that were not dependent on genetic, nucleotide-based replication. However, to date metabolism-first theories are not supported by experimental evidence. We propose to test this idea experimentally by observing the formose reaction compartmentalized in protocell analogues. Using high-throughput microfluidic techniques, we will react a simple formaldehyde feedstock in aqueous microdroplets, which can be split and fused to simulate protocell division and growth, respectively. We will identify the carbohydrates produced in the droplets, to determine if there is compositional heredity between generations of protocells. We will also investigate how the system responds to selection pressure: whether the sizes of the droplets or initial stochastic variations in their starting contents affect the nature and complexity of the carbohydrates produced. We hope to show that a network of chemical reactions is capable of heredity and evolvability, in the absence of canonical genetic macromolecules.

Over the last years much attention has been given to a new class of long non-coding transcripts that apparently does not code for proteins. While a few have been shown to be functional as RNAs, recent evidence from both genome-wide and targeted approaches suggests that a significant number of these transcripts harbor short open reading frames that are translated in vivo. Building on large-scale sequencing projects, the goal of my postdoctoral research is to discover and characterize novel secreted peptides that regulate embryonic development.

Aim 1 builds on preliminary research in the Schier lab that has identified a highly conserved, secreted short peptide expressed in a developmentally regulated manner in zebrafish embryos. Preliminary morpholino knockdown experiments suggest that this peptide is an important developmental regulator with potential roles in blood and/or mesoderm development. I will perform an in-depth characterization of the function of this particular peptide during embryogenesis.

In Aim 2 I will use polyribosome profiling, a new experimental technique for the genome-wide identification of translated regions, to discover novel short peptides expressed during embryogenesis. In addition to analyzing translated regions at different stages of embryogenesis in wild-type zebrafish embryos, I will identify peptides that are regulated by Nodal signaling, a pathway essential for mesendoderm induction. Combined with expression data from existing RNA-seq data and conservation analysis, I will phenotypically characterize promising candidates and identify novel signaling pathways that regulate development.

We have previously discovered that there is a characteristic chromatin profile for embryonic pluripotency and that this profile is established during the maternal-zygotic transition. While our studies have begun to clarify the in vivo relationship between chromatin state, the onset of zygotic transcription, and pluripotency, they have raised several important questions that are the focus of this proposal. First, what is the functional relationship between the appearance of specific histone modifications, the activation of the genome and embryonic pluripotency? To address this question I will interfere with histone modification marks in vivo, both genome-wide and gene-specifically, and analyze the effect on transcription and pluripotency. Second, how is the chromatin signature characteristic for pluripotency set up during development? Here, I will use a combined experimental and computational approach to predict whether sequence motifs and/or pre-existing histone modifications direct the recruitment of histone methyltransferases to their targets and test these predictions in vivo. Finally, it is unclear how developmental signaling pathways interact with chromatin to establish and maintain lineage-specific gene expression programs. I will analyze the chromatin profile of Nodal target genes during the transition from pluripotency to fate specification in the presence and absence of Nodal signaling in order to address this question. Together, my in vivo studies will clarify the role of chromatin modifications in the transitions between genome activation, pluripotency, and cell fate specification.

The immune system and the nervous system on the other have been considered autonomous systems operating independent of each other. However, a recent discovery described that neurons are able to attenuate the release of pro-inflammatory mediators from cells of the immune system, via the secretion of acetylcholine. This challenged the long maintained paradigm and established an important interaction between the immune and nervous system. While it has been shown that expression of the nicotinic acetylcholine receptor (nAChR) on macrophages is essential for reduced cytokine production, the contribution of this specific modulator on function and cytokine release of immune cells other than macrophages is unclear. Additionally, the contribution of this particular neuro-immune loop during the initiation, chronic or resolution phase of infections remains elusive. To reveal the relative contribution of acetylcholine as a specific immune modulator during infections I am going to investigate the effect of acetylcholine signaling on immune cells during different phases of skin infection. To address the relative contribution of T cells, innate lymphocytes and dendritic cells I will make use of mouse strains were deficiency of the nAChR is limited to those immune cells. Additionally, I am aiming to identify new factors of neuro-immune regulation by performing gene expression analysis on neurons from the dorsal root ganglia following skin infection. This approach aims to establish previously unrecognized neuro-immune interactions during various models of skin infection as a general mechanism of the fine-tuning of an ongoing immune response by the nervous system.

In the quest to understand biophysical principles that govern protein folding (i.e. the process through which polypeptide chains adopt their functional three dimensional structure), single molecule spectroscopy offers new insights into stochastic nature of structural or kinetic factors that drive this process, not detectable through conventional spectroscopic techniques that allow ensemble averaged measurements. To this end, I propose to use single molecule experiments to investigate the early stages of folding; where the nascent chains are just emerging from the ribosome, the machinery that produces proteins inside the cell through a process called translation. The goal of this proposal is to resolve the underlying heterogeneity in nascent protein conformations previously unaddressed in the myriad of ensemble approaches used to investigate co-translational folding. In particular, I will investigate the role of non-native competing protein-ribosome interactions. The methodologies developed and showcased here can easily be extended to studying other systems such as natively disordered proteins where stochastic properties of conformational heterogeneity has been postulate to contribute to promiscuity in function allowing natively disordered proteins to participate in various functions in side the cell. This proposal provides fundamental insights into the nature of forces that drive folding; especially at early stages where nascent chains face cellular milieu for the first time. I hope to be able to resolve this heterogeneity in vivo at single molecule level inside the cell to bridge the gap between what in vitro and in vivo studies of protein folding and function.

Cell membranes composed of thousands of lipid species, differing in their alkyl chains, headgroups and degree of saturation. Changes in lipid composition or even the absence of a single lipid have shown to lead to severe pathologies and death. The leading hypothesis which explains the role of lipids in membrane functionality is that the lipids segregate into distinct domains. These lipid domains can with high specificity incorporate or exclude proteins, hence inhibit or accelerate biological processes at the membrane surface. Knowledge of protein-domain interactions is essential to understand cell functionality, and yet today we cannot distinguish between the thousands of possible lipid domains, nor is there a method to determine with which domains a protein associates with.
This research would provide a new methodology, which will determine the lipid composition a protein has a high affinity to. The method utilizes a liposome library composed of ~1500 liposomes, each made of a different lipid composition. The library, which will be created by advanced microfluidics, has the largest and most diverse composition of lipid liposomes ever made. Protein-domain interactions will be measured on all liposomes in library. These results will be strengthened by in vivo assays, where the effect of manipulating the synthetic pathway of the lipids on protein localization will be monitored.
The results in this study may explain the diversity of lipid composition among different cells and cell organelles, and could have big implications on fields such as membrane targeted drugs, virus-membrane interactions, drug delivery, etc. They may also change our basic concepts of membrane function.

Neurodegenerative disorders, including prion diseases, are associated with protein aggregation. Considerable evidence indicates that the extent of neurotoxicity or infectivity of the oligomeric or aggregated proteins is strictly dependent on their structures. The proteins involved in these diseases are conformationally heterogeneous and, according to an emerging view, can switch back-and-forth between functional and various amyloidogenic/prionogenic conformations (strain conformations). Since the various conformations present in these heterogeneous ensembles lead to different aggregated forms, characterizing the monomer conformational space is critical to understand their pathogenecity. However, the characterization of the conformational heterogeneity of monomeric proteins by standard structural biological methods has proven to be extremely challenging. Single-molecule methodologies offer exciting opportunities to increase our understanding of protein conformational equilibrium. This project is aimed at using a novel approach that combines the two main methods for single-molecule manipulation, Optical Tweezers and Atomic Force Microscopy, to map the conformational energy landscapes of a mammalian prion protein (PrP) and yeast prion Sup35 protein, as well as their monomer conformations that promote the aggregation process. Mouse PrP is an ideal candidate for prion conformational studies due to the possibility of creating different infectious conformations in a test tube and of introducing them in mice to develop novel prion diseases. Similarly, the ability of Sup35NM, a prion domain of Sup35, to convert into prion states in a test tube and the possibility of introducing them in yeast, make the yeast prion [PSI+] system uniquely suited to investigate the mechanism by which a single polypeptide (Sup35NM), displaying a heterogeneous ensemble of conformations, can form initial nuclei and misfolds into self-propagating distinct amyloid conformations. The synergic integration of these single-molecule techniques will make it possible to explore conformational properties of both mammalian PrP and yeast prion that are inaccessible to more classical in-bulk ensemble-averaged methods, and to study the nature of the energy surfaces over which these molecules diffuse as they move between their different strain conformations and towards those that are responsible for triggering the aggregation processes and pathogenesis.

Ubiquitination is used by all eukaryotic cells to regulate the localization and turn-over of membrane proteins. Ubiquitination is mediated by ubiquitin ligases. This proposal brings together four researchers working in three continents to characterize a family of such ligases called MARCHs. The MARCHs play major immunoregulatory roles and are anchored to membranes, a unique feature among ubiquitin ligases. Indeed, they recognize their substrates via transmembrane (TM) region interactions, itself a poorly understood phenomenon. We will apply our expertise in immunology, cell biology, proteomics, solution NMR and computation biophysics to fulfill the following aims:

AIM 1: To identify MARCHs substrates

Aim 1.1: We will characterize the expression pattern of MARCH genes in the immune system.

Aim 1.2: We will use label-free quantitative proteomics of plasma membrane (PM) fractions to identify proteins with altered surface expression levels in cells deficient in MARCH 1, 2, 3, 4, 8 or 9 (MARCH substrates).

AIM 2: To characterize the structural basis of MARCH substrate recognition via TM interactions

Aim 2.1: We will determine the role of structural motifs in the TM regions of the MARCHs and their substrates in MARCH-substrate recognition.

Aim 2.2: We will characterize the three-dimensional arrangements of TM regions of the MARCHs and their substrates using solution NMR.

Aim 2.3: We will apply computational modeling to the study of interactions between the TM regions of the MARCHs and their substrates.

Aim 2.4: We will generate chimeric mice with bone marrow retrovirally transduced to express mutant forms of the MARCHs to validate the conclusions of the structural studies in vivo.

The knowledge we will gain with this project will be highly significant at multiple levels. It will contribute to understanding the structural basis of TM-mediated protein-protein recognition and the role of ubiquitination in regulation of membrane protein expression and localization, an activity present in all eukaryotic cells. As the MARCH substrates that we will identify are likely involved in immunoregulation, this project will also be relevant to immunological tolerance and the initiation of immunity. Finally, our project will lead to refinement of strategies for computational modeling of three-dimensional structures of membrane proteins.

Neuronal morphology and function relies on microtubules for transport of proteins and membranes; impairment of these transport functions may contribute to neurodegenerative disease. Currently, little is known about microtubule dynamics in the axon. The prevailing view is that all axonal microtubules are produced by the centrosome and then transported down the axon as stable polymers. However, very recently, it was shown that axons grow in the absence of centrosomes. Thus, acentrosomal microtubule nucleation may have a major role in axon extension, although the mechanism is unknown. Two missing tools have hindered our understanding of axonal microtubules: visualization of individual microtubule dynamics in axons, and molecular tools for understanding microtubule nucleation in acentrosomal arrays. My first aim will be to apply modern light microscopy methods to visualize microtubule dynamics using rat hippocampal cells and the squid giant axon. My second aim will be to further characterize the microtubule landscape in the axon in terms of stability and post-translational modifications and understand these modifications in the context of microtubule dynamics, transport and function. My third aim will be to identify the roles of two new microtubule interacting proteins - Augmin and Patronin in neurons. Molecular reagents for Augmin-g-TURC complex may provide new information about microtubule nucleation along the axon. In addition, I will also explore the role of Patronin a protein involved in capping microtubule minus-end. These new imaging and molecular assays should provide the first details about the dynamics and properties of axonal microtubules.

All phenomena of the living world shape, and are shaped by, the process of evolution. In perhaps no other area of biology this fundamental duality becomes as evident as in the complex communication systems that mediate mate recognition in the animal kingdom. Both neurobiologically and evolutionarily, the task of recognizing a potential mate for reproduction is truly a multiple-task including the fundamental challenge of sex and species recognition as well as the more sophisticated exercise of taxing the potential mate’s fitness, i.e. its likely reproductive value. Unsurprisingly thus, when it comes to their mating decisions animals usually rely on signals from multiple sensory information channels. One sensory system that is frequently involved in the mating negotiations of both vertebrates and invertebrates is the sense of hearing: From marine mammals to air-borne mosquitoes, acoustic courtship rituals have evolved that include the generation, reception and subsequent analysis of species-specific sound signals. A prominent example is the acoustic courtship of flies of the genus Drosophila. As part of their mating ritual, male flies sing a ‘love song’ to their females. These songs differ across fly species and are deemed to contribute to both species isolation and speciation in Drosophilid flies. We here attempt to elucidate the evolutionary, material and mechanistic bases of species-recognition in the Drosophila acoustic communication system. Applying state-of-the art techniques in biophysics, bioinformatics, genetics, bioengineering, neuroanatomy, behavioural analysis, and in vivo electrophysiology, across the 12 recently sequenced Drosophila species, we will explicitly probe for species-specific mechanisms underlying the emission, reception, and processing of sound in the fly. Our project represents an unprecedented multi-level approach, not only to acoustic courtship in Drosophila, but to animal communication, molecular evolution and speciation in general.

Faithful chromosome segregation is essential for the proliferation of all organisms. Although studies in model eukaryotic organisms have found that the kinetochore complex formed on centromeres facilitates their chromosome segregation, it is not known if this mechanism applies to all eukaryotes. To uncover novel principles of the molecular machinery that drives segregation, I will study chromosome segregation in Trypanosoma brucei because this protozoan parasite branched early in eukaryotic evolution and has various unusual features. Trypanosomes lack a centromeric histone H3 variant that is essential for kinetochore assembly in all other studied eukaryotes. Trypanosoma brucei possesses large chromosomes that have centromeres and small chromosomes that lack centromeres, yet both types of chromosomes undergo accurate segregation during mitosis. Very little is known about the molecular machinery that segregates these chromosomes. I propose to identify associated factors by PICh (Proteomics of Isolated Chromatin segments) and yeast one-hybrid assays. Identified proteins will be systematically characterized by GFP tagging and RNAi depletion analyses to test whether they have roles in chromosome segregation. I will also identify domains that confer mitotic stability of small chromosomes that lack centromeres. These efforts should lead to an understanding of the segregation machinery in this evolutionary distant eukaryote.

Immune cells play an essential role in protecting the body from invading pathogens by directing pathogen specific responses. These pathogen-tailored responses are largely executed by changes in expression of specific gene programs. Recent studies suggest that mutations in cis-regulatory elements in the toll-like receptors (TLR) pathway affect diverse set of disease. There is currently great need to develop a mechanistic and predictive model of how these dynamic gene responses are generated and controlled.
My proposal addresses this fundamental question with the aim of providing a systematic and dynamic view of the regulatory network underlying the transcriptional response to pathogens. Using a comprehensive, high throughput chromatin immunoprecipitation approach followed by sequencing (HT-ChIP-seq), I will map the genomic localization of all major transcriptional regulators. This physical binding data will be combined with differential gene expression responses under pathogen stimuli in WT cells and following systematic perturbations. The integrated data will allow for quantitative predictive models of gene regulation. I will validate the model by careful genetic perturbation and biochemical purifications of specific factors and complexes predicted by the model.
The successful completion of this project will provide a physical regulatory network under which to understand the impact of misregulated proteins, and a model to analyze the impact of regulatory mutations in the human population. With the help of the CDA award, I hope to have the funds as well as the scientific support of the HFSP community that will enable me to tackle this ambitious goal.