Spatial and temporal control of transcription activity is fundamental in animal development and disease. Establishment of precise on/off pattern of gene expression is regulated by the interaction between enhancers and promoters. Enhancers provide binding platform for a variety of sequence-specific transcriptional factors, thereby facilitating assembly of active transcriptional machinery at the promoters. Enhancers are often located at thousands base pairs away from upstream or downstream of target promoters. By looping out intervening sequences, enhancers directly interact with promoters during gene activation. However, the dynamics of this process has yet to be determined.
To explore the kinetics of enhancer looping, I will develop novel live imaging and visualize enhancer-promoter interaction in a living cell. Recently, real-time monitoring of transcription activity in a developing fly embryo has been developed. This relies on fluorescent labeling of nascent transcript by using specific interaction between GFP-tagged bacteriophage MS2 coat protein and its cognate MS2 stem loops. By introducing RFP-tagged PP7 coat protein and PP7 stem loops into this system, I will develop two-color live imaging. By creating series of transgenes that contain a single enhancer located between MS2 and PP7 reporter genes, I will analyze the kinetics of enhancer looping among two target promoters. Furthermore, by combining BAC transgenesis and live imaging, I will directly visualize interaction between distal enhancer and target promoter in cis and trans. This research project will allow first quantitative measurement of enhancer looping in a living cell.

Avian feather patterning has long been studied as a model of two-dimensional periodic pattern formation. Each feather emerges from the site where embryonic dermal cells aggregate. Previous studies have suggested that the dermal condensation at regular intervals is based on the spatial patterns of interacting morphogens. However my recent finding implied that the existence of cell-cell interactions between two types of dermal cells: one is the dermal cells of outer boundary of feather buds and the other is those of interior of buds. Time-lapse observation showed a unique behavior of dermal cells of outer boundary. At the beginning of feather patterning, the dermal cells was uniformly distributed throughout the region beneath epidermis. As skin develop, the cells actively moved to avoid the center of feather buds and localized in the outer boundary, suggesting segregating mechanisms between two types of dermal cells. This looks like a meshwork in the skin. The patterning process of mesh structures seems to be directly involved in the size of buds and distance between each buds. Thus, in this proposal, I aim to uncover the molecular basis of the pattern formation. To this end, I will use reconstitution assay in which dissociated dermal cells can regenerate complete feather buds, allowing the analysis of cell interaction and easy gene-transfer into dermal cells. We have candidate genes that regulate the spacing feather buds, so I will also examine whether these genes are involved in the interaction of dermal cells by gene suppression. This study will provide new insight into developmental biology.

The nuclear pore complex (NPC) is an essential nuclear transport channel which is functionally maintained in the rat brain through adulthood without being renewed. The aim of this project is to reveal the mechanism by which long-lived cells maintain the scaffold nucleoporins (Nups), essential components for NPC structural integrity, over the lifespan of the animal. Preliminary results suggest that long-lived cells (neurons) may modulate the expression level and phosphorylation status of membrane Nup Pom121 to make scaffold Nups more stably associated with the nuclear envelope, compared to short-lived cells (hepatocytes). To validate this, in Aim1, I will first deplete Pom121 from neurons and examine the effects on the scaffold stability and NPC functions (transport, permeability barrier) using advanced microscopy techniques such as super-resolution microscopy. This will take advantage of a neuron long-term culture system which will allow me to perform experiments on the order of months. Second, using rat brain and liver tissues, I will identify the brain-specific phosphorylation sites of Pom121 by mass spectrometry and examine the role in NPC maintenance by mutating identified sites.
Besides stabilization of the scaffold in the NE, neurons may prevent damage accumulation to scaffold Nups by modulating the expression and phosphorylation of peripheral Nups, which attach to the scaffold and thus may protect scaffold Nups from toxic metabolites. In Aim2, I will investigate the role of peripheral Nup50 in neuronal NPC maintenance as described in Aim1.
In summary, this proposal will provide novel insight into how cells keep their long-lived proteome healthy during aging.

Traits can be inherited in a non-Mendelian fashion by a variety of mechanisms including transgenerational epigenetic inheritance. Previous research has shown that mutation of a C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase, SPR-5, causes progressive sterility and extended longevity (Katz DJ et al Cell 2009, Greer EL et al Cell Reports 2014, Greer EL et al unpublished). Previous work has shown that the H3K4me2 mark and a novel DNA modification increase in successive generations of worms lacking spr-5. However, how this non-genetic information is transmitted from one generation to the next is still unknown. The goal of this proposal is to identify the mode and mechanism of epigenetic transgenerational inheritance that leads to progressive generational sterility in C. elegans with mutated spr-5. I hypothesize that dysregulation of SPR-5 causes an increase in H3K4me2 which is “remembered” from generation to generation by an epigenetic memory of DNA methylation. My goal is to identify whether H3K4me2 or this DNA methylation event are transmitted from one generation to the next in instances of transgenerational epigenetic inheritance and to identify regulators of DNA methylation. Understanding how epigenetic inheritance is dysregulated in spr-5 mutant worms will enhance our understanding of how epigenetic signals are propagated from generation to generation. In the long term, I will determine whether these moieties are conserved and whether they play a conserved role in regulating transgenerational epigenetic processes in mammals.

Over 100 post-transcriptional RNA modifications are known with N6-methyladenosine (m6A) being the most frequent. m6A has been reported to play a role in many human biological processes (e.g. obesity and cancer), but the detailed mechanisms remain unknown. Detailed study of the function of m6A requires the site-specific introduction of m6A into specific RNAs. However, this appears difficult using endogenous m6A modification enzymes because only a few such enzymes modify a wide range of RNAs. Here, I propose a methodology for introducing site-specific m6A modification in vivo by engineering of the C/D small nucleolar ribonucleoprotein (snoRNP). The C/D snoRNP catalyzes the 2’-O methylation of a substrate RNA and consists of four proteins and a C/D snoRNA, which recognizes the substrate RNA by base pairing. Therefore, the substrate specificity of C/D snoRNP can be easily changed by altering the C/D snoRNA sequence. In this research, I will engineer the catalytic protein of C/D snoRNP to catalyze m6A modification using rational design and directed evolution. First, the catalytic core of C/D snoRNP will be redesigned based on the structure of known m6A modification enzymes. Then, the engineered C/D snoRNP will be randomly mutated to construct a library to perform directed evolution. After directed evolution, the activity and substrate specificity of the engineered C/D snoRNP will be determined. Finally m6A modifications of target RNAs will be altered to assess the biological response. A similar approach could also be applied to other RNA modifications and this technique has the potential to be a fundamental tool to control and study various RNA modifications.

Follicular helper T (Tfh) cells play a key role for inducing antigen-specific responses. Although substantial numbers of Tfh cells reside in the gut-associated lymphoid tissues, it is not fully understood how Tfh cells develop and whether Tfh cells enter the memory pool. To clarify the mechanisms for Tfh differentiation and maintenance in the gut, I will investigate commensal-specific CD4 T cells throughout the course of bacterial colonization. For this purpose, I will focus on segmented filamentous bacteria-(SFB) induced responses, because SFB preferentially induces both Th17 and Tfh cells. Since Th17 cells show plasticity into Tfh cells, I will examine the role of RORgt in Tfh cell development and memory T cell formation, using our established transgenic mice bearing SFB-specific T cell receptor. Next, to identify genes responsible for Tfh cell development, genome-wide analyses will be performed comparing gene expression profiles between wild type and RORgt-deficient Tfh cells. In addition, a proteomic approach will be used for identification of Bcl6 binding partners. For this analysis knock-in mice bearing tagged Bcl6 will be newly generated using the CRISPR-Cas9 system. In addition, based on these data, knockout-based screening will be done in primary CD4 T cells. Focusing on the genes most strongly associated with Tfh cell development, I will generate mutant mice using the CRISPR-Cas9 system. Using these mice, I will investigate molecular mechanisms by which the targeted gene regulates the Tfh program in coordination with Bcl6 in vivo. Through this proposed project I will elucidate how Tfh cells develop and are maintained in response to luminal bacteria.

NAD+ (nicotinamide adenine dinucleotide) is required for redox reactions in various metabolic pathways and is a co-substrate for the sirtuins (SIRT1-7), a family of protein acetylases. Recent data that I have generated shows that protein O-GlcNAcylation is increased in white adipose tissue (WAT) and brown adipose tissue (BAT) during aging. Importantly, Increase in O-GlcNAcylation during aging was reversed by treatment with a molecule that raises intracellular NAD+ levels, nicotinanide mononucleotide (NMN). I intend to investigate role of NAD+, O-GlcNAcylation, and the role of Sirtuins, on adipocyte function and transdifferentiation in the context of aging via the following three aims:. Firstly, the role of NAD+ in regulating O-GlcNAcylation by modulation of flux through the hexosamine biosynthetic pathway (HBP) will be investigated. This is because O-GlcNAcylation depends on the availability of its donor, UDP-GlcNAc, a metabolite synthesized by the HBP that requires metabolites derived from glucose, amino acid (glutamine), fatty acid (acetyl-coA), and nucleotide metabolism. Secondly, I will determine whether sirtuins (SIRT1-7), which are NAD+-dependent deacetylases, regulate O-GlcNAcylation. Sirtuins regulate many important pathways and play a pivotal role in the maintenance of energy homeostasis. We hypothesize that sirtuins may regulate O-GlcNAcylation by changing the activity of OGT and/or OGA, the ability of their substrates to bind, or by changing their expression levels. Lastly, the physiological role of O-GlcNAcylation in the adipose tissue during aging will be evaluated. I intend to identify O-GlcNAcylated proteins which are regulated in an NAD+-dependent manner.

Primary cilia are microtubule-based organelles that project from the cell surface. Primary cilia are well established as important sensory and signaling structures, in which a plethora of signaling receptors, such as G protein-coupled receptors (GPCRs), are concentrated. A dysfunction of primary cilium results in several manifestations called a “ciliopathy”. The deficiencies include retinal degeneration, polycystic kidneys, and obesity, where the pathogenesis mechanisms remain largely unknown. While several classical studies indicated the relationship between ciliopathy-related obesity and leptin resistance in neurons, a very recent study clearly showed that the leptin-resistance arises as a consequence of obesity. Another unexplored mechanism that could contribute to obesity would be a defect in adipogenesis. In this study, I will try to verify this hypothesis. In one branch of the study, I will identify the ciliary GPCR that controls adipogenesis in 3T3-L1 preadipocytes, based on its ability to differentiate preadipocytes, ciliary localization, and dependence on the BBS and Tubby pathways, both of which regulate GPCR-trafficking and body weight control. To verify the relationship between the defect in the GPCR signaling and increased fat pad in vivo, I will examine the transplantation of preadipocytes with or without the GPCR in athymic mice. Moreover, I will identify the downstream signaling of the GPCR by gene expression-based, localization, and protein network analysis.
Collectively, this study will provide the new knowledge of the signaling pathway in primary cilia, and critical insight into how the deregulation of this signaling causes obesity.

Synthetic biology offers the possibility of new therapies based on functional cellular implants to cure intractable diseases, such as immune diseases and metabolic diseases. For example, the need for diabetics to inject insulin after every meal could potentially be avoided by introducing functional cellular implants that rapidly release insulin on demand. This would dramatically improve patients’ quality of life. However, it is difficult to rapidly control protein secretion in response to stimulation because current artificial cellular functions rely mainly on slow transcriptional control of transgenes within the cells.
My proposed project aims to create new cellular functions capable of rapidly modulating biological systems by secreting effector molecules outside the cells in response to external stimuli. It is established that a well-regulated exocytosis pathway exists in a wide variety of cells and can be artificially reconstructed in artificial liposomes. Therefore the project will focus on creating new, rapidly responsive cellular functions by artificially modulating the exocytosis pathway in living cells.
I intend to validate this design approach for innovative future cell-based therapies by constructing a new therapeutic model. Specifically, I propose to construct a model system for treating rejection after transplantation without the need for administration of immunosuppressive drugs, by incorporating artificial cellular functions into the surface layer of the transplant. These functions will serve to sense contact of active T cells and to rapidly secrete T-cell-blocking effector molecules only in the immediate vicinity of the transplant.

Endocytosis plays key roles in various cellular events. However, it remains largely unknown when, where and how endocytosis occurs in each cellular activity, mainly because it is impossible by conventional imaging techniques to visualize the structural dynamics of endocytosis in living cells. Here, I aim to unravel the nature of endocytosis by taking advantage of both two-photon microscopy and high-speed atomic force microscopy (AFM). I will optically identify endocytosis events using two-photon microscopy and visualize the events with nano-meter scale using high-speed AFM. Since endocytosis is involved in a variety of cellular activities and mediated by various molecules, I hypothesize that endocytosis consists of various structural forms. First, I will investigate the location, time-course and shape of endocytosis taking place in cell lines to classify the dynamic structures of endocytosis. Second, I will identify the molecules responsible for each type of endocytosis by gain- and loss-of-function analysis of candidate genes in cell lines. Third, I will reveal the functional relevance of each type of endocytosis by observing when, where and how endocytosis occurs in neurons during synaptic plasticity, in which endocytosis is known to play an essential role. Furthermore, I will clarify the spatio-temporal relationship between molecular activity, endocytosis events and structural changes of dendritic spines during synaptic plasticity by combining two-photon uncaging of caged glutamate, two-photon fluorescent life time imaging microscopy and high-speed AFM. This proposed study may provide new insights into the nature of endocytosis overlooked by conventional techniques.

Epithelial cells have an apical-basal polarity, which is regulated by polarity proteins. Lgl is an important polarity protein that localizes to basolateral membrane. The fly genetic studies suggest that Lgl functions not only as a polarity protein but also as a tumor suppressor.
In the breast cancer cells, the downregulation of Lgl is frequently observed but the pathophysiological significance of Lgl is not understood. Thus, this research project aims to reveal the molecular mechanisms on how Lgl contributes to the development of mammary carcinoma. Especially, this project will focus on the effects of Lgl on cell proliferation and try to reveal the molecular mechanisms on how Lgl regulates cell proliferation in mammary epithelium. To achieve the goal of this project, I designed several experiments.
First, to examine the effects of Lgl in mammary epithelial cells, Lgl will be knockdowned in mammary cells using RNAi techniques. The effects of Lgl-downregulation on cell proliferation will be examined in the culture condition of 3D space, which enable the mammary cells to form acinus structure in vitro. In addition, the effects of Lgl-downregulation on tumorigenicity will be examined by xenograft assay. Secondly, to confirm the results obtained from cultured cells, the phenotype of Lgl null mice will be examined. Finally, the molecular mechanisms on how Lgl-downregulation contributes to carcinogenesis will be addressed. Briefly, by using global analyses such as expression analyses with microarray and mass spectrometry, the downstream molecules of Lgl, which relates to cell proliferation, will be searched to understand the Lgl-mediated mammary carcinogenesis.

Axon degeneration is a tightly controlled process of axon self-destruction that plays both a physiological role during development and pathophysiological role during neurodegeneration. Following a physical insult, such as a nerve transection or crush, the axon segment distal to the injury site undergoes rapid degeneration, a process that is called Wallerian degeneration. In addition to its role in axon degeneration distal to an injury site, Wallerian and Wallerian-like degeneration are also prominent features of numerous neurodegenerative diseases. However, the molecular mechanisms underlying Wallerian degeneration are poorly understood.
To identify molecules that are critical for Wallerian degeneration, I will perform a genome-wide RNAi screen. The physiological significance of the most promising candidates will be evaluated using cultured sensory neurons and genetically modified mice. I will further characterize functions of the candidates with biochemical and cell biological methods and define where the candidates reside in the Wallerian degeneration pathway. Previously, Prof. Tessier-Lavigne team found that the genetic deletion of SARM1 protects against Wallerian degeneration. However, the molecular function of SARM1 remains elusive. As such, SARM1 will also be analyzed using these methods. Finally, I will examine the function of SARM1 and the other Wallerian degeneration molecules in animal models of neuropathy. I expect that these studies will advance our understanding of the mechanisms regulating axon degeneration and will also provide insight into how to therapeutically target this pathway for the treatment of neurodegenerative diseases.

It has long been assumed that because early mammalian development relies on almost immediate zygotic transcription, the requirement for translational control during embryogenesis and pattern formation may not be very significant. Our findings have, however, uncovered a surprising role for ribosome-mediated specificity in tissue patterning and gene expression. In particular, our studies show that expression of Homeobox (Hox) mRNAs is tightly regulated at the translation level to promote restricted patterns of protein expression within the developing vertebrate embryo. This strongly suggests that tight regulation in translational control of transcripts, largely at the level of 5’UTR activity, may provide a critical additional layer of regulation to the mammalian genome. At present, there is a strong bias in examining patterns of gene expression within the developing vertebrate embryo at the transcript level and we lack an even basic understanding of which transcripts are being translated into functional proteins. In this proposal, I will apply a new powerful technology that I have optimized for small embryos known as ribosome profiling to delineate the translational landscape of gene expression in mesoderm, one of the most important cell lineages specified during vertebrate development. This will provide unprecedented insight into the classes of mRNAs and the topography of regulatory elements positioned in their 5’UTRs that are regulated at the translation level. I will extend these studies into chick embryos where I will be able to subsequently functionally characterize regulatory elements that impart transcript-specific translation control.

The centrosome is the major microtubule organizing center in animals and fungi, being implicated in several human diseases. The control of centrosome number, structure and localization is poorly understood. I will take advantage of comparative genomic and synthetic biology approaches to study how those structures are assembled. Recent studies suggest that the common ancestor of animals and fungi had, similarly to extant animals, a centrosome composed of two centrioles. Fission yeast lost the centriole and the genes needed to assemble it, having a centrosome equivalent, the spindle pole body (SPB). Despite their difference in structure, animal and yeast centrosomes share components, and both assemble only once per cell cycle and close to an already existing centrosome. This suggests a conserved ancestral program regulates centrosome assembly. I will use the fission yeast system as a “test tube” to study centrosome assembly, by expressing and studying the behavior of Drosophila centriole proteins in fission yeast. I will subsequently validate my findings in Drosophila. Preliminary experiments performed by the host laboratory show that Drosophila centriole proteins interact with the SPB machinery and with each other, validating the premises of this project. This project combines my expertise on the fission yeast cell cycle with the host laboratory expertise on centrosomes and cytoskeleton. This approach will identify the minimum conditions to regulate and assemble the unique structure of centrosomes.

miRNAs are genome-encoded small RNAs that canonically repress translation of target mRNAs, which they recognize by base-pairing. In contrast, the non-canonical function of miRNAs in translational activation in G0-phase cells has been reported. Our knowledge of miRNA-mediated translational activation is still limited; in particular, it is unclear whether miRNA-mediated translational activation affects endogenous mRNAs significantly. The main aim of my research will be the genome-wide evaluation of miRNA-mediated translational activation in endogenous mRNAs. As quiescent and post-mitotic cells are pervasive in living organisms, our results will reveal important aspects of miRNA biology that have been overlooked in standard culture systems.
I will address endogenous activation targets using ribosome profiling. Ribosome profiling is a method based on deep sequencing of RNA fragments protected by ribosomes that measures the translation of each mRNA quantitatively. I will introduce miRNAs into G0-phase HeLa cells, synchronized by serum-starvation, and analyze how miRNA-mediated translational activation impacts endogenous mRNAs, how miRNAs activate translation, and how differently miRNAs recognize the target mRNAs.
One of my previous reports has suggested that the poly(A) tail of target mRNAs might be a key factor for miRNA-mediated translational activation. I will address the impact of polyadenylation by RNA-Seq analysis of poly(A)+ and poly(A)- fractions of rRNA-depleted RNA and high resolution poly(A) tail assay (Hire-PAT). This result will provide insights for the function of poly(A) tail in miRNA-mediated translational activation.

Recently, our laboratory has shown that cancer cells need both genetic mutations and epigenetic alterations for their continuous growth. This was revealed through a focused RNAi screen where all epigenetic regulators were systematically evaluated for their role in proliferation of acute myeloid leukemia (AML). I performed a new RNAi screen in acute lymphoblastic leukemia (ALL). Remarkably, this screen identified that TRIM33 (Tripartite motif-containing 33) is required for the growth of ALL but not for 10 other forms of cancer tested. TRIM33 harbors a PHD and a bromodomain which recognizes methylated and acetylated histones, respectively. While TRIM33 is uniquely required for growth of ALL cells, I found surprisingly that TRIM33 is expressed ubiquitously in many cell types. Therefore, TRIM33 promotes growth of ALL in a highly context-specific manner, which might be related to the underlying genetic/epigenetic cellular state. To understand the mechanism underlying this intriguing phenotype, I will establish a tractable mouse-model system in which we can manipulate cellular genetic/epigenetic contextual features to evaluate the key determinants of TRIM33-addiction. This powerful system will enable us a comprehensive evaluation of TRIM33 function in ALL, which will include: 1) identifying the target genes of TRIM33 2) revealing genome-wide distribution of TRIM33 3) purifyingTRIM33-containing complexes in multiple cell types, and 4) reconstituting TRIM33 functions in vitro. The central goal of this project will be to define the context-specific mechanism that accounts for the addiction of ALL cells to TRIM33, a novel therapeutic target in this untreatable disease.

To address the cellular and molecular requirements for Th17 differentiation during commensal colonization, I will focus SAA1 protein, which is strongly induced by SFB colonization. To monitor SFB colonization easily and surely, I propose to generate SAA1 luciferase mCherry knock in reporter mice and to use an ileal looping surgical approach that does not impair host circulation. Using this approach, we can understand when and where SAA1 is induced in response to SFB colonization. Although SFB may attach intestinal epithelial cells (IECs) and CX3CR1+ cells in intestine, which cells recognize SFB are not known. To determine the cellular requirement for SFB recognition, SAA induction and commensally driven Th17 differentiation, I will use specific cell linage depletion strategy that is well established. Using CX3CR1+ cells depleted mice, we can understand whether CX3CR1+ cells are necessary for SAA1 induction and Th17 differentiation or not.
To identify the genes which are required for Th17 differentiation in response to SFB colonization, I will try identifying the genes which are strongly induced by SFB. To eliminate secondary effects, I will purify IECs, DCs and CD4+ T cells from ileal loops of mice that are depleted CX3CR1+ cells and CD103+ DCs. Comparing these all gene profiles, we can understand the genes that are directly induced by SFB colonization in each cell linages. Thus, these genes are strong candidate that affect Th17 differentiation. In the last part of my study, I will elucidate whether the candidate genes are required for Th17 differentiation in response to SFB colonization by using conditional knock out mice.

While sleep is essential for survival, its functions remain elusive. Increasing evidence suggests a role for sleep in learning and memory consolidation. Although each learning task uses specific brain regions, sleep has been regarded as a whole brain phenomenon. Thus, sleep seemed to serve neural plasticity at the systems-level, for instance by shutting down external stimuli. However, a recent study reported that in sleep deprived rats, small areas of the cerebral cortex can fall into a sleep-like state while other areas do not (local sleep). When this occurs the animals appear behaviorally awake, and their scalp EEG is typical of waking. The observation provides us with a unique opportunity to explore whether local uses of specific brain regions lead to local sleep.
By implanting multi-unit arrays in brain regions of interest, we can record activity of individual neurons in freely behaving rats and ask whether greater plasticity in one area results in higher probability of local sleep in that area, and greater performance deficits in tasks relying on that area. First, we will study the effects on local sleep of electrically-induced long-term potentiation (LTP) in frontal cortex. Next, we will use physiological stimuli, tasks that rely on motor or frontal cortex. When local sleep is observed specifically in the used region, we will test the correlation between local sleep and performance in these tasks.
This study will allow us to assess the direct link between synaptic plasticity (due to LTP or learning a task) and local sleep, as well as the molecular mechanisms underlying the homeostatic sleep pressure and the impaired performance due to sleep deprivation.

Metastasis is a characteristic feature of malignant tumors and also an important factor to determine the prognosis of cancer patients. Preventing or eliminating metastasis is thus considered one of the most important steps to control cancers. Although previous microarray-based studies have identified crucial gene expression changes in tumor metastasis, the mechanisms that melanoma cells use to acquire metastatic potential remain poorly understood. Here, I propose to combine single nucleotide-level high-throughput analysis with computational and molecular pathological approaches to characterize the genetic changes that underlie melanoma progression from a slow-growing, surgically-curable lesion to aggressive metastatic disease. I will perform single nucleotide-level genome and transcriptome analysis on patient-derived melanomas that have metastatic potential and those that do not have metastatic potential to identify mutations associated with the acquisition of metastatic potential. Although previous methods such as gene expression profiling have focused only on gene expression changes, I will simultaneously analyze expression level and mutation status to pursue the primary causes of melanoma metastasis. I will also test whether mutations I identify functionally contribute melanoma metastasis in xenograft models of patient melanomas, and identify specific mechanisms that confer the potential for melanoma cells to form distant metastases. My work will bridge single nucleotide-level high-throughput analysis, computational modeling, and experimental validation and will reveal new insights into the biology of tumor metastasis that could enhance the management of melanoma.

During early mouse development, cells undergo reprogramming and programming events that are governed by epigenetic regulation. However, there is no knowledge as to how chromatin structure changes during these events. This knowledge is key to understanding developmental potential transitions and epigenetic reprogramming. Here I propose to apply an improved established method, which will provide the first information on global nucleosome positioning and DNA accessibility in the early mammalian embryo.
Secondly, I will determine the nucleosome positioning in the maternal versus the paternal genomes upon fertilisation, key to understand the earliest steps of genome reprogramming.
Thirdly, I will examine the functional relationship between the dynamics of nucleosome positioning by correlating positioning with histone modification by using chromatin-immunoprecipitation technique, a technique that has been recently established in embryos by the host laboratory. Lastly, I will manipulate selected chromatin modifiers known to be essential for early development and ask how nucleosome positioning is altered. This will reveal how nucleosome positioning and DNA accessibility are regulated as well as how this manipulation affects developmental processes.
Through these analyses, I will determine the local and global shifts of nucleosome positioning and DNA accessibility during early mammalian development. This will establish how transitions in chromosome structure occur during reprogramming and programming and will allow us to decipher the effects of chromatin organization on cell potency during developmental reprogramming and programming.