Reprogramming of endogenous glia into iNs by developmentally inspired transcription factors has been recently achieved in vivo. However, little is known about their physiological properties and recruitment into functional neuronal circuits, especially within the context of brain damage. The host lab has recently showed that injury-induced reactive NG2 glia can convert into immature iNs following retrovirus-mediated expression of Sox2 within the adult cerebral cortex. The deficiency in the acquisition of a mature neuronal phenotype may generate functional deficits. Therefore, I will promote the maturation of iNs by combining forced expression of Sox2 with strategies to enhance neuronal maturation: curtailing Sox2 expression, increasing mTOR signaling and pharmacologic ligand-mediated depolarization. I will also study the electrophysiological properties of NG2-derived iNs and compare them to those of native NG2 cells. This is important because NG2 glia even prior to reprogramming receive synaptic inputs from neurons and induced cells may directly inherit some of these connections. I will perform patch-clamp recordings in brain slices from NG2 glia and NG2-derived iNs to reveal changes in excitability, synaptic inputs, and synaptic plasticity following reprogramming. Finally, I will analyze the functional integration of the reprogrammed cells using in vivo 2-photon Ca2+ imaging. I will investigate the recruitment of iNs into functional neuronal circuits in the somatosensory cortex in response to whisker pad stimulation. The proposed study will unveil fundamental data regarding the capacity of NG2 glia-derived iNs to functionally integrate into severely damaged brain tissue.

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.

The genetics of Amyotrophic Lateral Sclerosis (ALS) have been intensively explored in the last two decades, identifying multiple disease-related mutations that underlie it. A paradigm shift in understanding ALS pathology has come from discovery of ALS-causing mutations in several genes encoding proteins with fundamental roles in RNA processing pathways. However, the scope, function and significance of RNA metabolism in affected neurons remains elusive, primarily because of the impossibility of examination of the affected neurons throughout disease course. Here, I propose to uncover the extent of aberrant RNA metabolism and its potential contribution to toxicity in ALS pathogenesis using neurons directly “trans-differentiated” from familial-ALS patient’s fibroblasts. I will use a new genome editing approach (CRISPR) to correct ALS-causing mutations and apply genome-wide sequencing technologies on mutated and corrected neurons to determine whether such neurons display a specific profile of altered RNAs. These data, including RNA transcription, splicing and editing, will allow the construction of high-resolution, disease-dependent molecular signatures that underlie the molecular and cellular basis of ALS progression. By exploiting this set of RNA alterations as multi-functional readouts, my final approach will be to perturb ALS-neurons using ~4500 available compounds with known mechanisms of action, thus discovering molecular pathways that reverse an initial RNA signature. Combining the complementary strategies outlined here, this proposal aims to discover and characterize previously unknown mechanisms through which RNA processing abnormalities provoke ALS pathogenesis

iRhoms are members of the rhomboid-like superfamily without any protease activity. In different contexts they either promote ER associated degradation (ERAD) of transmembrane client proteins, or their forward trafficking. To understand more about the molecular mechanisms of these recently discovered regulators, I will screen for proteins that interact with Drosophila iRhom using a new biotin tagging approach called BioID. This technique will systematically identify iRhom interactors in the Drosophila nervous system (where iRhom is highly enriched) . This technique once developed in flies, will be the first of its kind at an organismal level wherein all interactors (including ones which are transient) as well as the constituents of the intracellular neighbourhood of any protein of interest can be characterised. Using this approach, I expect to identify three classes of protein: iRhom clients, regulators, and components of the core ERAD and trafficking machinery. My primary focus will be on ERAD, as this is known to be iRhom’s central function in flies. I hope to understand why iRhom is largely restricted to the nervous system and, specifically, whether the neurons have specific quality control requirements. These interactions will also be explored in mammalian cell lines to understand whether they are conserved through evolution, as well as to address the question of how iRhoms act in different cellular processes. Finally, I will use genetics and a drug based approach in flies to look for a role of iRhom in various neurodegenerative diseases originating from ER stress.

Cellulosomes are protein networks used by anaerobic bacteria to digest cellulosic biomass. The protein networks consist of subunits that are held together by receptor-ligand pairs, some of which have similar affinities to the same binding sites. Intuitively, this structural property of cellulosomal subunits should give rise to a large number of cellulosomal compositions. In nature, however, cellulosomes are found to exhibit only a limited number of preferred compositions. It remains unclear how cells modulate the assembly of cellulosomes. Studies of cellulosomes are hampered by a lack of tools to manipulate genetic components of native cellulosome-producing bacteria. Furthermore, to resolve these issues, studies of cellulosome assembly require nanoscale measurement methods. Our team proposes integrating synthetic biology and single-molecule imaging for the study of cellulosome assembly.
Our long term goal is to integrate synthetic biology and single-molecule methods to understand control mechanisms of cellular dynamics. Based on our combined expertise in synthetic genetic circuits and cellulosomal interactions, we aim to address the hypothesis that architecture of genetic modules can regulate the timing of cellulosomal-subunit expression, thereby modulating the resultant cellulosome compositions. We will test this hypothesis by constructing synthetic genetic modules using cellulosomal subunits from Acetivibrio cellulolyticus and Clostridium thermocellum. We will also use stochastic models to predict the impact of genetic modules on cellulosome assembly. We will then test model predictions at the single-molecule level using cell-free systems and yeast in custom microfluidic devices. Our approach is innovative because it combines synthetic biology and single-molecule imaging to study cellulosome assembly. Our results will establish a foundation toward studying system dynamics of native cellulosome-producing bacteria. Furthermore, our results will unravel general principles of how genetic networks modulate the assembly of biological structures.

The objective of the proposed study is to investigate the molecular mechanisms underlying the regulation of assembly and disassembly axonemal tubulins in the unicellular alga Chlamydomonas reinhardtii. This study we will extend the knowledge on microtubules dynamics in non-mammalian sources. Also, it will provide molecular insight on the flagellar length regulation mechanism. In order to achieve these goals three specific aims were formulated: (i) To characterize the axonemal tubulin properties, including their polymerization and depolymerization processes (i.e. rate and frequency) and to study the effect of post-translational modifications; (ii) To explore the influence and mechanism of three potential microtubule-associated-proteins (CrKin13, CrEB1, FAP256) on the regulation of Chlamydomonas microtubules dynamics; (iii) To develop FRET-TIRF assay to monitor the microtubules dynamic and to improve the resolution of the process by decreasing the fluorescence background. More specifically, I will purify Chlamydomonas axonemal tubulin by using an innovative affinity column that was developed in the host-lab to facilitate the purification of native untagged tubulin from non-mammalian sources. Subsequently, I will investigate the dynamic properties of the axonemal tubulin and which factors influence the process, such as microtubule-associated-proteins or post-translational modifications, by using state-of-the art fluorescence microscopy and other molecular techniques. Also, I will examine the possibility to modify tubulin units with pairs of fluorophores to perform FRET in order to gain a better microscopic insight on microtubule dynamics.

Adaptation is a landmark property of living cells. Cells have evolved sophisticated regulatory networks to selectively switch their genes on or off depending on the environment. Such regulation allows them to robustly adapt their metabolism and survive in different environments. Failures in metabolic regulation can lead to disease, and regulation can also be exploited by pathogens to survive drug treatments.
While some metabolic responses are encoded in the architecture of a regulatory network, others are the result of evolutionary fine-tuning of the parameters governing molecular interactions. Little is known about the interplay between architecture and parameter fine-tuning, yet this knowledge could allow us to understand metabolic responses in broader contexts, including the emergence of antibiotic resistance, abnormal metabolism in disease, and the detection of weak spots for future therapeutics to disrupt the metabolism of pathogens.
We propose to combine control theory and synthetic biology to single out the genetic mechanisms that control metabolic adaptations. We will integrate theory and experiment to predict, measure and alter the metabolic responses of microbes to environmental shocks. Our focus will be the fatty acid biosynthetic pathway in Escherichia coli, which is a promising antibiotic target and is amenable to genetic manipulations. We will use these genetic manipulations to rewire the native regulation and systematically explore the roles of network architecture and parameters. Together we aim to gain a systemic grasp of the overlapping mechanisms that control metabolic adaptations, establishing the mathematical and experimental groundwork to quantitatively understand other complex regulatory networks found in nature.

The reflection superposition compound eye, found in decapod crustaceans represents a spectacularly complex natural optical device based on biominerals. The eye contains a multilayer reflector in the distal part of the eye to focus light onto the retina and a reflecting tapetum layer which increases light sensitivity. Using an array of physical techniques, including the use of synchrotron radiation we will determine the chemical identity of photonic crystals isolated from the eyes of the crayfish C. quadricarinatus and the prawn M. rosenbergii. We recently determined that magnesium lactate dihydrate, a previously unknown biomineral is present in the reflector material. Using cryo-SEM, we will determine the ultrastructural arrangement of photonic crystals in the distal and tapetal layers under life-like conditions. We will relate the crystal structure, crystal morphology and eye ultrastructural properties to understand better the overall optical function of the eye. In collaboration with Prof. Amir Sagi (Ben-Gurion University), we will study the mechanism of crystal formation by observing changes in the distal reflecting layer in the eye of M. rosenbergii during development from larvae to adult. By studying gene expression during ontogeny a list of 'candidate genes' will be drawn up and their possible role in the biomineralization pathway will be explored by a series of gene silencing experiments. A deeper understanding of the reflector materials in the eye could yield applications in material optics and uncover new paradigms in biologically controlled crystal formation which will have a wide impact in the fields of biomineralization, materials science and pharmacy.

Immune-mediated diseases, including inflammatory bowel disease (IBD), spread rapidly around the globe on the heels of the transition to modern lifestyles. One predominate factor driving this epidemic is the decline in diversity of the human microbiome as a result of our highly hygienic lifestyles. The human gut harbors vast communities of bacteria, protists, helminths and other microbes that have co-evolved with humans over millions of years. Absence of exposure to helminths and altered bacterial communities in the gut (called dysbiosis) are both strong risk factors for immune-mediated disease development. Protists are poorly studied, but likely also important for human health. Humans ancestrally harbored diverse protists communities, and today healthy people are more likely to harbor diverse protists, especially Blastocystis, than those with IBD. The underlying mechanism remains unknown, but likely involves changes in bacterial community composition. Intriguingly, re-introducing helminths or correcting bacterial dysbiosis are both promising disease treatments; animal studies and small trials report successful outcomes in upwards of 60% of cases. However, subsequent unsuccessful clinical trials and reports that helminth therapy can exacerbate pathogenic infections dampen enthusiasm and point to a fundamental gap in our knowledge of how helminths impact the gut ecosystem and how interactions with the other gut microbes such as Blastocystis might affect treatment outcomes. Here, we propose to revisit helminth therapy from an ecological perspective to ask what role the dynamics in the overall gut communities play in the success or failure of these treatments. We will conduct a series of experiments in a rat model system to address two central aims: (i) to characterize the dynamics of the host immune response and the microbiome in the face of disturbance caused by induced colitis and the introduction of the helminth Hymenolepis diminuta and/or Blastocystis ST1, and (ii) disentangle therapeutic impact arising from direct interaction of helminths with host from impacts mediated by shifts in microbiota. We hypothesize that shifting the microbiota to a new stable state may be key to long-term treatment success and believe our complementary expertise uniquely enables us to address this complex research question.

It has generally been assumed that a small number of molecules is sufficient to explain most if not all aspects of early development. This idea has been challenged by our recent discovery that toddler/apela encodes a novel, highly conserved secreted peptide with essential functions during embryogenesis. Genome-wide technologies have allowed us to identify hundreds of other uncharacterized peptides and translated short open reading frames (ORFs) that are expressed during embryogenesis.

These recent findings pose two fundamental questions: (1) What are the functions of these newly discovered small peptides? Small peptides are particularly promising candidates because they are likely to have been missed in prior random mutagenesis screens. (2) What is the role of the expanded universe of translated ORFs? Rather than encoding functional peptides, translation could be neutral, or it could be the translation process itself that serves a function.

The goal of the proposed research is to identify functions for novel peptides and short translated ORFs during early development, using zebrafish as a model system. Aim 1 will investigate the molecular and cellular mechanism of Toddler/Apelin receptor signaling. Aim 2 will investigate the functions of other novel peptides and aim at unraveling regulatory functions for short translated ORFs during developmental transitions.

These studies will identify the roles and mechanisms of novel bioactive peptides in early development. The proposed research has the potential to identify molecules with possible applications in tissue repair and in vitro differentiation, and could uncover novel principles of translational regulation.

Glioblastoma multiforme (GBM), the most prevalent form of primary brain tumors, severely debilitates patients by infiltrating and destroying the brain parenchyma. The molecular mechanisms governing GBM invasion are poorly understood, rendering GBM incurable. Previous work has revealed that 30-50% of GBM tumors overexpress a distinct mesenchymal-like gene signature, which is further enriched in GBM undergoing recurrence, raising the question whether specific mesenchymal genes regulate GBM progression. We propose to tackle this by implementing a three-dimensional (3D) organotypic brain tissue model for the dynamic study of GBM invasion ex vivo. Using glioblastoma neural stem-like (GNS) cell populations, we will perform loss-of-function complemented with gain-of-function genetic screens to identify mesenchymal genes critical for GNS cell invasiveness and GBM tumor progression. To molecularly dissect cell-intrinsic networks in GNS invasion, we will conduct epistasis experiments with pro-invasive genes and downstream pathway effectors. We will also examine cell-extrinsic effects of pro-invasive genes on the neural microenvironment. Potential paracrine feedback by microenvironment-secreted factors, deregulated by invading GNS cells, will be tested in antibody-based blocking experiments and validated using brain tissue from mice genetically inactivated for targeted factors. Finally, the prognostic power of pro-invasive mesenchymal genes in predicting GBM patient outcomes will be assessed. The findings from this proposal, if successful, should provide a molecular handle on GBM invasion by implicating specific mesenchymal genes as key therapeutic targets.

Obesity is a serious medical problem that poses an enormous public health burden. Understanding how the brain integrate and control satiety and hunger signals are vital for unraveling the causes of obesity. Many brain regions are known to regulate feeding behavior including the hippocampus, a brain region usually associated with memory. However, while some studies show a role for the hippocampus to control food intake, the identity of the specific cells that modulate this behavior are not known. I propose to identify and profile the hippocampal cells that are activated during a feeding paradigm in mice. To gain greater insight on how the hippocampus regulate feeding in mice, I will initially focus on the subiculum which was found recently to send projections to the nucleus accumbens (NAc). This will be accomplished using two new profiling technologies that allow the identification of specific molecular markers of neural populations based on changes in their activity (PhosphoTrap) or based on their pattern of projections (RetroTrap). Once these specific cells are identified, I will modulate the activity of these cells using optogenetics, DREADDs (designer receptors exclusively activated by designer drugs) or radiowaves and assay possible effects on feeding behavior in wild-type and leptin-deficient obese (ob/ob) mice. I will also use electrophysiology assays to monitor the effects of activating or inhibiting subiculum cells on the activity of neurons in the nucleus accumbens. This project will generate new tools, provide novel evidence on how the hippocampus controls feeding behavior and potentially identify new targets for genetic manipulation and drug design.

Theory predicts that when a system is close to a catastrophic transition it will take unusually long times to recover from small perturbations. This effect, called critical slowing down, may predict the impending
catastrophe better than other indicators. For example, it indicates when an ecosystem is near to its collapse, while other parameters such as population sizes may only show the collapse when it is already occurring.

We propose to use critical slowing down to predict the death of an animal. The theory is powerful enough to address this question but has never been applied to individual animals, which involve new challenges such as inter-individual variability, restricted access to the state variables and highly interconnected and heterogeneous subsystems.

We will test our predictions in the nematode Caenorhabditis elegans, which is experimentally tractable yet at the same time is a complex multicellular organism with distinct organs and systems. We will focus on osmoregulation, as perturbations are easy to apply, the effects of osmotic shocks are easy to measure, and a sufficiently severe osmotic shock will induce death. We predict that quantitative measurements of osmoregulation in individual animals will reveal an increase in time to recover from osmotic shocks as the animal approaches death, regardless of whether the death is caused by osmotic stress or other reasons, such as heat stress or even aging. We will try to show not only that the effect exists on average across replicates, but also that it has diagnostic power on individual animals, identifying those individuals less capable of withstanding a given condition.

Understanding tissue morphogenesis and function is of fundamental importance in developmental biology. Resolving the underlying mechanisms relies on tracking dynamic cellular behavior in toto and requires new microscopes able to image the developing embryo over a range of spatiotemporal domains. Selective plane illumination microscopy (SPIM) represents the state of the art in time-lapse imaging. However, optical penetration is fundamentally limited in turbid media and multi-view imaging unnecessarily exposes the sample and yields large datasets that are often sparse in information content. Typically, the ballistic regime defines the superficial region available to conventional optical imaging. This limitation will be overcome via a range of imaging modalities available in parallel. Self-healing beams and multi-photon excitation with adaptive wavefront correction will allow even deep tissues to be imaged without loss of contrast. New transgenic lines will be developed for multi-color infrared imaging of cardiac morphogenesis to produce 4D biophysical models of the heart. Quantitative analysis performed in real time will allow highly selective imaging of evolving regions of interest thus minimizing sample exposure and maximizing sample throughput. This smart SPIM will be used to map and image not only vascularization but also earlier developmental events such as germ layer coordination during gastrulation with isotropic resolution. I envisage these strategies will provide the basis for the next generation of smart SPIM and allow real-time system level study of zebrafish embryogenesis.

Cell migration is a fundamental processes of life, yet also one of the molecularly most complex ones. The majority of mechanistic insight of cell migration stems from observations on coverslips. While efforts are underway to observe cell migration in entire organisms, from fruit flies to mice, the molecular detail and statistical power that can be achieved are limited. Instead, I propose here to use ex vivo, but physiologically relevant samples of human cell migration that are amenable to systematic and quantitative high-resolution imaging. I hypothesize that some modes of migration in 3D share mechanical similarities with 2D migration, whereas other modes will exhibit completely novel mechanisms. To test this, I will first identify a finite set of migration modes as a function of the mechanical properties of the external cellular matrix and correlate them with the dynamics of actomyosin organization, cell cortex contractility, and adhesion patterns. To measure actomyosin dynamics I will adapt an imaging modality known as fluorescent speckle microscopy to high-resolution light-sheet imaging. I will be trained by, and in constant collaboration with, colleagues in the Danuser lab at UT Southwestern who are experts in fluorescent probe design and optical engineering. I will contribute to this team with my expertise in image signal processing and computer vision. Using speckle microscopy we will then determine the dynamic interactions between actin filaments and myosin motors, map out spatial organization of micro-strains in the cortex, and relate these parameters to the formation of cell adhesions, cell protrusions and retractions, and the efficiency of whole cell migration.

Satellite cells (SCs) are stem cells required for growth, maintenance, and regeneration of skeletal muscle. In response to molecular triggers from injuries or disease, subsets of SCs activate and have the ability to self renew, proliferate as myoblasts, or undergo myogenic differentiation to regenerate damaged tissue. Central to the specification and function of SCs is the PAX3 transcription factor, which activates factors that mediate migration of muscle progenitor cells during development. In adult SCs however, PAX3 expression is down-regulated in all but a few muscle groups, such as the diaphragm muscle. Pax3 expression is controlled via alternative polyadenylation (APA) of its 3'untranslated region (3'UTR), which alters the ability of microRNA-206 (miR-206) to bind and knock-down Pax3 transcripts. Recently, it was discovered that the poly-A binding protein nuclear 1 (PABPN1) regulates the length of 3’UTRs in human cells by suppressing APA. Moreover, mutations in PABPN1 are linked to the late onset myopathy, oculopharyngeal muscular dystrophy (OPMD). Here, we hypothesize that PABPN1 mediates Pax3 3’UTR alternative polyadenylation. Transient expression of PAX3 is associated with SC activation and corresponds with a proliferative state of myoblasts. Thus, PABPN1 may also regulate SC function. I will first assess the role of PABPN1 in Pax3 regulation and satellite cell proliferation ex vivo. I will then determine the binding profile of PABPN1 to Pax3 pre-mRNA in satellite cells from different muscle groups. Finally, I will investigate the affect of PABPN1 knockout or mutation in mouse models of satellite cell activation.

In order to maintain the integrity of the proteome, eukaryotic cells have evolved a complex array of spatial protein quality control (PQC) mechanisms that recognize misfolded proteins and triage them for refolding, degradation or--if these processes fail--sequestration. Soluble misfolded proteins in yeast are rapidly sequestered in dynamic punctate inclusions called Q-bodies; these coalesce into larger structures, where the misfolded proteins are degraded by the ubiquitin-proteasome system or targeted to a juxtanuclear quality control compartment. By contrast, a separate pathway--involving permanent sequestration into a peripheral insoluble protein deposit--seems to exist for proteins that cannot be degraded, such as disease-linked amyloid proteins. To date, studies have described spatial PQC using conventional optical microscopy, thereby providing limited information as to the ultrastructural basis of this system. I will use correlative light-and-electron microscopy and super-resolution fluorescent microscopy to pinpoint the location of fluorescently-tagged misfolded proteins at various stages of spatial PQC with respect to the subcellular architecture in mammalian cells. Combining this approach with cutting-edge proteomic tools that allow analysis of specific PQC compartments, together with various genetic, biochemical and cell biological methods of modulating protein function, will provide unprecedented mechanistic detail into the role of various PQC machineries in these pathways. My integrated approach should considerably advance our understanding of misfolded protein triage in mammalian cells, both under physiological conditions and in response to cellular stresses.

Our ability to identify disease-associated genomic alterations by next generation sequencing and to genetically model those alterations using novel genome editing tools will revolutionize biomedical research. Studying phenotypic extremes and the role of rare variants in human disease such as cancer will provide us with key insights into basic biology and might even unravel novel therapeutic strategies. Cancer sequencing projects are currently uncovering hundreds of genes associated with cancer, which show a classic long tail distribution revealing the enormous heterogeneity not only between different cancers but even within a tumor itself. Mining this information for therapies and personalized cancer medicine is predicated on weeding out ‘bystander’ mutation and identifying ‘driver’ mutations and molecular pathways responsible for tumor initiation, progression and metastasis. However, the complex nature of cancer involving multiple genes, several cell types and stromal cues necessitates the use of model organisms to faithfully recapitulate physiological and pathological processes, which has impeded a systematic and comprehensive characterization of rare variants and the search for synthetic lethal interactions. I propose to develop direct in vivo genome editing technologies to specifically modify and assess function of hundreds of putative cancer genes within intact organs of a living mouse in a mosaic fashion. By integrating human cancer genomics and mouse modeling with in vivo screening, I hope to (1) rapidly discover and functionally characterize potent but low penetrance cancer driver mutations and to (2) elucidate novel cancer vulnerabilities.

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.

One question standing upfront in the developmental biology field is how the developmental transition from unipotency to totipotency takes place post fertilization. It is believed, that totipotency results from extensive epigenetic reprogramming of the germline state into an embryonic state. My host lab and others have recently highlighted that both the highly asymmetric mature gametes contributes to this developmental reprogramming and hence the zygotic genome activation (ZGA). Genes specifically expressed at the onset of the ZGA, factors controlling their activation and the parental contribution of such regulatory factors remain unknown. To achieve this, we propose to perform a computational analyses combined with in-vivo functional characterization. Our preliminary analysis on published data sets, by filtering and measuring the intronic transcripts in early embryos, have already gave us a list of early transcribed genes and the transcription factors (TFs) potentially determining the regulatory states of the cells. Furthermore, I propose to investigate the influence of germline inheritance of chromatin states on ZGA, by generating expression data from genetically hybrid embryos enabling the distinction between parental genomes. This will provide an unprecedented insight into the parental contribution of selected ZGA regulators. Subsequently, I will perform a functional characterization of these potential ZGA regulators by applying loss- and gain-of- function studies in early embryos. I will extend my studies and use CRISPR/Cas system to generate transgenic mouse for further in-depth studies.