Stavros Lomvardas - US grants

DESCRIPTION (Provided by the applicant) Abstract: Our brain displays an astonishing degree of plasticity. Experiences from a constantly changing environment generate, modify and eliminate synapses and alter the function of our neurons. Extensive research over the last three decades has demonstrated that long term potentiation is a process that requires enduring changes in gene expression. Although transcription factors mediate most of these changes, it is the covalent modifications on DNA and chromatin that render this changes long lasting. Among these, "so called" epigenetic changes, DNA methylation is the only one that cannot be enzymatically reversed. DNA methylation on CpG islands is a well established mechanism of gene silencing. Here, we show that we discovered a novel epigenetic modification, the methylation of CpA dinucleotides. Using a novel, genome-wide method to detect CpA methylation in primary neurons, we made the remarkable observation that CpA methylation appears only on actively transcribed genes. Moreover, our preliminary data suggest that this modification can be modulated by neuronal activity;exactly like the transcription status of the genes that it marks. An irreversible modification that can enhance, or modulate gene expression could have profound consequences in neuronal plasticity. Therefore, we propose experiments that will dissect the role of CpA methylation in gene expression and neuronal function. Public Health Relevance: In this proposal we describe a novel epigenetic modification, the activity dependent methylation of CpA dinucleotides in primary neurons. We propose experimental strategies that will reveal the role of this modification in gene expression and neuronal function. CpA methylation, as a regulatory mechanism, could have critical impact on a plethora of neuronal functions, including axon targeting and synaptic plasticity and specificity. Taken into account that DNA methylation is pharmacologically amenable, our findings could have significant clinical consequences. A large spectrum of neurological or neurodevelopmental disorders, ranging from dementia to autism spectrum disorders, could be caused by the inability of a neuron to transform synaptic activity into long lasting changes in gene expression. Therefore, understanding the role of this epigenetic modification in this process should have a broad scientific, medical and socioeconomic effect.

DESCRIPTION (provided by applicant): Olfactory receptor (OR) genes compose a family of ~1,300 genes that are expressed in a, seemingly, stochastic and monoallelic fashion in the mouse olfactory epithelium. Each olfactory sensory neuron expresses only one OR gene from the whole repertoire and this singularity is essential for the proper wiring and function of the olfactory system. Here, we present preliminary data that a repressive epigenetic modification, trimethylation of lysine 9 of histone H3, may be responsible for the silencing of all the OR alleles that are not expressed in a sensory neuron. We propose experiments that will monitor the spatial and temporal coordinates of this modification in the olfactory epithelium and will reveal the molecular components that orchestrate it. These experiments are essential for understanding the role of this repressive mechanism in OR choice and will provide targets for future genetic analyses of epigenetic silencing in the nose. PUBLIC HEALTH RELEVANCE: This proposal aims to analyze the epigenetic silencing of olfactory receptor genes during the development of the olfactory epithelium. Olfaction is a primal human sense and understanding the epigenetic regulation of olfactory receptor genes will uncover principles for the generation of mammalian neuronal diversity and function.

DESCRIPTION (provided by applicant): Autism is a neurodevelopmental disorder that develops in the first three years of life. It is probably caused by diverse genetic mutations that remain to be identified. Rett syndrome is a devastating form of autism spectrum disorders, and it is one of the few in which the underlying genetic cause, mutations on the MeCP2 gene, has been identified. MECP2 is a methyl-DNA binding protein that is thought to function as a transcriptional repressor. Despite immense efforts, only a few genes have been shown to be regulated by MECP2 in vivo. Here, we report that MECP2 regulates olfactory receptor expression, in an unusual way. In MeCP2 knockout mice olfactory receptor neurons express multiple olfactory receptors, in contrast to the wild type mice where only one olfactory receptor allele is expressed in each neuron. Interestingly, the olfactory receptor alleles that appear coexpressed in the MeCP2 knockout neurons are always members of the same chromosomal cluster. This observation suggests that MeCP2 functions as an insulator that could be regulating the monogenic expression of gene families that are organized in chromosomal clusters like the olfactory receptor genes. The violation of the one receptor per neuron rule provides a robust molecular assay for MeCP2 activity. We propose to use this assay in a high throughput screen of chemicals that can reverse the molecular consequences of MeCP2 deletion in dissociated olfactory neurons. These compounds could eventually be the basis for a pharmacological treatment of Rett syndrome symptoms. PUBLIC HEALTH RELEVANCE: This application proposes the use of a robust molecular phenotype that we discovered in MeCP2 knockout olfactory neurons, for a high throughput screen for compounds that restore the deficiencies caused by MeCP2 deletion. MeCP2 mutations are the main cause of Rett syndrome; therefore our application has significant relevance to the health and quality of life of Rett syndrome patients and their families.

DESCRIPTION (provided by applicant): Project Summary and Relevance Since the proposal of the nucleosome hypothesis, more than 30 years ago, an extensive body of work has demonstrated the critical role of chromatin in gene regulation. In vitro and in vivo experiments from different model systems have unequivocally shown that covalent histone modifications provide a signal for the recruitment of chromatin modifying proteins that activate or silence gene expression. High throughput approaches recognized that these covalent marks have different genomic distribution, some are enriched on promoter sequences, others mark enhancers, whereas a few decorate the transcriptionally inactive and repetitive portion of our genome. Moreover, genetic and pharmacological experiments showed that these modifications, despite being termed epigenetic, appear extremely dynamic; the enzymes that apply them are in constant competition with the enzymes that erase them. Therefore, each nucleus and even each DNA locus adopts distinct epigenetic states in space and time. The dynamic nature of the epigenome poses a significant challenge towards the understanding of the exact order of events that culminate in gene expression or silencing during development and differentiation. Overcoming this challenge becomes even more critical by the realization that the epigenome has a profound effect in human health and that perturbations thereof result in disease. Therefore, new technologies need to be developed for the monitoring of the epigenome in a single cell resolution in living cells. Here, we propose the use of the powerful bimolecular Fluorescence Complementation technology towards the live imaging of the mammalian epigenome. Our experiments aim to visualize the extend, and nuclear distribution of different histone marks in differentiating cells and to assign the position of these marks in relation to specifically labeled DNA loci. Moreover, we will use a novel imaging system, soft X-ray tomography that combines remarkable spatial resolution with the benefits of fluorescent microscopy. With this technology we will gain structural insight of different epigenetic domains in the fully hydrated mammalian nucleus. The information generated by the combination of these innovative approaches will allow a better understanding of epigenetic regulation in model organisms and will promote the development of therapeutic epigenetic compounds. Moreover, these experiments will lay the foundation of an epigenomic analysis of the genetically intractable human being and will provide sensitive tools for the future for the diagnosis or even prognosis of diseases linked to epigenetic perturbations.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The expression of a single odorant receptor (OR) gene from a large gene family in individual sensory neurons is an essential feature of the organization and function of the olfactory system. Individual olfactory sensory neurons in mice express only one of 1300 odorant receptor genes (Chess et al., 1994;Malnic et al., 1999). The choice of a specific odorant receptor defines the functional identity of a sensory neuron, and the receptor also provides an instructive cue that dictates the site of projection in the brain (Wang et al., 1998;Feinstein and Mombaerts, 2004;Barnea et al., 2004). Thus, the expression of a single receptor gene in a sensory neuron is an essential feature of olfactory perception. The goal of my research is to understand the mechanism of olfactory receptor gene choice. Olfactory sensory neurons (OSNs) express in a seemingly stochastic, monogenic and monoallelic fashion one out of 1400 olfactory receptor (OR) genes. We recently showed that OR loci are marked with the hallmarks of constitutive heterochromatin, trimethylation of lysines 9 and 20 of histones H3 and H4 respectively, in differentiating and fully differentiated OSNs. The deposition of these epigenetic marks on OR loci results in the formation of a chromatin structure with remarkable biochemical properties, such as extreme levels of compaction and unprecedented resistance to Dnase I digestion (Magklara et., al. submitted). Most likely, this unusual chromatin conformation assures that in each OSN the non-selected OR genes will become transcriptionally silent in the most effective and complete manner, as the "one receptor per neuron" rule is the cornerstone of mammalian olfaction. This chromatin-mediated silencing is fortified by the aggregation of the heterochromatic OR clusters in a few, distinct nuclear foci of transcriptionally silent chromatin.

DESCRIPTION (provided by applicant): The transition from acute to chronic neuropathic pain following nerve injury, in many respects, results from a maladaptive plasticity of the nervous system, i.e. it is a disease of the nervous system. The plasticity is manifest at molecular, structural, biochemical and physiological levels, all leading to a condition in which there is ongoing, intense spontaneous pain, pain in response to normally innocuous stimuli (allodynia) and exaggerated pain in response to normally painful stimuli (hyperalgesia). The initiation and maintenance of neuropathic pain involves remodeling of injured nerve circuits, changes in synaptic strength as the pain message is processed through the spinal cord and brain as and perturbations in signaling processes, at all levels of the pain pathway. Many of these changes, however, are the product and/or the cause of long lasting alterations in gene expression. As the most stable modulation of gene expression programs is under epigenetic regulatory control, here we will perform studies to test the hypothesis that nerve injury induces significant alterations to the neuronal epigenome, and most importantly that some of these changes contribute to the generation and persistence of the neuropathic pain condition. In other words, we will dissect the epigenetic landscape of chronic neuropathic pain. Our initial studies will focus on changes that occur in the spinal cord following nerve injury, in standard rodent models of neuropathic pain. Our objective is to obtain a better understanding of the molecular underpinnings of this maladaptive process, which is a critical first step to identifying new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): Opioids are essential to the management of acute and persistent pain, but their chronic use is associated with significant adverse effects, includin tolerance and dependence. There is now considerable information as to the physiological and molecular changes associated with chronic opioid use, but treatments that mitigate the problems are limited and generally ineffective. Unquestionably, the long lasting cellular changes that contribute to the development of tolerance and the manifestations of withdrawal are governed, at least partly, by stable changes in gene expression that are induced by pathways directly engaged during opioid receptor signaling. On the other hand, as manifestations of chronic opioid use, including psychological dependence, may persist long after the termination of opioid intake, we hypothesize that there are additional, maladaptive epigenetic mechanisms that retain transcriptional responses after the removal of the initial stimulus. Here, we propose t identify not only the transcriptional changes associated with tolerance and withdrawal, but also the epigenetic changes that we hypothesize induce and sustain them. We will use mouse models of morphine tolerance and withdrawal and we will focus our analysis on two brain regions, the locus ceruleus (LC) and the ventral tegmental area (VTA), both of which have been implicated in critical features of long term opioid use. We will perform genome wide, next generation sequence-based analysis of changes in the transcriptome (RNA-seq) and the epigenetic landscape (ChIP-seq) of FAC-sorted, pure neuronal populations. Importantly, we will extend our analysis to the 3-dimensional organization of the epigenome, which as is now recognized, provides an additional layer of epigenetic regulation that is likely more stable than chromatin and DNA post-translational modifications. To achieve the high-resolution, 3-dimensional and quantitative imaging of primary neurons isolated from the LC and VTA, we will use soft X-ray tomography (SXT). SXT, as we have recently demonstrated, is ideal for the study of nuclear architecture in primary neurons, and provides the most sensitive imaging method for generating an unbiased identification of the changes in chromatin organization and compaction associated with chronic opioid (morphine) use. This approach will be complemented by a genome wide chromatin conformation capture approach (Hi-C) and DNA FISH experiments. Thus, our proposed experiments will not only map the nuclear and epigenetic landscape of completely uncharted neuronal populations but will also have the potential to provide novel and fundamental insights into the epigenetic processes associated with tolerance and dependence. Together our findings will lay the foundation for the development of novel pharmacological interventions that may not only improve the management of pain by opioids, but also reduce the incidence of adverse side effects, including opioid abuse.

DESCRIPTION (provided by applicant): The realization that epigenetic control of gene expression can override regulatory information encoded in DNA provides the exciting opportunity to stably alter gene expression programs in vivo with the use of epigenetic modifiers. However, this aspiration is challenged by limitations in our ability to alter the epigenetic state of specific target genes in restricted cell types in a temporally regulated fashio. For this reason, we propose to combine novel genetic approaches that afford tight spatiotemporal control in vivo with innovative biochemical tools that allow the targeting of specific genomic loci in a sequence-specific manner. We will modify an assay that we previously designed for the inducible labeling of specific neuronal populations, named Tango, towards the controlled expression of synthetic TALE (Transcription Activator Like Effectors)-fusion proteins that will bind to target genomic loci and alter their epigenetic properties. As a model for these proof-of-principle experiments we will use the genetically, epigenetically and biochemically tractable mouse olfactory system. As we previously showed, the monogenic and monoallelic expression of olfactory receptor (OR) genes in olfactory sensory neurons (OSNs) is epigenetically regulated, both at the level of post-translational histone modifications and at the level of nuclear organization and distribution of active and silent OR alleles. Therefore, we propose to express TALE-fusion proteins with specificity for OR genes and their regulating enhancers in an inducible fashion in specific OSN subpopulations using variations of the TANGO system. This way we will alter the epigenetic state of active or silent ORs, and induce their re-positioning to distinct nuclear territories with the goal of stably altering their expresson pattern. This strategy of chemically or optically controlled epigenetic manipulations will be directly applicable to any other cell type in the mouse, and compatible with viral delivery methods that will make our approach applicable to future therapeutic interventions for human disease.

DESCRIPTION (provided by applicant): Olfactory receptor (OR) choice, the transcriptional activation of one out of thousands of available mammalian OR alleles is a poorly understood process. We previously demonstrated that in response to OR translation, the ER-resident kinase Perk phosphorylates the translation initiation factor eif2a, eliciting a signal that culminates in the stabilization of OR choice. Genetic experiments suggest that this feedback signal depends upon the transient but general attenuation of translation and the specific upregulation of translation of the nuclear isoform of transcription factor ATF5. Production of nuclear ATF5 enhances the transcription of Adenylyl cyclase 3 (Adcy3), which relieves the OR-induced ER stress and represses the expression of histone demethylase LSD1, allowing the terminal differentiation of olfactory neurons and making the expression of the chosen OR permanent. These observations pose significant questions regarding the molecular principles of this signaling pathway. Here, we propose experiments that seek to provide answers to these questions and to offer mechanistic insight into this novel use of the unfolded protein response pathway. Specifically, we propose biochemical and genetic experiments aiming to reveal whether OR proteins interact directly with Perk and to map the exact peptides responsible for Perk activation. Furthermore, we aim to elucidate the mechanism of action of ATF5 as transcriptional regulator and to explore the function of two distinct nuclear isoforms that, according to our preliminary genetic analysis, play different roles in the regulation of OR choice and the differentiation of olfactory neurons. Finally, we propose experiments that aim to dissect the concluding step of this signaling pathway, which is the relief of ER stress and the termination of OR-induced Perk signaling. Timely termination of this arm of the unfolded protein response is as critical for the stabilization of OR expression as the initiation of this pathway an we hypothesize that OR-specific chaperones play a critical role in preventing OR-Perk interactions in the ER. Genetic and biochemical experiments will reveal the identity of these proteins and examine their role in the OR-elicited feedback. Our experiments will provide novel insight into a process that has remained enigmatic since the discovery the largest mammalian gene family and will reveal regulatory principles that likely apply to many other chemoreceptor families mediating communication with the outside world.

DESCRIPTION (provided by applicant): Olfactory receptor (OR) choice, the transcriptional activation of one out of thousands of available mammalian OR alleles, is a poorly understood process. We previously demonstrated that the nuclear intra- and inter- chromosomal aggregation of OR genes in a few, OR-specific, heterochromatic foci contributes to the efficient OR silencing, preserving the monogenic and monoallelic nature of OR expression. Here, we examine the hypothesis that complex interchromosomal associations are responsible also for the transcriptional activation of a single OR allele. Using high throughput epigenetic and genetic approaches we identified a set of novel OR enhancers that support reporter expression in significant fractions of olfactory sensory neurons in zebrafish and mice. Our preliminary data suggest that these enhancers might work in concert for the activation of OR expression, creating a structural and functional singularity in the nuclei of olfactory sensory neurons. We propose a series of experiments that will map and quantify the interchromosomal associations of the newly identified enhancers using imaging and Hi-C approaches. Moreover, we propose genetic loss-, and gain-of- function experiments that will test whether OR enhancers act synergistically towards the activation of a single OR allele. Finally, we seek to investigate the contribution of the unusual epigenetic signature of OR enhancers in their function and nuclear organization. Our experiments will provide novel insight into the role of nuclear architecture in gene expression and will uncover molecular mechanisms involved in the generation of cellular diversity in vivo.

? DESCRIPTION (provided by applicant): The stochastic and monoallelic expression of one out of a thousand olfactory receptor (OR) genes in mammals is a complex process governed by the spatial compartmentalization of active and silent OR alleles in olfactory sensory neurons (OSNs). During OSN differentiation OR loci from multiple chromosomes converge into distinct, OSN-specific nuclear foci characterized by the hallmarks of constitutive heterochromatin. Absent from these unusual nuclear bodies is the OR allele that is transcriptionally active in each OSN, which typically resides on euchromatic nuclear compartments and is surrounded by numerous enhancer elements recruited from several chromosomes. This intricate network of interchromosomal interactions is responsible for both the robust transcription of the chosen OR allele and the complete silencing of the repressed ones. The extraordinary number of OR family members and the unprecedented extent of long-range genomic interactions that culminate to the remarkable organization of the OR nucleome, make the olfactory system ideal for they study of the molecular principles that organize the mammalian nuclear architecture in vivo. For a comprehensive interrogation of the OR nucleome, we assembled a multidisciplinary team seeking to combine novel genetic manipulations with a one of a kind imaging system, a state of the art proteomics facility, and innovative genomic analyses. With CRISPR, phiC31 integrase and in utero DNA electroporation we will tag OR loci and enhancers, making the OR subgenome accessible by three novel experimental strategies: High resolution imaging by correlated soft X-ray tomography and cryo- SIM; biochemical purification by sequence specific tagging with Halo and APEX followed by sophisticated mass spectrometry; and genomic analysis of long range interactions occurring during OSN differentiation using two different DNA modifying enzymes and single molecule real time sequencing. This ambitious experimental project not only will reveal molecular mechanisms that govern the nuclear organization of OR genes but also generally applicable principles and powerful technologies for the study of the mammalian nucleome in vivo.

Research Summary The molecular mechanisms by which a finite genome generates an almost infinite ensemble of cellular identities are poorly understood. The mammalian olfactory epithelium provides an extreme example of cellular heterogeneity in which the functional identity of each olfactory sensory neuron (OSN) is determined by the identity of the olfactory receptor (OR) it expresses. In the mouse each OSN expresses only one out of more than a thousand OR genes in seemingly stochastic and monoallelic fashion. Although some of the regulatory layers governing this remarkable process were recently elucidated, important questions remain open. For example, we previously showed that the transcriptionally active OR allele associates with an intricate interchromosomal hub that contains intergenic OR enhancers from many chromosomes. Here, we seek to elucidate the molecular signatures that distinguish intergenic OR enhancers and promoters from the rest of the genome, allowing the formation of specific genomic interactions that culminate in robust OR transcription. Using ATAC-seq and ChIP-seq approaches in FACsorted olfactory neurons, we identified a novel genetic signature that is highly enriched on intergenic OR enhancers and likely promotes the cooperative binding of transcription factors (TFs) that occupy most OR enhancers. Cooperative TF binding on this novel motif likely permits the synergistic recruitment of two distinct types of adaptor proteins to intergenic OR enhancers, creating a unique molecular surface that may dictate the specificity on enhancer-enhancer and enhancer- promoter interactions in the OR subgenome. We therefore propose genetic experiments that will test these predictions and will elucidate the specificity by which intergenic OR enhancers become recognized by TFs, the specificity by which OR enhancers interact with each other and the specificity by which they recruit OR promoters. Because the proteins we propose to investigate are expressed in many neuronal tissues, the regulatory principles that will produced by these studies are likely to impact our general understanding of the combinatorial control of neuronal specification.

Project Summary The molecular mechanisms that cause spontaneous neurodegeneration remain poorly understood. Here, we propose a novel hypothesis for the molecular events that lead to the generation of misfolded proteins and their accumulation in neurotoxic protein aggregates. Prompted by our recent discoveries on the mechanisms of olfactory receptor (OR) gene regulation, we propose that olfactory neurons could be used as early molecular sensors for neurodegenerative disorders. This hypothesis is supported by two key observations. First, Lsd1, the histone demethylase that activates OR transcription, releases hydrogen peroxide in nuclei of olfactory neurons, causing increased DNA oxidation locally, at OR loci, and genome-wide. 8-oxoguanine, the modified base generated by Lsd1-mediated oxidation, is not efficiently repaired in olfactory neurons and can cause frequent G to A conversion during transcription. Consequently, olfactory neurons may have high incidence of missense mutations in their mRNAs, some of which are likely to generate mutant proteins that are prone to misfolding and aggregation. Second, olfactory neurons cannot clear misfolded and aggregated proteins from their endoplasmic reticulum (ER) because they co-opted the unfolded protein response (UPR) pathway towards OR regulation instead of ER homeostasis. This unique combination of regulatory ?side-effects?, i.e. increased mutagenesis rates through DNA oxidation and impaired ability to remove misfolded proteins from the ER, may provide an ideal cellular environment for the generation of misfolded proteins and the accumulation of protein aggregates. Thus, we propose experiments that will further explore this hypothesis and will provide molecular insight to spontaneous neurodegeneration. With the development of novel, whole genome sequencing approaches with base-pair resolution, we will map the location of Lsd1-dependent 8-oxoguanine accumulation and we will quantitate its mutagenic potential during transcription. High throughput in vitro screens combined with mouse genetics will evaluate the contribution of frequent missense mutation in the misfolding and aggregation of proteins previously linked to neurodegeneration. Genetic approaches will determine how the alternative UPR pathway that evolved for OR regulation contributes to the onset of neurodegeneration. Our experiments have paradigm-shifting potential towards the understanding of spontaneous neurodegeneration and may lead to the development of novel molecular tools for its timely diagnosis or reliable prognosis, which could provide decisive advantage towards the prevention and treatment of Alzheimer?s disease.

Different cell types in a given animal, such as heart cells and brain cells display different behaviors because they express different sets of genes. Yet, they all have DNA with essentially the same sequence and thus the same set of genes. How is it that the same DNA is used to generate different cell types? Which genes are on and which genes are off is controlled by how their underlying DNA sequences are packaged. DNA is packaged by wrapping it around specific proteins called histones to generate bead-like structures called nucleosomes. Strings of nucleosomes are then further folded to condense the underlying DNA and make it less accessible. Structures called heterochromatin are thought to be particularly effective at compacting strings of nucleosomes and turning off the underlying genes. A few years ago it was discovered that proteins named HP1 proteins, which are core components of heterochromatin, can sequester DNA into droplets that are separated from the surrounding solution in a different phase. This discovery provides a novel way to think about DNA packaging while also raising new fundamental questions such as: how are these droplet-based DNA compartments regulated by cellular signals and; how do changes in droplet mediated DNA organization impact biology at the level of a whole animal? To address these questions the PIs have assembled a multi-disciplinary team that brings together expertise in mouse biology, cutting-edge imaging technology and advanced biophysical methods. Another key goal of the project is to provide middle school students from underrepresented communities hands-on experience in carrying out experiments with packaged DNA. The research will: (i) shed light on how small collections of molecules can drive heritable changes at the level of a whole animal and; (ii) introduce middle-school students to the wonders of scientific discovery.

A major form of heritable gene regulation is driven by heterochromatin, which silences specific subsets of genes and is essential for cellular differentiation, environmental adaptation and organismal physiology. The discovery that heterochromatin can form by phase-separation based mechanisms have led to a new paradigm for imagining genome organization, in which phase-separation enables genome sequestration. Given the novelty of the findings many fundamental questions remain unanswered such as: (i) what are the physico-chemical rules underlying phase-separation by heterochromatin; (ii) what types of emergent properties are conferred by phase-separation and; (iii) what are the physiological consequences? Addressing these questions requires working at the intersection of multiple disciplines. Therefore this project is organized within the physiological context of mouse olfactory receptor regulation as studied by Dr. Lomvardas and uses new imaging technologies pioneered by Dr. Larabell and biophysical tools developed by Dr. Narlikar. The project integrates experimental enquiry across multiple scales, from atomic-level studies of HP1 behavior to assessment of whole mouse phenotypes. Specifically the PIs will study olfactory receptor (OR) expression in mice, which is controlled by specialized heterochromatic compartments (ORH). ORH is spatially distinct from heterochromatin formed near centromeres (PH), strongly indicative of two different phase-separated states. ORH and PH are enriched for different HP1 paralogs, HP1 and HP1, respectively. Using a combination of mouse genetics, soft-Xray tomography and quantitative phase-separation methods the PIs will investigate (i) whether ORH and PH have different physico-chemical properties that prevent mixing and enable distinct physiological functions and, (ii) whether these different properties may arise from differences in the substructures formed within HP1 vs. HP1 phases. The PIs anticipate these studies will illuminate how atomic scale differences in sequence between HP1; and HP1; result in meso-scale differences in droplet structure, which in turn have a defined physiological impact. A key goal of the project is also to provide middle school students a hands-on experience in heterochromatin based phase-separation experiments through an annual summer workshop.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary/Abstract In this Competitive Revision proposal, we seek to investigate the non-cell autonomous effects of Covid- 19 infections in olfaction. Our preliminary data suggest that induction of pro-inflammatory/antiviral pathways result in disruption of inter-chromosomal genomic interactions, and downregulation of Olfactory Receptor (OR) gene expression. Since antiviral responses are expected be elicited upon Covid-19 infection, we hypothesize that disruptions in nuclear architecture and OR expression account for the reported olfactory deficits in infected patients. Thus, we propose to analyze human autopsies of the olfactory epithelium, to decipher whether Covid- 19 infections disrupt genomic interactions required for OR transcription. We will complement our studies in human autopsies with experiments using mice infected with SARS-CoV-2. RNA-seq, in situ HiC and immunohistochemistry experiments in human and mice will reveal the molecular mechanisms by which Covid- 19 induces olfactory dysfunction. Our experiments will provide critical insight to the mechanisms by which the virus hijacks molecular and physiological processes of the host cell, opening new potential avenues for the prevention, diagnosis and treatment of Covid-19 infection.

Abstract The monogenic, monoallelic, and seemingly stochastic transcriptional choice of one out of > 1000 olfactory receptor (OR) genes remained elusive for decades after the discovery of the largest mammalian gene family. However, in the past few years we obtained significant understanding on the molecular underpinnings of this enigmatic gene regulatory process. Specifically, we showed that OR gene clusters become heterochromatic at the early stages of olfactory sensory neuron (OSN) differentiation and then they aggregate in distinct nuclear compartments that assure their stable repression. As a result of this interchromosomal convergence, intergenic OR enhancers (known as Greek Islands) that are found in most OR gene clusters come in close nuclear proximity and form a multi-chromosomal super-enhancer that in each OSN associates with the transcriptionally active OR allele. The formation of the Greek Island hub is dependent upon the recruitment of the adaptor protein Ldb1, which is essential for the stable interchromosomal interactions between Greek Islands and for OR transcription. This intricate network of activating and repressive interchromosomal interactions, together with a feedback signal elicited by the expression of the chosen OR, likely generate the regulatory framework for transcriptional singularity. However, what remains unknown is the process by which an OR allele is recruited to the Greek Island hub and the mechanism that assures that only one OR allele will remain stably associated with a multi-chromosomal structure that contains numerous enhancer elements. Our preliminary data suggest that developmentally transient OR transcription and production of nascent OR mRNAs contribute to the recruitment of an OR allele to the Greek Island hub and to OR gene choice. Thus, we propose genetic experiments that will determine which sequences of the sense OR mRNA are required and sufficient for recruitment of trans OR enhancers, what proteins recognized the nascent OR mRNA and what contribution these proteins have to the assembly of a multi-enhancer/OR complex. These experiments not only will shed light to the enigmatic process of OR gene choice, but will also provide general molecular principles for the mechanisms that mediate genomic compartmentalization during cellular differentiation.

Abstract The monogenic, monoallelic, and seemingly stochastic transcriptional choice of one out of > 1000 olfactory receptor (OR) genes remained elusive for decades after the discovery of the largest mammalian gene family. However, in the past few years we obtained significant understanding on the molecular underpinnings of this enigmatic gene regulatory process. Specifically, we showed that OR gene clusters become heterochromatic at the early stages of olfactory sensory neuron (OSN) differentiation and then they aggregate in distinct nuclear compartments that assure their stable repression. As a result of this interchromosomal convergence, intergenic OR enhancers (known as Greek Islands) that are found in most OR gene clusters come in close nuclear proximity and form a multi-chromosomal super-enhancer that in each OSN associates with the transcriptionally active OR allele. The formation of the Greek Island hub is dependent upon the recruitment of the adaptor protein Ldb1, which is essential for the stable interchromosomal interactions between Greek Islands and for OR transcription. This intricate network of activating and repressive interchromosomal interactions, together with a feedback signal elicited by the expression of the chosen OR, likely generate the regulatory framework for transcriptional singularity. However, what remains unknown how this seemingly stochastic process operates under deterministic restrictions related to the spatial location of the OSN along the dorso-ventral and apico-basal axes of the MOE. These restrictions, known as zonal pattern of OR expression, restrict the expression of each OR gene in one of five zones of expression. Here we identified putative mechanisms of zonal restriction, by uncovering the molecular mechanisms that enable only zone 5 ORs to be expressed in zone 5. We show that transcription factors of the NFI family enable the transcriptional activation of zone 5 ORs, by mediating the recruitment of these ORs to the interchromosomal OR compartment. Moreover, we show that the repressive histone modification H3K79me3 prevents the expression of out of zone ORs, possibly under the control of NFI factors, as well. We propose experiments that will decipher which NFI factors are required and sufficient for specification of zone 5 transcription programs, and experiment that will determine how NFI proteins accomplish these zonal restrictions. Our experiments will reveal novel mechanisms of regulation of nuclear architecture, and will uncover generally applicable principles for the regulation of developmental patterning.

Project Summary Detection and identification of the astronomical number of volatile chemicals that we perceive as odors depends upon the monogenic and monoallelic expression of olfactory receptor (OR) genes in olfactory sensory neurons (OSNs). An intricate network of OSN-specific interchromosomal interactions coordinates the transcriptional activation of only 1 OR allele out of >2000 OR alleles distributed across 18 different chromosomes. Genomic interactions between silent OR genes assemble heterochromatic multi-chromosomal compartments that keep OR genes transcriptionally inactive, whereas genomic interactions between intergenic OR enhancers result in a multi-chromosomal enhancer hub that activates singular OR transcription. Here, we propose to combine Dip-C, a variation of single cell HiC, with viral-based cell tagging technologies, towards the identification of genome folding intermediates across OSN differentiation lineages. This ?cradle to crate? genomic analysis will follow individual OSN progenitors and their barcoded progeny, allowing a complete cartography of genomic interactions made by every OR allele en route to transcriptional activation. Single molecule DNA FISH experiments will complement the proposed genomic studies, providing high resolution insight to the genomic choreography that orchestrates singular OR gene choice during development. Finally, we seek to explore how the trajectories of OSN genomic folding become altered and eventually disrupted in a humanized model for Alzheimer?s disease (AD). Olfactory dysfunction, hyposmia, and anosmia constitute well- established prodromal symptoms of AD, but the molecular etiology of this intriguing connection is not known. Using a humanized model for AD we discovered that interchromosomal OR compartments dissipate prior to the onset of neurodegeneration, resulting in strong downregulation of OR transcription. Thus, we propose to apply our single cell interrogation of genome folding transition in the context of AD and to establish the baseline of nuclear architecture in human OSNs. Deciphering how OR compartments assemble in health, and how they become disrupted in disease, may provide the basis for novel prognostic and diagnostic tools for AD, and molecular assays for in vivo screening of AD therapeutics.