ABSTRACT
Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.
Subject(s)
Axons/metabolism , Endoplasmic Reticulum Stress , Receptors, Odorant , Animals , Mice , Olfactory Bulb , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/genetics , Receptors, Odorant/metabolism , Transcription Factors/metabolismABSTRACT
SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.
Subject(s)
Anosmia , COVID-19 , Animals , Cricetinae , Down-Regulation , Humans , Receptors, Odorant , SARS-CoV-2 , SmellABSTRACT
Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.
Subject(s)
Cadherins/genetics , DNA Demethylation , RNA, Antisense/metabolism , RNA, Long Noncoding/genetics , Animals , Binding Sites , CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Cadherins/metabolism , Cell Line , Enhancer Elements, Genetic , Exons , Female , Humans , Mice , Mice, Transgenic , Multigene Family , Neurons/cytology , Neurons/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA, Antisense/genetics , Transcription, GeneticABSTRACT
Genetic material is not randomly organized within the nucleus of a cell. How this organization occurs and why it matters are questions that Cell editor Marta Koch posed to Mitchell Guttman, Job Dekker, and Stavros Lomvardas. Excerpts from this Conversation are presented below, and an audio file of the full discussion is available with the article online.
Subject(s)
Cell Nucleus/chemistry , Chromosomes/chemistry , DNA/chemistry , Animals , Cell Nucleus/genetics , Cell Nucleus/physiology , Chromosomes/genetics , Chromosomes/metabolism , DNA/genetics , DNA/metabolism , Genomics , National Institutes of Health (U.S.) , United StatesABSTRACT
Olfactory receptor (OR) choice provides an extreme example of allelic competition for transcriptional dominance, where every olfactory neuron stably transcribes one of approximately 2,000 or more OR alleles1,2. OR gene choice is mediated by a multichromosomal enhancer hub that activates transcription at a single OR3,4, followed by OR-translation-dependent feedback that stabilizes this choice5,6. Here, using single-cell genomics, we show formation of many competing hubs with variable enhancer composition, only one of which retains euchromatic features and transcriptional competence. Furthermore, we provide evidence that OR transcription recruits enhancers and reinforces enhancer hub activity locally, whereas OR RNA inhibits transcription of competing ORs over distance, promoting transition to transcriptional singularity. Whereas OR transcription is sufficient to break the symmetry between equipotent enhancer hubs, OR translation stabilizes transcription at the prevailing hub, indicating that there may be sequential non-coding and coding mechanisms that are implemented by OR alleles for transcriptional prevalence. We propose that coding OR mRNAs possess non-coding functions that influence nuclear architecture, enhance their own transcription and inhibit transcription from their competitors, with generalizable implications for probabilistic cell fate decisions.
Subject(s)
Olfactory Receptor Neurons , RNA , Receptors, Odorant , Alleles , Cell Lineage , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/genetics , Receptors, Odorant/metabolism , Regulatory Sequences, Nucleic Acid/genetics , RNA/genetics , Transcription, Genetic , Genomics , Single-Cell AnalysisABSTRACT
The transcriptional activation of one out of ?2800 olfactory receptor (OR) alleles is a poorly understood process. Here, we identify a plethora of putative OR enhancers and study their in vivo activity in olfactory neurons. Distinguished by an unusual epigenetic signature, candidate OR enhancers are characterized by extensive interchromosomal interactions associated with OR transcription and share a similar pattern of transcription factor footprints. In particular, we establish the role of the transcription factor Bptf as a facilitator of both enhancer interactions and OR transcription. Our observations agree with the model whereby OR transcription occurs in the context of multiple interacting enhancers. Disruption of these interchromosomal interactions results in weak and multigenic OR expression, suggesting that the rare coincidence of numerous enhancers over a stochastically chosen OR may account for the singularity and robustness in OR transcription.
Subject(s)
Enhancer Elements, Genetic , Receptors, Odorant/genetics , Transcriptional Activation , Animals , Animals, Genetically Modified , Antigens, Nuclear/metabolism , Mice , Nerve Tissue Proteins/metabolism , Nucleoproteins/metabolism , Olfactory Receptor Neurons/metabolism , Transcription Factors/metabolism , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
The sense of smell collects vital information about the environment by detecting a multitude of chemical odorants. Breadth and sensitivity are provided by a huge number of chemosensory receptor proteins, including more than 1,400 olfactory receptors (ORs). Organizing the sensory information generated by these receptors so that it can be processed and evaluated by the central nervous system is a major challenge. This challenge is overcome by monogenic and monoallelic expression of OR genes. The single OR expressed by each olfactory sensory neuron determines the neuron's odor sensitivity and the axonal connections it will make to downstream neurons in the olfactory bulb. The expression of a single OR per neuron is accomplished by coupling a slow chromatin-mediated activation process to a fast negative-feedback signal that prevents activation of additional ORs. Singular OR activation is likely orchestrated by a network of interchromosomal enhancer interactions and large-scale changes in nuclear architecture.
Subject(s)
Olfactory Receptor Neurons/physiology , Animals , Axons/physiology , Humans , Odorants , Smell/physiologyABSTRACT
Olfactory receptor (OR) expression requires the transcriptional activation of 1 out of 1,000s of OR alleles and a feedback signal that preserves this transcriptional choice. The mechanism by which olfactory sensory neurons (OSNs) detect ORs to signal to the nucleus remains elusive. Here, we show that OR proteins generate this feedback by activating the unfolded protein response (UPR). OR expression induces Perk-mediated phosphorylation of the translation initiation factor eif2α causing selective translation of activating transcription factor 5 (ATF5). ATF5 induces the transcription of adenylyl cyclase 3 (Adcy3), which relieves the UPR. Our data provide a role for the UPR in defining neuronal identity and cell fate commitment and support a two-step model for the feedback signal: (1) OR protein, as a stress stimulus, alters the translational landscape of the OSN and induces Adcy3 expression; (2), Adcy3 relieves that stress, restores global translation, and makes OR choice permanent.
Subject(s)
Feedback, Physiological , Neurons/metabolism , Receptors, Odorant/metabolism , Unfolded Protein Response , Activating Transcription Factors/genetics , Activating Transcription Factors/metabolism , Adenylyl Cyclases/metabolism , Animals , Endoplasmic Reticulum/metabolism , Eukaryotic Initiation Factor-2/metabolism , Mice , Mice, Knockout , Neurons/cytology , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/genetics , eIF-2 Kinase/metabolismABSTRACT
The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the OR-dependent expression of adenylyl cyclase 3 (Adcy3) as requirements for initiation and stabilization of OR expression. As a transcriptional coactivator, LSD1 is necessary for desilencing and initiating OR transcription, but as a transcriptional corepressor, it is incompatible with maintenance of OR expression, and its downregulation is imperative for stable OR choice. Adcy3, a sensor of OR expression and a transmitter of an OR-elicited feedback, mediates the downregulation of LSD1 and promotes the differentiation of olfactory sensory neurons (OSNs). This novel, three-node signaling cascade locks the epigenetic state of the chosen OR, stabilizes its singular expression, and prevents the transcriptional activation of additional OR alleles for the life of the neuron.
Subject(s)
Adenylyl Cyclases/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Oxidoreductases, N-Demethylating/metabolism , Receptors, Odorant/genetics , Sensory Receptor Cells/metabolism , Animals , Down-Regulation , Histone Demethylases , Mice , Mice, Knockout , Nasal Mucosa/metabolism , Olfactory Receptor Neurons/metabolismABSTRACT
Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally synchronous with the downregulation of lamin b receptor (LBR) and can be reversed by ectopic expression of LBR in mature olfactory neurons. LBR-induced reorganization of nuclear architecture and disruption of OR aggregates perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression.
Subject(s)
Cell Nucleus/chemistry , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/genetics , Animals , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Down-Regulation , Gene Expression Regulation , Heterochromatin/metabolism , In Situ Hybridization, Fluorescence , Mice , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic , Lamin B ReceptorABSTRACT
Constitutive heterochromatin is traditionally viewed as the static form of heterochromatin that silences pericentromeric and telomeric repeats in a cell cycle- and differentiation-independent manner. Here, we show that, in the mouse olfactory epithelium, olfactory receptor (OR) genes are marked in a highly dynamic fashion with the molecular hallmarks of constitutive heterochromatin, H3K9me3 and H4K20me3. The cell type and developmentally dependent deposition of these marks along the OR clusters are, most likely, reversed during the process of OR choice to allow for monogenic and monoallelic OR expression. In contrast to the current view of OR choice, our data suggest that OR silencing takes place before OR expression, indicating that it is not the product of an OR-elicited feedback signal. Our findings suggest that chromatin-mediated silencing lays a molecular foundation upon which singular and stochastic selection for gene expression can be applied.
Subject(s)
Chromatin Assembly and Disassembly , Gene Silencing , Olfactory Mucosa/metabolism , Receptors, Odorant/genetics , Animals , Chromatin Immunoprecipitation , Gene Expression , Heterochromatin , Histone Code , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence AnalysisABSTRACT
The genome is partitioned into topologically associated domains and genomic compartments with shared chromatin valence. This architecture is constrained by the DNA polymer, which precludes interactions between genes on different chromosomes. Here we report a marked divergence from this pattern of nuclear organization that occurs in mouse olfactory sensory neurons. Chromatin conformation capture using in situ Hi-C on fluorescence-activated cell-sorted olfactory sensory neurons and their progenitors shows that olfactory receptor gene clusters from 18 chromosomes make specific and robust interchromosomal contacts that increase with differentiation of the cells. These contacts are orchestrated by intergenic olfactory receptor enhancers, the 'Greek islands', which first contribute to the formation of olfactory receptor compartments and then form a multi-chromosomal super-enhancer that associates with the single active olfactory receptor gene. The Greek-island-bound transcription factor LHX2 and adaptor protein LDB1 regulate the assembly and maintenance of olfactory receptor compartments, Greek island hubs and olfactory receptor transcription, providing mechanistic insights into and functional support for the role of trans interactions in gene expression.
Subject(s)
Chromosomes, Mammalian/genetics , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , LIM Domain Proteins/metabolism , LIM-Homeodomain Proteins/metabolism , Receptors, Odorant/genetics , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cell Differentiation/genetics , Chromatin/genetics , Chromatin/metabolism , Chromosome Positioning/genetics , Chromosomes, Mammalian/metabolism , Female , Male , Mice , Multigene Family/genetics , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/metabolismABSTRACT
The senses provide a means by which data on the physical and chemical properties of the environment may be collected and meaningfully interpreted. Sensation begins at the periphery, where a multitude of different sensory cell types are activated by environmental stimuli as different as photons and odorant molecules. Stimulus sensitivity is due to expression of different cell surface sensory receptors, and therefore the receptive field of each sense is defined by the aggregate of expressed receptors in each sensory tissue. Here, we review current understanding on patterns of expression and modes of regulation of sensory receptors.
Subject(s)
Receptors, G-Protein-Coupled/genetics , Receptors, Odorant/genetics , Receptors, Odorant/physiology , Sensory Receptor Cells/physiology , Vomeronasal Organ/physiology , Animals , Receptors, G-Protein-Coupled/physiologyABSTRACT
This corrects the article DOI: 10.1038/nature23884.
ABSTRACT
The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
Subject(s)
Cell Nucleus/genetics , Cell Nucleus/physiology , Genome , Models, Molecular , Molecular Imaging/methods , Spatio-Temporal Analysis , Animals , Cell Line , Chromatin/genetics , Chromatin/metabolism , Chromosomes/chemistry , Chromosomes/genetics , Chromosomes/metabolism , Genomics/methods , Genomics/organization & administration , Goals , Humans , Information Dissemination , Mice , Models, Biological , Reproducibility of Results , Single-Cell AnalysisABSTRACT
Sprinkled throughout the genome are a million regulatory sequences called transcriptional enhancers that activate gene promoters in the right cells, at the right time. Enhancers endow the brain with its incredible diversity of cell types and also translate neural activity into gene induction. Thanks to rapid advances in genomic technologies, it is now possible to identify thousands of enhancers rapidly, test their transcriptional function en masse, and address their neurobiological functions via genome editing. Enhancers also promise to be a great technological opportunity for neuroscience, offering the potential for cell-type-specific genetic labeling and manipulation without the need for transgenesis. The objective of this review and the accompanying 2015 SfN mini-symposium is to highlight the use of new and emerging genomic technologies to probe enhancer function in the nervous system. SIGNIFICANCE STATEMENT: Transcriptional enhancers turn on genes in the right cells, at the right time. Enhancers are also the genomic sequences that encode the incredible diversity of cell types in the brain and enable the brain to turn genes on in response to new experiences. New technology enables enhancers to be found and manipulated. The study of enhancers promises to inform our understanding of brain development and function. The application of enhancer technology holds promise in accelerating basic neuroscience research and enabling gene therapies to be targeted to specific cell types in the brain.
Subject(s)
Central Nervous System/physiology , Enhancer Elements, Genetic/genetics , Genomics , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Animals , Central Nervous System/cytology , Gene Expression/genetics , Humans , Models, BiologicalABSTRACT
The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene regulation and cellular identity. To determine how 5hmC influences gene expression in an in vivo neuronal population, we assessed the patterning and function of the base along the developmental lineage of the main olfactory epithelium-from multipotent stem cells through neuronal progenitors to mature olfactory sensory neurons (mOSNs). We find that 5hmC increases over gene bodies during mOSN development with substantial patterning occuring between the progenitor and mOSN stages. Although gene-body 5hmC levels correlate with gene expression in all three developmental cell types, this association is particularly pronounced within mOSNs. Overexpression of Tet3 in mOSNs markedly alters gene-body 5hmC levels and gene expression in a manner consistent with a positive role for 5hmC in transcription. Moreover, Tet3 overexpression disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system. Our results suggest a physiologically significant role for gene-body 5hmC in transcriptional facilitation and the maintenance of cellular identity independent of its function as an intermediate to demethylation.
Subject(s)
Cytosine/analogs & derivatives , Gene Expression Profiling , Gene Expression Regulation, Developmental , Olfactory Receptor Neurons/metabolism , 5-Methylcytosine/analogs & derivatives , Animals , Cell Differentiation/genetics , Cytosine/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dioxygenases , Fluorescent Antibody Technique , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Olfactory Bulb/cytology , Olfactory Bulb/metabolism , Olfactory Mucosa/cytology , Olfactory Mucosa/metabolism , Olfactory Receptor Neurons/cytology , Olfactory Receptor Neurons/growth & development , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Transcriptionally repressive histone lysine methylation is used by eukaryotes to tightly control cell fate. Here we explore the importance of this form of regulation in the control of clustered genes in the genome. Two distinctly regulated gene families with important roles in vertebrates are discussed, namely the Hox genes and olfactory receptor genes. Major recent advances in these two fields are compared and contrasted, with an emphasis on the roles of the two different forms of histone trimethylation. We discuss how this repression may impact both the transcriptional output of these loci and the way higher-order chromatin organization is related to their unique control.
Subject(s)
Gene Expression Regulation , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Repressor Proteins/metabolism , Transcription, Genetic/genetics , Animals , Down-Regulation , Genetic Determinism , Histone Methyltransferases , Humans , Methylation , Protein Processing, Post-Translational , Stochastic ProcessesABSTRACT
Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers, Dync1i1 exon 15, has active enhancer marks and physically interacts with Dlx5/6 promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated with DLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.