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1.
Cell ; 185(21): 3896-3912.e22, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36167070

RESUMO

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.


Assuntos
Axônios/metabolismo , Estresse do Retículo Endoplasmático , Receptores Odorantes , Animais , Camundongos , Bulbo Olfatório , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Fatores de Transcrição/metabolismo
2.
Cell ; 177(3): 639-653.e15, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30955885

RESUMO

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.


Assuntos
Caderinas/genética , Desmetilação do DNA , RNA Antissenso/metabolismo , RNA Longo não Codificante/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Caderinas/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos , Éxons , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Família Multigênica , Neurônios/citologia , Neurônios/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Antissenso/genética , Transcrição Gênica
3.
Nature ; 625(7993): 181-188, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38123679

RESUMO

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.


Assuntos
Neurônios Receptores Olfatórios , RNA , Receptores Odorantes , Alelos , Linhagem da Célula , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , RNA/genética , Transcrição Gênica , Genômica , Análise de Célula Única
4.
Development ; 151(10)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38813842

RESUMO

This Review elucidates the regulatory principles of random monoallelic expression by focusing on two well-studied examples: the X-chromosome inactivation regulator Xist and the olfactory receptor gene family. Although the choice of a single X chromosome or olfactory receptor occurs in different developmental contexts, common gene regulatory principles guide monoallelic expression in both systems. In both cases, an event breaks the symmetry between genetically and epigenetically identical copies of the gene, leading to the expression of one single random allele, stabilized through negative feedback control. Although many regulatory steps that govern the establishment and maintenance of monoallelic expression have been identified, key pieces of the puzzle are still missing. We provide an overview of the current knowledge and models for the monoallelic expression of Xist and olfactory receptors. We discuss their similarities and differences, and highlight open questions and approaches that could guide the study of other monoallelically expressed genes.


Assuntos
Alelos , RNA Longo não Codificante , Receptores Odorantes , Inativação do Cromossomo X , Animais , Humanos , Inativação do Cromossomo X/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
5.
PLoS Genet ; 17(2): e1009338, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33600447

RESUMO

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Mecanismo Genético de Compensação de Dose , Feminino , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Masculino , Neurônios/citologia , RNA-Seq/métodos , Fatores Sexuais
6.
Genes Dev ; 28(5): 463-78, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24589776

RESUMO

Many loci maintain parent-of-origin DNA methylation only briefly after fertilization during mammalian development: Whether this form of transient genomic imprinting can impact the early embryonic transcriptome or even have life-long consequences on genome regulation and possibly phenotypes is currently unknown. Here, we report a maternal germline differentially methylated region (DMR) at the mouse Gpr1/Zdbf2 (DBF-type zinc finger-containing protein 2) locus, which controls the paternal-specific expression of long isoforms of Zdbf2 (Liz) in the early embryo. This DMR loses parental specificity by gain of DNA methylation at implantation in the embryo but is maintained in extraembryonic tissues. As a consequence of this transient, tissue-specific maternal imprinting, Liz expression is restricted to the pluripotent embryo, extraembryonic tissues, and pluripotent male germ cells. We found that Liz potentially functions as both Zdbf2-coding RNA and cis-regulatory RNA. Importantly, Liz-mediated events allow a switch from maternal to paternal imprinted DNA methylation and from Liz to canonical Zdbf2 promoter use during embryonic differentiation, which are stably maintained through somatic life and conserved in humans. The Gpr1/Zdbf2 locus lacks classical imprinting histone modifications, but analysis of mutant embryonic stem cells reveals fine-tuned regulation of Zdbf2 dosage through DNA and H3K27 methylation interplay. Together, our work underlines the developmental and evolutionary need to ensure proper Liz/Zdbf2 dosage as a driving force for dynamic genomic imprinting at the Gpr1/Zdbf2 locus.


Assuntos
Metilação de DNA , Impressão Genômica/genética , Mamíferos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Evolução Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Humanos , Masculino , Mamíferos/embriologia , Mamíferos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Espermatogênese/genética
7.
Mol Cell ; 47(6): 909-20, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22902559

RESUMO

Identifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life.


Assuntos
Caderinas/genética , Metilação de DNA , Embrião de Mamíferos/metabolismo , Impressão Genômica , Células Germinativas/metabolismo , Oócitos/metabolismo , Animais , Blastocisto/metabolismo , Embrião de Mamíferos/citologia , Fertilização , Testes Genéticos , Camundongos , Pseudogenes , Análise de Sequência de DNA
8.
Development ; 140(3): 562-72, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23293288

RESUMO

A stimulatory DNA methyltransferase co-factor, Dnmt3L, has evolved in mammals to assist the process of de novo methylation, as genetically demonstrated in the germline. The function of Dnmt3L in the early embryo remains unresolved. By combining developmental and genetic approaches, we find that mouse embryos begin development with a maternal store of Dnmt3L, which is rapidly degraded and does not participate in embryonic de novo methylation. A zygotic-specific promoter of Dnmt3l is activated following gametic methylation loss and the potential recruitment of pluripotency factors just before implantation. Importantly, we find that zygotic Dnmt3L deficiency slows down the rate of de novo methylation in the embryo by affecting methylation density at some, but not all, genomic sequences. Dnmt3L is not strictly required, however, as methylation patterns are eventually established in its absence, in the context of increased Dnmt3A protein availability. This study proves that the postimplantation embryo is more plastic than the germline in terms of DNA methylation mechanistic choices and, importantly, that de novo methylation can be achieved in vivo without Dnmt3L.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Animais , Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Proteólise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Espermatozoides/citologia , Espermatozoides/metabolismo , Transcrição Gênica
9.
Nat Commun ; 9(1): 2189, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29875359

RESUMO

Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the "immature" splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.


Assuntos
Processamento Alternativo , Sistema Nervoso Central/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Animais , Diferenciação Celular/genética , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Modelos Neurológicos , Neurônios/citologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Tempo
10.
Neuron ; 83(4): 823-38, 2014 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-25123312

RESUMO

During differentiation, neurons exhibit a reorganization of DNA modification patterns across their genomes. The de novo DNA methyltransferase Dnmt3a is implicated in this process, but the effects of its absence have not been fully characterized in a purified neuronal population. To better understand how DNA modifications contribute to neuronal function, we performed a comprehensive analysis of the epigenetic and transcriptional landscapes of Dnmt3a-deficient mature olfactory sensory neurons (mOSNs), the primary sensory neurons of the olfactory epithelium. Dnmt3a is required for both 5-methylcytosine and 5-hydroxymethylcytosine patterning within accessible genomic regions, including hundreds of neurodevelopmental genes and neural enhancers. Loss of Dnmt3a results in the global disruption of gene expression via activation of silent genes and reduction of mOSN-expressed transcripts. Importantly, the DNA modification state and inducibility of odorant-activated genes are markedly impaired in Dnmt3a knockouts, suggesting a crucial role for this enzyme in establishing an epigenetic landscape compatible with neuronal plasticity.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Percepção Olfatória/genética , Neurônios Receptores Olfatórios/metabolismo , Olfato/genética , Animais , Células Cultivadas , Metilação de DNA/genética , DNA Metiltransferase 3A , Epigenômica , Camundongos , Plasticidade Neuronal/genética , Neurônios Receptores Olfatórios/crescimento & desenvolvimento
11.
Curr Top Dev Biol ; 104: 293-328, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23587245

RESUMO

The early mammalian embryo is marked by genome-wide parental epigenetic asymmetries, which are directly inherited from the sperm and the oocyte, but are also amplified a few hours after fertilization. The yin-yang of these complementary parental programs is essential for proper development, as uniparental embryos are not viable. The majority of these parental asymmetries are erased, as the embryonic genome assumes its own chromatin signature toward pluripotency and then differentiation, reducing the risk for haploinsufficiency. At a few loci, however, parent-of-origin information persists through development, via maintenance and protective complexes. In this review, we discuss the parental asymmetries that are inherited from the gametes, the forces involved in their elimination, reinforcement or protection, and how this influences the embryonic program. We highlight the gradual loss of all parental asymmetries occurring throughout development, except at imprinted loci, which maintain distinct parent-of-origin chromatin and transcriptional characteristics for life. A deeper understanding of the nongenetic contributions of each germline is important to provide insight into the origin of non-Mendelian inheritance of phenotypic traits, as well as the risk of incompatibilities between parental genomes.


Assuntos
Epigênese Genética , Mamíferos/embriologia , Mamíferos/genética , Pais , Caracteres Sexuais , Animais , Núcleo Celular/metabolismo , Implantação do Embrião/genética , Humanos , Padrões de Herança/genética
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