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1.
Cell ; 174(6): 1571-1585.e11, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-30193114

RESUMEN

Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.


Asunto(s)
Relojes Circadianos/fisiología , Metaboloma , Animales , Dieta Alta en Grasa , Metabolismo Energético , Hígado/metabolismo , Masculino , Redes y Vías Metabólicas , Metabolómica , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Corteza Prefrontal/metabolismo , Núcleo Supraquiasmático/metabolismo , Proteína Desacopladora 1/metabolismo
2.
EMBO J ; 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38951609

RESUMEN

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

3.
Int J Mol Sci ; 24(18)2023 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-37762112

RESUMEN

Since its initial involvement in numerous neurodegenerative pathologies in 2006, either as a principal actor or as a cofactor, new pathologies implicating transactive response (TAR) DNA-binding protein 43 (TDP-43) are regularly emerging also beyond the neuronal system. This reflects the fact that TDP-43 functions are particularly complex and broad in a great variety of human cells. In neurodegenerative diseases, this protein is often pathologically delocalized to the cytoplasm, where it irreversibly aggregates and is subjected to various post-translational modifications such as phosphorylation, polyubiquitination, and cleavage. Until a few years ago, the research emphasis has been focused particularly on the impacts of this aggregation and/or on its widely described role in complex RNA splicing, whether related to loss- or gain-of-function mechanisms. Interestingly, recent studies have strengthened the knowledge of TDP-43 activity at the chromatin level and its implication in the regulation of DNA transcription and stability. These discoveries have highlighted new features regarding its own transcriptional regulation and suggested additional mechanistic and disease models for the effects of TPD-43. In this review, we aim to give a comprehensive view of the potential epigenetic (de)regulations driven by (and driving) this multitask DNA/RNA-binding protein.


Asunto(s)
Cromatina , Proteínas de Unión al ADN , Humanos , Citoplasma , Proteínas de Unión al ADN/genética , Epigénesis Genética , Epigenómica
4.
Cell Mol Life Sci ; 78(13): 5245-5256, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-33990851

RESUMEN

Retrotransposons are genetic elements present across all eukaryotic genomes. While their role in evolution is considered as a potentially beneficial natural source of genetic variation, their activity is classically considered detrimental due to their potentially harmful effects on genome stability. However, studies are increasingly shedding light on the regulatory function and beneficial role of somatic retroelement reactivation in non-pathological contexts. Here, we review recent findings unveiling the regulatory potential of retrotransposons, including their role in noncoding RNA transcription, as modulators of mammalian transcriptional and epigenome landscapes. We also discuss technical challenges in deciphering the multifaceted activity of retrotransposable elements, highlighting an unforeseen central role of this neglected portion of the genome both in early development and in adult life.


Asunto(s)
Epigenoma , Evolución Molecular , Inestabilidad Genómica , Mamíferos/genética , ARN no Traducido/genética , Retroelementos , Animales , Humanos , Mamíferos/crecimiento & desarrollo
5.
Int J Mol Sci ; 23(5)2022 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-35269678

RESUMEN

Approach and avoidance (A/A) tendencies are stable behavioral traits in responding to rewarding and fearful stimuli. They represent the superordinate division of emotion, and individual differences in such traits are associated with disease susceptibility. The neural circuitry underlying A/A traits is retained to be the cortico-limbic pathway including the amygdala, the central hub for the emotional processing. Furthermore, A/A-specific individual differences are associated with the activity of the endocannabinoid system (ECS) and especially of CB1 receptors whose density and functionality in amygdala differ according to A/A traits. ECS markedly interacts with the immune system (IS). However, how the interplay between ECS and IS is associated with A/A individual differences is still ill-defined. To fill this gap, here we analyzed the interaction between the gene expression of ECS and immune system (IS) in relation to individual differences. To unveil the deep architecture of ECS-IS interaction, we performed cell-specific transcriptomics analysis. Differential gene expression profiling, functional enrichment, and protein-protein interaction network analyses were performed in amygdala pyramidal neurons of mice showing different A/A behavioral tendencies. Several altered pro-inflammatory pathways were identified as associated with individual differences in A/A traits, indicating the chronic activation of the adaptive immune response sustained by the interplay between endocannabinoids and the IS. Furthermore, results showed that the interaction between the two systems modulates synaptic plasticity and neuronal metabolism in individual difference-specific manner. Deepening our knowledge about ECS/IS interaction may provide useful targets for treatment and prevention of psychopathology associated with A/A traits.


Asunto(s)
Endocannabinoides , Transcriptoma , Amígdala del Cerebelo/metabolismo , Animales , Endocannabinoides/metabolismo , Ratones , Plasticidad Neuronal , Neuronas/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo
6.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33467450

RESUMEN

Fear extinction requires coordinated neural activity within the amygdala and medial prefrontal cortex (mPFC). Any behavior has a transcriptomic signature that is modified by environmental experiences, and specific genes are involved in functional plasticity and synaptic wiring during fear extinction. Here, we investigated the effects of optogenetic manipulations of prelimbic (PrL) pyramidal neurons and amygdala gene expression to analyze the specific transcriptional pathways associated to adaptive and maladaptive fear extinction. To this aim, transgenic mice were (or not) fear-conditioned and during the extinction phase they received optogenetic (or sham) stimulations over photo-activable PrL pyramidal neurons. At the end of behavioral testing, electrophysiological (neural cellular excitability and Excitatory Post-Synaptic Currents) and morphological (spinogenesis) correlates were evaluated in the PrL pyramidal neurons. Furthermore, transcriptomic cell-specific RNA-analyses (differential gene expression profiling and functional enrichment analyses) were performed in amygdala pyramidal neurons. Our results show that the optogenetic activation of PrL pyramidal neurons in fear-conditioned mice induces fear extinction deficits, reflected in an increase of cellular excitability, excitatory neurotransmission, and spinogenesis of PrL pyramidal neurons, and associated to strong modifications of the transcriptome of amygdala pyramidal neurons. Understanding the electrophysiological, morphological, and transcriptomic architecture of fear extinction may facilitate the comprehension of fear-related disorders.


Asunto(s)
Amígdala del Cerebelo/fisiología , Condicionamiento Clásico/fisiología , Extinción Psicológica/fisiología , Miedo/fisiología , Células Piramidales/fisiología , Transcriptoma/genética , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/metabolismo , Animales , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/fisiología , Miedo/psicología , Masculino , Memoria/fisiología , Ratones Transgénicos , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Optogenética/métodos , Corteza Prefrontal/citología , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiología , Células Piramidales/metabolismo , Transmisión Sináptica/fisiología
7.
Annu Rev Genet ; 46: 561-89, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22994356

RESUMEN

The first genes composing the Polycomb group (PcG) were identified 50 years ago in Drosophila melanogaster as essential developmental functions that regulate the correct segmental expression of homeotic selector genes. In the past two decades, what was initially described as a large family of chromatin-associated proteins involved in the maintenance of transcriptional repression to maintain cellular memory of homeotic genes turned out to be a highly conserved and sophisticated network of epigenetic regulators that play key roles in multiple aspects of cell physiology and identity, including regulation of all developmental genes, cell differentiation, stem and somatic cell reprogramming and response to environmental stimuli. These myriad phenotypes further spread interest for the contribution that PcG proteins revealed in the pathogenesis and progression of cancer and other complex diseases. Recent novel insights have increasingly clarified the molecular regulatory mechanisms at the basis of PcG-mediated epigenetic silencing and opened new visions about PcG functions in cells. In this review, we focus on the multiple modes of action of the PcG complexes and describe their biological roles.


Asunto(s)
Proteínas de Drosophila/metabolismo , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Cromosomas de Insectos/genética , Cromosomas de Insectos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Activación Enzimática , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb/genética , Mapeo de Interacción de Proteínas , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética , Ubiquitinación
9.
Bioessays ; 40(4): e1700137, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29522656

RESUMEN

Cells and tissues are continuously exposed to a changing microenvironment, hence the necessity of a flexible modulation of gene expression that in complex organism have been achieved through specialized chromatin mechanisms. Chromatin-based cell memory enables cells to maintain their identity by fixing lineage specific transcriptional programs, ensuring their faithful transmission through cell division; in particular PcG-based memory system evolved to maintain the silenced state of developmental and cell cycle genes. In evolution the complexity of this system have increased, particularly in vertebrates, indicating combinatorial and dynamic properties of Polycomb proteins, in some cases even overflowing outside the cell nucleus. Therefore, their function may not be limited to the imposition of rigid states of genetic programs, but on the ability to recognize signals and allow plastic transcriptional changes in response to different stimuli. Here, we discuss the most novel PcG mediated memory functions in facing and responding to the challenges posed by a fluctuating environment.


Asunto(s)
Epigénesis Genética/genética , Proteínas del Grupo Polycomb/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Proteínas del Grupo Polycomb/genética
10.
Nature ; 480(7377): 391-5, 2011 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-22056986

RESUMEN

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.


Asunto(s)
Proteínas Argonautas/metabolismo , Cromatina/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , ARN Helicasas/metabolismo , Interferencia de ARN , Ribonucleasa III/metabolismo , Transcripción Genética , Animales , Proteínas Argonautas/deficiencia , Proteínas Argonautas/genética , Cromatina/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Helicasas/deficiencia , ARN Helicasas/genética , ARN Polimerasa II/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Factores de Transcripción
11.
PLoS Genet ; 9(2): e1003283, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23437006

RESUMEN

Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, we find that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets we show that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. Our work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time.


Asunto(s)
Cromatina , Replicación del ADN/genética , Proteínas de Drosophila , Epigénesis Genética/genética , Proteínas de Homeodominio , Proteínas del Grupo Polycomb , Factores de Transcripción , Animales , División Celular , Línea Celular , Cromatina/genética , Cromatina/ultraestructura , Proteínas de Unión al ADN , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Silenciador del Gen , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
12.
Nat Genet ; 38(6): 626-35, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16645617

RESUMEN

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.


Asunto(s)
Evolución Molecular , Regiones Promotoras Genéticas , Regiones no Traducidas 3' , Animales , Secuencia de Bases , ADN , Genoma , Proteoma , TATA Box
13.
Nat Cell Biol ; 9(10): 1167-74, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17828248

RESUMEN

In Drosophila, the function of the Polycomb group genes (PcGs) and their target sequences (Polycomb response elements (PREs)) is to convey mitotic heritability of transcription programmes--in particular, gene silencing. As part of the mechanisms involved, PREs are thought to mediate this transcriptional memory function by building up higher-order structures in the nucleus. To address this question, we analysed in vivo the three-dimensional structure of the homeotic locus bithorax complex (BX-C) by combining chromosome conformation capture (3C) with fluorescent in situ hybridization (FISH) and FISH immunostaining (FISH-I) analysis. We found that, in the repressed state, all major elements that have been shown to bind PcG proteins, including PREs and core promoters, interact at a distance, giving rise to a topologically complex structure. We show that this structure is important for epigenetic silencing of the BX-C, as we find that major changes in higher-order structures must occur to stably maintain alternative transcription states, whereas histone modification and reduced levels of PcG proteins determine an epigenetic switch that is only partially heritable.


Asunto(s)
Cromosomas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Animales , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/genética , Hibridación Fluorescente in Situ , Modelos Biológicos , Proteínas del Grupo Polycomb , Unión Proteica , Proteínas Represoras/genética , Transcripción Genética
14.
PLoS Genet ; 7(11): e1002370, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22072989

RESUMEN

Polycomb group (PcG) proteins are part of a conserved cell memory system that conveys epigenetic inheritance of silenced transcriptional states through cell division. Despite the considerable amount of information about PcG mechanisms controlling gene silencing, how PcG proteins maintain repressive chromatin during epigenome duplication is still unclear. Here we identified a specific time window, the early S phase, in which PcG proteins are recruited at BX-C PRE target sites in concomitance with H3K27me3 repressive mark deposition. Notably, these events precede and are uncoupled from PRE replication timing, which occurs in late S phase when most epigenetic signatures are reduced. These findings shed light on one of the key mechanisms for PcG-mediated epigenetic inheritance during S phase, suggesting a conserved model in which the PcG-dependent H3K27me3 mark is inherited by dilution and not by de novo methylation occurring at the time of replication.


Asunto(s)
División Celular/genética , Replicación del ADN/genética , Silenciador del Gen , Proteínas Represoras/metabolismo , Fase S/genética , Animales , Cromatina/genética , Desarrollo Embrionario/genética , Epigénesis Genética , Células HEK293 , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb , Proteínas Represoras/genética
15.
Nat Methods ; 7(7): 528-34, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20543846

RESUMEN

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5' ends that vary in size. Methods to identify the 5' ends of transcripts will facilitate the discovery of new promoters and 5' ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano-cap analysis of gene expression (nanoCAGE), a method that captures the 5' ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5' ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.


Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Nanotecnología/métodos , Regiones Promotoras Genéticas/fisiología , ARN/metabolismo , Genoma Humano , Humanos , ARN/genética
16.
Front Cell Dev Biol ; 11: 1123975, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36760365

RESUMEN

Non-coding DNA accounts for approximately 98.5% of the human genome. Once labeled as "junk DNA", this portion of the genome has undergone a progressive re-evaluation and it is now clear that some of its transcriptional products, belonging to the non-coding RNAs (ncRNAs), are key players in cell regulatory networks. A growing body of evidence demonstrates the crucial impact of regulatory ncRNAs on mammalian gene expression. Here, we focus on the defined relationship between chromatin-interacting RNAs, particularly long non-coding RNA (lncRNA), enhancer RNA (eRNA), non-coding natural antisense transcript (ncNAT), and circular RNA (circRNA) and epigenome, a common ground where both protein and RNA species converge to regulate cellular functions. Through several examples, this review provides an overview of the variety of targets, interactors, and mechanisms involved in the RNA-mediated modulation of loci-specific epigenetic states, a fundamental evolutive strategy to orchestrate mammalian gene expression in a timely and reversible manner. We will discuss how RNA-mediated epigenetic regulation impacts development and tissue homeostasis and how its alteration contributes to the onset and progression of many different human diseases, particularly cancer.

17.
Methods Mol Biol ; 2655: 31-39, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37212986

RESUMEN

Upon cellular reprogramming, the activity of polycomb repressive complex 2 (PRC2), together with histone demethylases, is essential for the suppression of cell lineage-specific gene expression programs, for resetting of epigenetic memory and for the reacquisition of pluripotency.PRC2 requires interaction with RNAs for the correct protein complex assembly and recruitment on chromatin. Moreover, PRC2 components can be found in different cell compartments and their intracellular dynamics is part of their functional activity. Several loss-of-function studies revealed that many lncRNAs expressed upon reprogramming are essential for the silencing of lineage-specific genes and the function of chromatin modifiers. Compartment-specific UV-RIP technique is a method that will help understanding which is the nature of those interactions, with no interference from indirect interactions typical of methods involving the use of chemical cross-linkers or performed in native conditions with non-stringent buffers. This technique will shed lights on the specificity of lncRNA interaction and PRC2 stability/activity on chromatin and whether PRC2-lncRNA interaction occurs in specific cell compartments.


Asunto(s)
Complejo Represivo Polycomb 2 , ARN Largo no Codificante , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina/genética
18.
Methods Mol Biol ; 2655: 101-116, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37212992

RESUMEN

The Polycomb repressive complex 2 (PRC2) is a well-characterized chromatin regulator of transcription programs acting through H3K27me3 deposition. In mammals, there are two main versions of PRC2 complexes: PRC2-EZH2, which is prevalent in cycling cells, and PRC2-EZH1 where EZH1 replaces EZH2 in post-mitotic tissues. Stoichiometry of PRC2 complex is dynamically modulated during cellular differentiation and various stress conditions. Therefore, unraveling unique architecture of PRC2 complexes under specific biological context through comprehensive and quantitative characterization could provide insight into the underlying mechanistic molecular mechanism in regulation of transcription process. In this chapter, we describe an efficient method which combines tandem-affinity purification (TAP) with label-free quantitative proteomics strategy for studying PRC2-EZH1 complex architecture alterations and identifying novel protein regulators in post-mitotic C2C12 skeletal muscle cells.


Asunto(s)
Histonas , Complejo Represivo Polycomb 2 , Animales , Complejo Represivo Polycomb 2/genética , Histonas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Cromatina , Mamíferos/metabolismo
19.
J Vis Exp ; (193)2023 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-37010313

RESUMEN

Histone post-translational modifications (PTMs) and other epigenetic modifications regulate the chromatin accessibility of genes to the transcriptional machinery, thus affecting an organism's capacity to respond to environmental stimuli. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has been widely utilized to identify and map protein-DNA interactions in the fields of epigenetics and gene regulation. However, the field of cnidarian epigenetics is hampered by a lack of applicable protocols, partly due to the unique features of model organisms such as the symbiotic sea anemone Exaiptasia diaphana, whose high water content and mucus amounts obstruct molecular methods. Here, a specialized ChIP procedure is presented, which facilitates the investigation of protein-DNA interactions in E. diaphana gene regulation. The cross-linking and chromatin extraction steps were optimized for efficient immunoprecipitation and then validated by performing ChIP using an antibody against the histone mark H3K4me3. Subsequently, the specificity and effectiveness of the ChIP assay were confirmed by measuring the relative occupancy of H3K4me3 around several constitutively activated gene loci using quantitative PCR and by next-generation sequencing for genome-wide scale analysis. This optimized ChIP protocol for the symbiotic sea anemone E. diaphana facilitates the investigation of the protein-DNA interactions involved in organismal responses to environmental changes that affect symbiotic cnidarians, such as corals.


Asunto(s)
Anémonas de Mar , Animales , Anémonas de Mar/genética , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Secuenciación de Inmunoprecipitación de Cromatina/métodos , ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos
20.
Cell Rep ; 42(9): 113066, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37656620

RESUMEN

Fear-related disorders arise from inefficient fear extinction and have immeasurable social and economic costs. Here, we characterize mouse phenotypes that spontaneously show fear-independent behavioral traits predicting adaptive or maladaptive fear extinction. We find that, already before fear conditioning, specific morphological, electrophysiological, and transcriptomic patterns of cortical and amygdala pyramidal neurons predispose to fear-related disorders. Finally, by using an optogenetic approach, we show the possibility to rescue inefficient fear extinction by activating infralimbic pyramidal neurons and to impair fear extinction by activating prelimbic pyramidal neurons.


Asunto(s)
Miedo , Corteza Prefrontal , Ratones , Animales , Corteza Prefrontal/fisiología , Miedo/fisiología , Transcriptoma/genética , Extinción Psicológica/fisiología , Amígdala del Cerebelo/fisiología , Células Piramidales/fisiología
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