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1.
EMBO J ; 42(12): e110286, 2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37082862

RESUMEN

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher-order chromatin structure have remained elusive. Here, we applied FLIM-FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called "nanocompaction." Both in naive embryonic stem cells (ESCs) and in ESC-derived epiblast-like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1ß, indicating the existence of a dynamic HP1-dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular-proliferation marker Ki-67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki-67.


Asunto(s)
Proteínas Cromosómicas no Histona , Heterocromatina , Heterocromatina/genética , Antígeno Ki-67/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/química , Cromatina , Células Madre Embrionarias
2.
J Cell Sci ; 134(3)2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33526472

RESUMEN

PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1ß (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1ß-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1ß on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.


Asunto(s)
Cromatina , Complejo de la Endopetidasa Proteasomal , Autoantígenos , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Heterocromatina/genética , Humanos , Complejo de la Endopetidasa Proteasomal/genética
3.
Biochem Soc Trans ; 49(4): 1867-1879, 2021 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-34338292

RESUMEN

Different classes of non-coding RNA (ncRNA) influence the organization of chromatin. Imprinted gene domains constitute a paradigm for exploring functional long ncRNAs (lncRNAs). Almost all express an lncRNA in a parent-of-origin dependent manner. The mono-allelic expression of these lncRNAs represses close by and distant protein-coding genes, through diverse mechanisms. Some control genes on other chromosomes as well. Interestingly, several imprinted chromosomal domains show a developmentally regulated, chromatin-based mechanism of imprinting with apparent similarities to X-chromosome inactivation. At these domains, the mono-allelic lncRNAs show a relatively stable, focal accumulation in cis. This facilitates the recruitment of Polycomb repressive complexes, lysine methyltranferases and other nuclear proteins - in part through direct RNA-protein interactions. Recent chromosome conformation capture and microscopy studies indicate that the focal aggregation of lncRNA and interacting proteins could play an architectural role as well, and correlates with close positioning of target genes. Higher-order chromatin structure is strongly influenced by CTCF/cohesin complexes, whose allelic association patterns and actions may be influenced by lncRNAs as well. Here, we review the gene-repressive roles of imprinted non-coding RNAs, particularly of lncRNAs, and discuss emerging links with chromatin architecture.


Asunto(s)
Cromatina/química , Impresión Genómica , Dominios Proteicos , ARN no Traducido/química , Animales , Humanos , Conformación Proteica , Inactivación del Cromosoma X
4.
Proc Natl Acad Sci U S A ; 114(23): E4676-E4685, 2017 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-28533375

RESUMEN

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.


Asunto(s)
Enfermedad de Huntington/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Adulto , Anciano , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Enfermedad de Huntington/genética , Proteína 1 Asociada A ECH Tipo Kelch/química , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/prevención & control , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/química , Células-Madre Neurales/metabolismo , Fármacos Neuroprotectores/farmacología , Conformación Proteica/efectos de los fármacos , Ratas , Transducción de Señal
5.
Proc Natl Acad Sci U S A ; 110(38): 15259-64, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-23986495

RESUMEN

The transcription factor NF-E2 p45-related factor 2 (Nrf2), a master regulator of cytoprotective genes, is controlled by dimeric Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor protein for Cullin3/RING-box protein 1 ubiquitin ligase, which normally targets Nrf2 for ubiquitination and degradation but loses this ability in response to electrophiles and oxidants (inducers). By using recombinant proteins and populations of cells, some of the general features of the regulation of Nrf2 by Keap1 have been outlined. However, how the two proteins interact at a single-cell level is presently unknown. We now report the development of a quantitative Förster resonance energy transfer-based system using multiphoton fluorescence lifetime imaging microscopy and its application for investigating the interaction between Nrf2 and Keap1 in single live cells. By using this approach, we found that under homeostatic conditions, the interaction between Keap1 and Nrf2 follows a cycle in which the complex sequentially adopts two distinct conformations: "open," in which Nrf2 interacts with a single molecule of Keap1, followed by "closed," in which Nrf2 binds to both members of the Keap1 dimer. Inducers disrupt this cycle by causing accumulation of the complex in the closed conformation without release of Nrf2. As a consequence, free Keap1 is not regenerated, and newly synthesized Nrf2 is stabilized. On the basis of these findings, we propose a model we have named the "cyclic sequential attachment and regeneration model of Keap1-mediated degradation of Nrf2." This previously unanticipated dynamism allows rapid transcriptional responses to environmental changes and can accommodate multiple modes of regulation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas del Citoesqueleto/química , Regulación de la Expresión Génica/fisiología , Complejos Multiproteicos/química , Factor 2 Relacionado con NF-E2/química , Conformación Proteica , Estrés Fisiológico/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Clonación Molecular , Proteínas del Citoesqueleto/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Microscopía Fluorescente/métodos , Complejos Multiproteicos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteolisis
6.
Nat Genet ; 55(8): 1359-1369, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37400615

RESUMEN

Metazoan promoters are enriched in secondary DNA structure-forming motifs, such as G-quadruplexes (G4s). Here we describe 'G4access', an approach to isolate and sequence G4s associated with open chromatin via nuclease digestion. G4access is antibody- and crosslinking-independent and enriches for computationally predicted G4s (pG4s), most of which are confirmed in vitro. Using G4access in human and mouse cells, we identify cell-type-specific G4 enrichment correlated with nucleosome exclusion and promoter transcription. G4access allows measurement of variations in G4 repertoire usage following G4 ligand treatment, HDAC and G4 helicases inhibitors. Applying G4access to cells from reciprocal hybrid mouse crosses suggests a role for G4s in the control of active imprinting regions. Consistently, we also observed that G4access peaks are unmethylated, while methylation at pG4s correlates with nucleosome repositioning on DNA. Overall, our study provides a new tool for studying G4s in cellular dynamics and highlights their association with open chromatin, transcription and their antagonism to DNA methylation.


Asunto(s)
Cromatina , G-Cuádruplex , Animales , Humanos , Ratones , Cromatina/genética , Nucleosomas/genética , ADN/genética , Regiones Promotoras Genéticas
7.
EMBO Rep ; 11(6): 445-51, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20467437

RESUMEN

Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5' and 3' alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas Fluorescentes Verdes , Células HeLa , Respuesta al Choque Térmico , Ribonucleoproteína Heterogénea-Nuclear Grupo M/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Nucleares/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas Recombinantes de Fusión
8.
Cells ; 9(9)2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32911802

RESUMEN

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.


Asunto(s)
Cromatina/metabolismo , Células Germinativas/metabolismo , Animales , Caenorhabditis elegans
9.
J Cell Biol ; 166(6): 787-800, 2004 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-15353547

RESUMEN

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.


Asunto(s)
Nucléolo Celular/metabolismo , Cinética , Mitosis , Colorantes Fluorescentes , Células HeLa , Humanos , Imagenología Tridimensional , Inmunohistoquímica , Modelos Biológicos , Membrana Nuclear/metabolismo , Región Organizadora del Nucléolo/metabolismo , Pruebas de Precipitina , ARN Polimerasa I/metabolismo
10.
Genome Biol ; 20(1): 272, 2019 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-31831055

RESUMEN

BACKGROUND: Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints-the Igf2-H19 and Dlk1-Dio3 domains-and assessed the involvement of the insulator protein CTCF in mouse cells. RESULTS: Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation. CONCLUSIONS: Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Impresión Genómica , Animales , Proteínas de Unión al Calcio/genética , Factor II del Crecimiento Similar a la Insulina/genética , Yoduro Peroxidasa/genética , Ratones , ARN Largo no Codificante/genética
11.
Cell Rep ; 23(2): 337-348, 2018 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-29641995

RESUMEN

Although many long non-coding RNAs (lncRNAs) are imprinted, their roles often remain unknown. The Dlk1-Dio3 domain expresses the lncRNA Meg3 and multiple microRNAs and small nucleolar RNAs (snoRNAs) on the maternal chromosome and constitutes an epigenetic model for development. The domain's Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signaling and regulates diverse developmental processes. Using a hybrid embryonic stem cell (ESC) system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves transcriptional upregulation on the paternal chromosome. The maternal Dlk1 gene remains poised. Its protection against activation is controlled in cis by Meg3 expression and also requires the H3-Lys-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. We find that Meg3 lncRNA is partially retained in cis and overlaps the maternal Dlk1 in embryonic cells. Combined, our data evoke an imprinting model in which allelic lncRNA expression prevents gene activation in cis.


Asunto(s)
Impresión Genómica , ARN Largo no Codificante/metabolismo , Alelos , Animales , Sistemas CRISPR-Cas/genética , Proteínas de Unión al Calcio , Diferenciación Celular , Línea Celular , Metilación de ADN , Células Madre Embrionarias , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Activación Transcripcional , Regulación hacia Arriba
12.
Nat Commun ; 9(1): 3704, 2018 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-30209253

RESUMEN

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. Mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, chromatin relaxation is controlled by SET8-dependent methylation of histone H4 on lysine 20. In the absence of either SET8 or H4K20 residue, substantial genome-wide chromatin decompaction occurs allowing excessive loading of the origin recognition complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.


Asunto(s)
Cromatina/metabolismo , Replicación del ADN/fisiología , Histonas/metabolismo , Línea Celular Tumoral , Cromatina/genética , Daño del ADN/genética , Daño del ADN/fisiología , Replicación del ADN/genética , Citometría de Flujo , Histonas/genética , Humanos , Microscopía Fluorescente , ARN Interferente Pequeño/genética
13.
Cell Rep ; 18(7): 1791-1803, 2017 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-28199849

RESUMEN

How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.


Asunto(s)
Adenosina Trifosfatasas/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Animales , Caenorhabditis elegans/genética , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Cromosomas/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Heterocromatina/metabolismo , Histonas/genética , Microscopía Fluorescente/métodos
14.
Elife ; 5: e13722, 2016 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-26949251

RESUMEN

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.


Asunto(s)
Proliferación Celular , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Antígeno Ki-67/metabolismo , Animales , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Xenopus
15.
Biotechnol Adv ; 32(6): 1133-44, 2014 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24681086

RESUMEN

The transcription factor NF-E2 p45-related factor 2 (Nrf2) and its negative regulator Kelch-like ECH associated protein 1 (Keap1) control the expression of nearly 500 genes with diverse cytoprotective functions. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, normally continuously targets Nrf2 for degradation, but loses this ability in response to electrophiles and oxidants (termed inducers). Consequently, Nrf2 accumulates and activates transcription of its downstream target genes. Many inducers are phytochemicals, and cruciferous vegetables represent one of the richest sources of inducer activity among the most commonly used edible plants. Here we summarize the discovery of the isothiocyanate sulforaphane as a potent inducer which reacts with cysteine sensors of Keap1, leading to activation of Nrf2. We then describe the development of a quantitative Förster resonance energy transfer (FRET)-based methodology combined with multiphoton fluorescence lifetime imaging microscopy (FLIM) to investigate the interactions between Keap1 and Nrf2 in single live cells, and the effect of sulforaphane, and other cysteine-reactive inducers, on the dynamics of the Keap1-Nrf2 protein complex. We present the experimental evidence for the "cyclic sequential attachment and regeneration" or "conformation cycling" model of Keap1-mediated Nrf2 degradation. Finally, we discuss the implications of this mode of regulation of Nrf2 for achieving a fine balance under normal physiological conditions, and the consequences and mechanisms of disrupting this balance for tumor biology.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Subunidad p45 del Factor de Transcripción NF-E2 , Transducción de Señal , Análisis de la Célula Individual/métodos , Animales , Sitios de Unión , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Ratas
16.
Dev Cell ; 31(1): 19-33, 2014 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-25263792

RESUMEN

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.


Asunto(s)
Replicación del ADN , Impresión Genómica , Péptidos y Proteínas de Señalización Intercelular/genética , Yoduro Peroxidasa/genética , Región de Control de Posición , ARN no Traducido/genética , Animales , Blastocisto/citología , Blastocisto/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular , Metilación de ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Ratones , ARN no Traducido/metabolismo
17.
Epigenomics ; 5(6): 715-28, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24283884

RESUMEN

Mammalian genes controlled by genomic imprinting play important roles in development and diverse postnatal processes. A growing number of congenital disorders have been linked to genomic imprinting. Each of these is caused by perturbed gene expression at one principal imprinted domain. Some imprinting disorders, including the Prader-Willi and Angelman syndromes, are caused almost exclusively by genetic mutations. In several others, including the Beckwith-Wiedemann and Silver-Russell growth syndromes, and transient neonatal diabetes mellitus, imprinted expression is perturbed mostly by epigenetic alterations at 'imprinting control regions' and at other specific regulatory sequences. In a minority of these patients, DNA methylation is altered at multiple imprinted loci, suggesting that common trans-acting factors are affected. Here, we review the epimutations involved in congenital imprinting disorders and the associated clinical features. Trans-acting factors known to be causally involved are discussed and other trans-acting factors that are potentially implicated are also presented.


Asunto(s)
Anomalías Congénitas/genética , Epigénesis Genética , Enfermedades Genéticas Congénitas/genética , Impresión Genómica , Transactivadores/metabolismo , Animales , Metilación de ADN , Diabetes Mellitus/genética , Sitios Genéticos , Genoma Humano , Humanos , Recién Nacido , Mutación
18.
PLoS One ; 8(6): e67689, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23840764

RESUMEN

The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.


Asunto(s)
Adenosina Trifosfato/metabolismo , Cationes Bivalentes/metabolismo , Núcleo Celular/metabolismo , Cromatina/química , Nucleosomas/metabolismo , Poliaminas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Microscopía Fluorescente
19.
PLoS One ; 7(11): e48084, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23152763

RESUMEN

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA.


Asunto(s)
Cromatina/metabolismo , Empalme del ARN/fisiología , Ribonucleoproteínas/metabolismo , Acetilación , Empalme Alternativo/efectos de los fármacos , Animales , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cromatina/efectos de los fármacos , Homólogo de la Proteína Chromobox 5 , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , Empalme del ARN/efectos de los fármacos , ARN Largo no Codificante/metabolismo , Ribonucleoproteínas/genética
20.
J Cell Biol ; 187(4): 481-96, 2009 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-19948497

RESUMEN

We present a quantitative Förster resonance energy transfer (FRET)-based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLa(H2B-2FP)). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM-FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.


Asunto(s)
Cromatina/metabolismo , Anafase , Cromatina/química , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Microscopía Fluorescente , Nucleosomas/química , Nucleosomas/metabolismo , Pliegue de Proteína , Factores de Tiempo
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