RESUMEN
Enhancer RNAs (eRNAs) are long non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of activated enhancers, the molecular features required for eRNA function and the mechanism of how eRNAs impinge on target gene transcription have not been established. Thus, using eRNA-dependent RNA polymerase II (Pol II) pause release as a model, we here investigate the requirement of sequence, structure and length of eRNAs for their ability to stimulate Pol II pause release by detaching NELF from paused Pol II. We find eRNAs not to exert their function through common structural or sequence motifs. Instead, eRNAs that exhibit a length >200 nucleotides and that contain unpaired guanosines make multiple, allosteric contacts with NELF subunits -A and -E to trigger efficient NELF release. By revealing the molecular determinants of eRNA function, our study establishes eRNAs as an important player in Pol II pause release, and it provides new insight into the regulation of metazoan transcription.
Asunto(s)
ARN Polimerasa II , ARN Largo no Codificante , Animales , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
CCCTC-binding factor (CTCF) critically contributes to 3D chromatin organization by determining topologically associated domain (TAD) borders. Although CTCF primarily binds at TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking the putative CTCF-binding sites remains largely elusive. Here, we show that the ATP-dependent chromatin remodeler, chromodomain helicase DNA-binding 4 (CHD4), regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 short interspersed nuclear elements (SINEs) in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible and aberrant CTCF recruitment occurs within TADs, resulting in disorganization of local TADs. RNA-binding intrinsically disordered domains (IDRs) of CHD4 are required to prevent this aberrant CTCF binding, and CHD4 is critical for the repression of B2 SINE transcripts. These results collectively reveal that a CHD4-mediated mechanism ensures appropriate CTCF binding and associated TAD organization in mESCs.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , ADN Helicasas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Sitios de Unión , Técnicas de Cultivo de Célula , RatonesRESUMEN
Bromodomain and extraterminal proteins (BET) are epigenetic readers that play critical roles in gene regulation. Pharmacologic inhibition of the bromodomain present in all BET family members is a promising therapeutic strategy for various diseases, but its impact on individual family members has not been well understood. Using a transcriptional induction paradigm in neurons, we have systematically demonstrated that three major BET family proteins (BRD2/3/4) participated in transcription with different recruitment kinetics, interdependency, and sensitivity to a bromodomain inhibitor, JQ1. In a mouse model of fragile X syndrome (FXS), BRD2/3 and BRD4 showed oppositely altered expression and chromatin binding, correlating with transcriptional dysregulation. Acute inhibition of CBP/p300 histone acetyltransferase (HAT) activity restored the altered binding patterns of BRD2 and BRD4 and rescued memory impairment in FXS. Our study emphasizes the importance of understanding the BET coordination controlled by a balanced action between HATs with different substrate specificity.
Asunto(s)
Síndrome del Cromosoma X Frágil , Proteínas Nucleares , Animales , Síndrome del Cromosoma X Frágil/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Neuronal activity-induced gene transcription is an important cellular mechanism for long-term plasticity. In this issue of Neuron, Tyssowski et al. (2018) provide new genome-wide features of the activity-transcription coupling mechanism that have deepened our molecular understanding of activity pattern-dependent synaptic plasticity.
Asunto(s)
Plasticidad Neuronal , NeuronasRESUMEN
Homeostatic scaling allows neurons to maintain stable activity patterns by globally altering their synaptic strength in response to changing activity levels. Suppression of activity by the blocking of action potentials increases synaptic strength through an upregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Although this synaptic upscaling was shown to require transcription, the molecular nature of the intrinsic transcription program underlying this process and its functional significance have been unclear. Using RNA-seq, we identified 73 genes that were specifically upregulated in response to activity suppression. In particular, Neuronal pentraxin-1 (Nptx1) increased within 6 hr of activity blockade, and knockdown of this gene blocked the increase in synaptic strength. Nptx1 induction is mediated by calcium influx through the T-type voltage-gated calcium channel, as well as two transcription factors, SRF and ELK1. Altogether, these results uncover a transcriptional program that specifically operates when neuronal activity is suppressed to globally coordinate the increase in synaptic strength.
Asunto(s)
Neuronas/fisiología , Sinapsis/fisiología , Transcripción Genética/fisiología , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Canales de Calcio Tipo T/metabolismo , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Homeostasis/fisiología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, ß-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the ß-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wild-type iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered ß-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development.
Asunto(s)
Displasia Ectodérmica/metabolismo , Displasia Ectodérmica/patología , Insuficiencia de Crecimiento/metabolismo , Insuficiencia de Crecimiento/patología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Cuerpos Embrioides/metabolismo , Facies , Humanos , Masculino , Neuronas/patología , Transporte de Proteínas , beta Catenina/metabolismoRESUMEN
LIN28-mediated processing of the microRNA (miRNA) let-7 has emerged as a multilevel program that controls self-renewal in embryonic stem cells. LIN28A is believed to act primarily in the cytoplasm together with TUT4/7 to prevent final maturation of let-7 by Dicer, whereas LIN28B has been suggested to preferentially act on nuclear processing of let-7. Here, we find that SET7/9 monomethylation in a putative nucleolar localization region of LIN28A increases its nuclear retention and protein stability. In the nucleoli of human embryonic stem cells, methylated LIN28A sequesters pri-let-7 and blocks its processing independently of TUT4/7. The nuclear form of LIN28A regulates transcriptional changes in MYC-pathway targets, thereby maintaining stemness programs and inhibiting expression of early lineage-specific markers. These findings provide insight into the molecular mechanism underlying the posttranslational methylation of nuclear LIN28A and its ability to modulate pluripotency by repressing let-7 miRNA expression in human embryonic stem cells.
Asunto(s)
Nucléolo Celular/metabolismo , Células Madre Embrionarias/fisiología , MicroARNs/metabolismo , Células Madre Pluripotentes/fisiología , Proteínas de Unión al ARN/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Genes myc/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , MicroARNs/genética , Multimerización de Proteína , Transporte de Proteínas , Proteínas de Unión al ARN/genéticaRESUMEN
Histone-modifying enzymes play a pivotal role in gene expression and repression. In human, DOT1L (Dot1-like) is the only known histone H3 lysine 79 methyltransferase. hDOT1L is associated with transcriptional activation, but the general mechanism connecting hDOT1L to active transcription remains largely unknown. Here, we report that hDOT1L interacts with the phosphorylated C-terminal domain of actively transcribing RNA polymerase II (RNAPII) through a region conserved uniquely in multicellular DOT1 proteins. Genome-wide profiling analyses indicate that the occupancy of hDOT1L largely overlaps with that of RNAPII at actively transcribed genes, especially surrounding transcriptional start sites, in embryonic carcinoma NCCIT cells. We also find that C-terminal domain binding or H3K79 methylations by hDOT1L is important for the expression of target genes such as NANOG and OCT4 and a marker for pluripotency in NCCIT cells. Our results indicate that a functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to actively transcribed genes.
Asunto(s)
Regulación de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Estudio de Asociación del Genoma Completo , Células HEK293 , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Metilación , Metiltransferasas/genética , Unión Proteica/fisiología , ARN Polimerasa II/genéticaRESUMEN
H2B monoubiquitylation (H2Bub1), which is required for multiple methylations of both H3K4 and H3K79, has been implicated in gene expression in numerous organisms ranging from yeast to human. However, the molecular crosstalk between H2Bub1 and other modifications, especially the methylations of H3K4 and H3K79, remains unclear in vertebrates. To better understand the functional role of H2Bub1, we measured genome-wide histone modification patterns in human cells. Our results suggest that H2Bub1 has dual roles, one that is H3 methylation dependent, and another that is H3 methylation independent. First, H2Bub1 is a 5'-enriched active transcription mark and co-occupies with H3K79 methylations in actively transcribed regions. Second, this study shows for the first time that H2Bub1 plays a histone H3 methylations-independent role in chromatin architecture. Furthermore, the results of this work indicate that H2Bub1 is largely positioned at the exon-intron boundaries of highly expressed exons, and it demonstrates increased occupancy in skipped exons compared with flanking exons in the human and mouse genomes. Our findings collectively suggest that a potentiating mechanism links H2Bub1 to both H3K79 methylations in actively transcribed regions and the exon-intron structure of highly expressed exons via the regulation of nucleosome dynamics during transcription elongation.
Asunto(s)
Cromatina/genética , Exones , Histonas/metabolismo , Intrones , Transcripción Genética , Animales , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Drosophila/genética , Regulación de la Expresión Génica , Genoma Humano , Histonas/genética , Humanos , Metilación , Ratones , Neoplasias de Células Germinales y Embrionarias/genética , UbiquitinaciónRESUMEN
Transcriptional factor 4 (TCF4), encoding a basic helix-loop-helix transcriptional factor, has recently been demonstrated as a causative gene for Pitt-Hopkins syndrome, a neurodevelopmental disease. Examination of gastric cancers using the restriction landmark genomic scanning technique revealed methylation at a NotI enzyme site in TCF4 intron 8 and further identified CpG dinucleotide hypermethylation in TCF4 exon 1, strongly associated with gene silencing in gastric cancer cell lines. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A restored TCF4 expression in TCF4-silenced gastric cancer cell lines. Real-time reverse transcription-polymerase chain reaction analysis of 77 paired primary gastric tumor samples revealed that 38% of analyzed tumors had a >2-fold decrease in TCF4 expression compared with adjacent normal-appearing tissue, and the decrease significantly correlated with increased CpG methylation in TCF4 exon 1. Clinicopathologic data showed that decreased TCF4 expression occurred significantly more frequently in intestinal-type (22/37, 59%) than in diffuse-type (7/37, 19%) gastric cancers (P = 0.0004) and likewise more frequently in early (12/18, 67%) than in advanced (17/59, 29%) gastric cancers (P = 0.004). CpG methylation markedly increased with patient age among normal-appearing tissues, suggesting that CpG methylation in gastric mucosa may be one of the earliest events in carcinogenesis of intestinal-type gastric cancers. Furthermore, ectopic expression of TCF4 decreased cell growth in a gastric cancer cell line, and the knock down of TCF4 using small interfering RNA increased cell migration. Based on these results, we propose that the observed frequent epigenetic-mediated TCF4 silencing plays a role in tumor formation and progression.
Asunto(s)
Envejecimiento/fisiología , Islas de CpG/fisiología , Proteínas de Unión al ADN/genética , Exones , Mucosa Gástrica/fisiología , Silenciador del Gen , Neoplasias Intestinales/genética , Neoplasias Gástricas/genética , Factores de Transcripción TCF/genética , Factores de Transcripción/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Línea Celular Tumoral , Clonación Molecular , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Mucosa Gástrica/patología , Humanos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Factor de Transcripción 4RESUMEN
Recent finding has shown that LIMS2 (also known as PINCH2) functions as a natural regulator of the LIMS1-ILK-parvin complex formation and is associated with cell spreading and migration via integrins at focal adhesions. Here, we report for the first time the epigenetic silencing of LIMS2 in gastric tumors. Downregulation of LIMS2 was detected in 91% (10 of 11) of gastric cancer cell lines by real-time quantitative RT-PCR and 80% (8 of 10) of the LIMS2-downregulated cell lines were associated with CpG island hypermethylation at a 5'-upstream region of LIMS2. Furthermore, LIMS2 was restored in its non-expressing cell lines after treatment with 5-Aza-dC and/or trichostatin A. Loss of expression of LIMS2 was also detected in 53% (51 of 96) of primary gastric tumors. This decrease in expression level significantly correlated with an increase of the CpG island hypermethylation. In addition, the methylation status in any normal-appearing gastric tissues was gradually increased in an age-dependent manner, suggesting that the positive methylation in normal-appearing gastric mucosa can be due to 'field cancerization effect' as an early event in gastric carcinogenesis. Moreover, the transient transfection of LIMS2-siRNA significantly stimulated cell migration in gastric cancer cells but had no effects on cell growth. These results suggest that the frequent inactivation of LIMS2 by epigenetic alteration in gastric cancer may be important in tumor progression events, such as invasion and metastasis. Thus, LIMS2 may be useful as a molecular biomarker and a therapeutic target by increasing its expression and activity in gastric cancer.