RESUMEN
Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.
Asunto(s)
Cromatina , Cromosomas , Animales , Ratones , Cromatina/genética , Cromosomas/metabolismo , Transcripción Genética , Diferenciación Celular/genética , Genoma , Ensamble y Desensamble de Cromatina , Factor de Unión a CCCTC/genéticaRESUMEN
The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.
RESUMEN
The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.
RESUMEN
The purported structures of the peyssonenynes A and B isolated from Peyssonnelia caulifera, and considered to be geometric isomers at the acetoxyenediyne moiety, have been synthesized. The E and Z geometries of the synthetic compounds were secured by the magnitude of the (3)J(H9-C7) values measured using the EXSIDE band-variant of the gradient HSQC pulse sequence and by the chemical shifts of C(6). Comparison of the NMR data of the synthetic and natural products revealed that only those of the Z isomers matched, which correspond to peyssonenyne A. Using HPLC analysis it was found that peyssonenyne B must correspond to the sn-2 positional isomer of the Z sn-1/3 counterpart. The four synthetic sn-1/3 diastereomers are roughly equipotent as DNMT1 inhibitors when evaluated on a radioactive methyl transfer enzymatic assay after immunoprecipitation from K562 human leukemia cells with anti-DNMT1 antibody.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Enediinos/síntesis química , Inhibidores Enzimáticos/síntesis química , Ácidos Grasos Insaturados/síntesis química , ADN (Citosina-5-)-Metiltransferasa 1 , Enediinos/antagonistas & inhibidores , Enediinos/farmacología , Ácidos Grasos Insaturados/antagonistas & inhibidores , Ácidos Grasos Insaturados/farmacología , Humanos , Células K562 , Estructura Molecular , Relación Estructura-ActividadRESUMEN
RAR and RXR agonists: A collection of pyrazine-based RAR/RXR ligands were prepared by a series of palladium catalyzed cross-coupling reactions and characterized. Structure-activity relationships were elucidated. Retinoic acid receptor (RAR) alpha/beta-subtype-selective and retinoid X receptor (RXR) inverse agonist activities are described for pyrazine acrylic acid arotinoid, 14 d. Heterocyclic arotinoids derived from central-region dihalogenated pyrazine scaffolds have been synthesized by consecutive halogen and/or position-selective palladium-catalyzed cross-coupling reactions. Pyrazines were further functionalized as alkyl ethers or methylamines prior to the last Pd-catalyzed reactions. Transient transactivation studies with the retinoic acid receptor (RAR) alpha, beta, and gamma subtypes and with retinoid X receptor (RXR) alpha revealed distinct agonist, antagonist, and inverse agonist activities for these compounds. Of interest are the RARalpha,beta-selective inverse agonists with pyrazine acrylic acid structures, in particular 14 c, which is RARbeta-selective, and 14 d, a pan-RAR/RXR inverse agonist with more affinity for the RAR subtypes that enhance the interaction of RAR with cognate corepressors.
Asunto(s)
Pirazinas/química , Receptores de Ácido Retinoico/agonistas , Receptor alfa X Retinoide/agonistas , Retinoides/química , Agonismo Inverso de Drogas , Ligandos , Pirazinas/síntesis química , Receptores de Ácido Retinoico/metabolismo , Receptor alfa X Retinoide/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap-based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes â¼10 d to implement.
Asunto(s)
ARN/genética , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Perfilación de la Expresión Génica , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación de Ácido Nucleico/métodos , ARN/análisis , TranscriptomaRESUMEN
Recent evidence has suggested the existence of sense-antisense transcription in mammals, but the existence of double-stranded RNAs endowed with biological function has remained elusive. Herein we show that hundreds of putative natural double-stranded RNAs (ndsRNAs) are expressed from interspersed genomic locations and respond to cellular cues. We demonstrate that a subset of ndsRNAs localize in the nucleus and, in their double-stranded form, interact with nuclear proteins. Detailed characterization of an ndsRNA (nds-2a) revealed that this molecule displays differential localization throughout the cell cycle and directly interacts with RCC1 and RAN and, through the latter, with the mitotic RANGAP1-SUMO1-RANBP2 complex. Notably, altering nds-2a levels led to postmitotic abnormalities, mitotic catastrophe and cell death, thus supporting a mitosis-related role. Altogether, our study reveals a hitherto-unrecognized class of RNAs that potentially participate in major biological processes in human cells.
Asunto(s)
Regulación de la Expresión Génica , ARN Bicatenario/aislamiento & purificación , ARN Bicatenario/metabolismo , Animales , Ciclo Celular , Línea Celular , Núcleo Celular/química , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , Análisis de Secuencia de ADNRESUMEN
In the absence of ligand, some nuclear receptors, including retinoic acid receptor (RAR), act as transcriptional repressors by recruiting corepressor complexes to target genes. This constitutive repression is crucial in metazoan reproduction, development and homeostasis. However, its specific molecular determinants had remained obscure. Using structural, biochemical and cell-based assays, we show that the basal repressive activity of RAR is conferred by an extended beta-strand that forms an antiparallel beta-sheet with specific corepressor residues. Agonist binding induces a beta-strand-to-alpha-helix transition that allows for helix H11 formation, which in turn provokes corepressor release, repositioning of helix H12 and coactivator recruitment. Several lines of evidence suggest that this structural switch could be implicated in the intrinsic repressor function of other nuclear receptors. Finally, we report on the molecular mechanism by which inverse agonists strengthen corepressor interaction and enhance gene silencing by RAR.
Asunto(s)
Regulación de la Expresión Génica , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Co-Represor 1 de Receptor Nuclear/química , Co-Represor 1 de Receptor Nuclear/metabolismo , Mutación Puntual , Estructura Secundaria de Proteína , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido RetinoicoRESUMEN
The synthesis and biological evaluation of the entire series of C3-halogenated derivatives and bulkier substituents at the C8'' position of the parent stilbene-based RARbeta-selective agonist BMS641 4 c was undertaken. The synthesis uses an E-selective Horner-Wadsworth-Emmons (HWE) condensation of C8-substituted C5-dimethyl dihydronaphthaldehyde and the benzylic phosphonates derived from the C3-halogenated benzoates to construct the stilbene skeleton. Transactivation studies revealed the synergistic effect of small halogen atoms at C3 (F, Cl) and the moderately bulky phenyl group at C8'' (in 4 b and 4 c) to achieve RARbeta selectivity. Our results, supported by computational studies, provide a structural rationale for the mixed agonist-antagonist activities of these arotinoids, which are potent agonists of the RARbeta subtype and antagonists of the RARalpha paralogue. Moreover, transitions from partial agonists to inverse agonists and antagonists can be accomplished with the incorporation of the same halogen atoms into the structures of known modulators BMS701 (5 a) and BMS493 (6 a), which have bulkier substituents than phenyl (p-tolyl and phenylethynyl, respectively) at C8''. Conversely, incorporation of halogen atoms in 6 a converted the ligand from an RARbeta inverse agonist (6 b) to an antagonist (6 c) or an agonist (6 d). Amazingly, 6 a-c commonly acted as inverse agonists for RARalpha, while 6 d and 6 e acted as regular RARalpha antagonists, not affecting co-repressor interaction. In the case of the mixed agonist/antagonist 5 a, C3-halogenation yields inverse RARalpha and RARbeta agonists (5 b-d) with the exception of iodinated 5 e, which is a regular antagonist for both these receptors. Because RARbeta gene expression is frequently deleted or epigenetically silenced in several tumor cells, the novel repertoire of receptor and function-selective RAR agonists, mixed agonist/antagonists, regular antagonists, and inverse agonists will be useful in the elucidation of the mechanism of tumor suppression by retinoids.