RESUMEN
Nuclear factors rapidly scan the genome for their targets, but the role of nuclear organization in such search is uncharted. Here we analyzed how multiple factors explore chromatin, combining live-cell single-molecule tracking with multifocal structured illumination of DNA density. We find that factors displaying higher bound fractions sample DNA-dense regions more exhaustively. Focusing on the tumor-suppressor p53, we demonstrate that it searches for targets by alternating between rapid diffusion in the interchromatin compartment and compact sampling of chromatin dense regions. Efficient targeting requires balanced interactions with chromatin: fusing p53 with an exogenous intrinsically disordered region potentiates p53-mediated target gene activation at low concentrations, but leads to condensates at higher levels, derailing its search and downregulating transcription. Our findings highlight the role of disordered regions on factors search and showcase a powerful method to generate traffic maps of the eukaryotic nucleus to dissect how its organization guides nuclear factors action.
Asunto(s)
Cromatina , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN/metabolismo , Cromosomas/metabolismo , Activación Transcripcional , Núcleo Celular/genética , Núcleo Celular/metabolismoRESUMEN
Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.
RESUMEN
In eukaryotes, transcription is a discontinuous process with mRNA being generated in bursts, after the binding of transcription factors (TFs) to regulatory elements on the genome. Live-cell single-molecule microscopy has highlighted that transcriptional bursting can be controlled by tuning TF/DNA binding kinetics. Yet the timescales of these two processes seem disconnected with TF/DNA interactions typically lasting orders of magnitude shorter than transcriptional bursts. To test models that could reconcile these discrepancies, reliable measurements of TF binding kinetics are needed, also accounting for the current limitations in performing these single-molecule measurements at specific regulatory elements. Here, we review the recent studies linking TF binding kinetics to transcriptional bursting and outline some current and future challenges that need to be addressed to provide a microscopic description of transcriptional regulation kinetics.
