RESUMEN
The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Nucleosomas/metabolismo , Animales , Cromatina/química , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , ADN/química , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Cinética , Metilación , Microscopía Fluorescente , Conformación Molecular , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/genética , Unión ProteicaRESUMEN
We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.