Abstract
Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.
Highlights
The nucleosome is the basic repeating unit of chromatin
By combining our previously described extension of nucleosome single molecule FRET experiments (smFRET) to bulk concentrations (‘‘quasi-bulk smFRET’’ [14]) with an innovative bulk fluorescence resonance energy transfer (FRET) assay that is sensitive down to concentrations near those used for single molecule spectroscopy, we have developed such a strategy
Each indicates the simultaneous presence of multiple molecules in the focus, when smFRET experiments no longer reflect the true heterogeneity in the ensemble
Summary
The nucleosome is the basic repeating unit of chromatin. It regulates DNA accessibility, and its structural variability has profound influence on genetic function. The nucleosome consists of approximately 150 bp of DNA wrapped around a histone protein octamer containing two copies of each of the histones H2A, H2B, H3 and H4 [1]. The string of nucleosomes is further organized into the chromatin fiber and higher order structures. Structural changes in nucleosomes alter the local chromatin morphology, which modulates the accessibility of DNA to nuclear proteins such as transcription factors or polymerases. To understand the complex role of chromatin structure in gene regulation, we first need to elucidate the structural transitions that occur within nucleosomes
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