SummaryLysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

Temperature influences the distribution, range, and phenology of plants. The key transcriptional activators of heat shock response in eukaryotes, the heat shock factors (HSFs), have undergone large-scale gene amplification in plants. While HSFs are central in heat stress responses, their role in the response to ambient temperature changes is less well understood. We show here that the warm ambient temperature transcriptome is dependent upon the HSFA1 clade of Arabidopsis HSFs, which cause a rapid and dynamic eviction of H2A.Z nucleosomes at target genes. A transcriptional cascade results in the activation of multiple downstream stress-responsive transcription factors, triggering large-scale changes to the transcriptome in response to elevated temperature. H2A.Z nucleosomes are enriched at temperature-responsive genes at non-inducible temperature, and thus likely confer inducibility of gene expression and higher responsive dynamics. We propose that the antagonistic effects of H2A.Z and HSF1 provide a mechanism to activate gene expression rapidly and precisely in response to temperature, while preventing leaky transcription in the absence of an activation signal.

Accessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of approximately 0.2-0.6 at nucleosome ends and approximately 0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin.

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