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J Mol Cell Cardiol. 2012 Oct;53(4):552-8. doi: 10.1016/j.yjmcc.2012.07.009. Epub 2012 Jul 28.

Features of endogenous cardiomyocyte chromatin revealed by super-resolution STED microscopy.

Author information

1
Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, USA.

Abstract

Despite the extensive knowledge of the functional unit of chromatin-the nucleosome-for which structural information exists at the atomic level, little is known about the endogenous structure of eukaryotic genomes. Chromosomal capture techniques and genome-wide chromatin immunoprecipitation and next generation sequencing have provided complementary insight into global features of chromatin structure, but these methods do not directly measure structural features of the genome in situ. This lack of insight is particularly troublesome in terminally differentiated cells which must reorganize their genomes for large scale gene expression changes in the absence of cell division. For example, cardiomyocytes, which are fully committed and reside in interphase, are capable of massive gene expression changes in response to physiological stimuli, but the global changes in chromatin structure that enable such transcriptional changes are unknown. The present study addressed this problem utilizing super-resolution stimulated emission depletion (STED) microscopy to directly measure chromatin features in mammalian cells. We demonstrate that immunolabeling of histone H3 coupled with STED imaging reveals chromatin domains on a scale of 40-70 nm, several folds better than the resolution of conventional confocal microscopy. An analytical workflow is established to detect changes in chromatin structure following acute stimuli and used to investigate rearrangements in cardiomyocyte genomes following agonists that induce cellular hypertrophy. This approach is readily adaptable to investigation of other nuclear features using a similar antibody-based labeling technique and enables direct measurements of chromatin domain changes in response to physiological stimuli.

PMID:
22846883
PMCID:
PMC3704345
DOI:
10.1016/j.yjmcc.2012.07.009
[Indexed for MEDLINE]
Free PMC Article

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