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Single-cell quantitation of histones and histone post-translational modifications across the cell cycle by high-throughput imaging

Linda Zane, Fleur Chapus, Gianluca Pegoraro, Tom Misteli
doi: https://doi.org/10.1101/070219
Linda Zane
1Cell Biology of Genomes, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Fleur Chapus
1Cell Biology of Genomes, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Gianluca Pegoraro
2NCI High-Throughput Imaging Facility, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Tom Misteli
1Cell Biology of Genomes, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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ABSTRACT

Post-translational modifications (PTM) of histone proteins are critical determinants of genome function. Analysis of histone PTM levels has traditionally been performed using biochemical bulk methods to measure individual modifications. These approaches are generally unable to detect differences in histone and histone PTM levels during the cell cycle, do not provide information on variability amongst individual cells, and they are not suitable for multiplexing to comprehensively analyze large numbers of histone modifications in single experiments. We have developed HiHiMap (High-throughput Histone Mapping), an automated high-throughput immunofluorescence technique to determine histone and histone PTM levels across the cell cycle at the single-cell level in a highly parallel format. Our approach uses imaging-based quantification of DNA content and cyclin A levels to stage individual cells in the cell cycle combined with determination of the corresponding histone and histone PMT levels in the same cells. We have applied HiHiMap to a set of 22 histone variants and histone modifications. As proof-of-principle for a biological application, we use HiHiMap to the histone and histone PTMs landscape in primary, immortalized and oncogenically transformed cells. We find differences in the behavior of a specific set of histone PTM during the the cell cycle in transformed cells compared to normal and immortalized cells. The method developed here is widely applicable to the systematic study of histone modifications in physiological and pathological settings at the single-cell level.

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Posted August 18, 2016.
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Single-cell quantitation of histones and histone post-translational modifications across the cell cycle by high-throughput imaging
Linda Zane, Fleur Chapus, Gianluca Pegoraro, Tom Misteli
bioRxiv 070219; doi: https://doi.org/10.1101/070219
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Single-cell quantitation of histones and histone post-translational modifications across the cell cycle by high-throughput imaging
Linda Zane, Fleur Chapus, Gianluca Pegoraro, Tom Misteli
bioRxiv 070219; doi: https://doi.org/10.1101/070219

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