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BMC Cell Biology

, 17:33

Nuclear function and transport


BackgroundGenomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. Chromatin is not a static structure, but is known to be dynamic and vary between cells. In order to monitor the organisation of chromatin in live cells we have engineered fluorescent fusion proteins which recognize specific operator sequences to tag pairs of syntenic gene loci. The separation of these loci was then tracked in three dimensions over time using fluorescence microscopy.

ResultsWe established a work flow for measuring the distance between two fluorescently tagged, syntenic gene loci with a mean measurement error of 63 nm. In general, physical separation was observed to increase with increasing genomic separations. However, the extent to which chromatin is compressed varies for different genomic regions. No correlation was observed between compaction and the distribution of chromatin markers from genomic datasets or with contacts identified using capture based approaches. Variation in spatial separation was also observed within cells over time and between cells. Differences in the conformation of individual loci can persist for minutes in individual cells. Separation of reporter loci was found to be similar in related and unrelated daughter cell pairs.

ConclusionsThe directly observed physical separation of reporter loci in live cells is highly dynamic both over time and from cell to cell. However, consistent differences in separation are observed over some chromosomal regions that do not correlate with factors known to influence chromatin states. We conclude that as yet unidentified parameters influence chromatin configuration. We also find that while heterogeneity in chromatin states can be maintained for minutes between cells, it is not inherited through cell division. This may contribute to cell-to-cell transcriptional heterogeneity.

KeywordsChromatin structure Fluorescence microscopy Live cell imaging Epigenetic inheritance AbbreviationsChIPChromatin immunoprecipitation

FOVField of view

K-S testKolmogorov-Smirnov test

LMSLaser milled slide

MSDCMean square distance change

OMXOptical microscope experimental

PSFPoint spread function

RIRefractive index

TADTopologically associated domain

Electronic supplementary materialThe online version of this article doi:10.1186-s12860-016-0111-y contains supplementary material, which is available to authorized users.

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Autor: David Dickerson - Marek Gierliński - Vijender Singh - Etsushi Kitamura - Graeme Ball - Tomoyuki U. Tanaka - Tom Owen-Hugh

Fuente: https://link.springer.com/

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