Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0111-y) contains supplementary material, which is available to authorized users.
Highlights
Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation
A workflow has been established to study the separation of fluorescently tagged reporter loci in live cells
The mean separation of reporter loci was observed to increase with increasing intervening genomic sequence
Summary
Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. 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 tracked in three dimensions over time using fluorescence microscopy. Studies of native chromatin provide evidence for well organised 30 nm fibres in only a few specialised cases [2, 8, 9]. Growing evidence from close-to-native-state methodologies favours the existence of relatively disordered arrays of nucleosomes in both mitotic [10,11,12,13] and interphase chromosomes [8, 12,13,14,15]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
