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High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division

View Article: PubMed Central - PubMed

ABSTRACT

Background: Genomes 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.

Results: We 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.

Conclusions: The 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 material: The online version of this article (doi:10.1186/s12860-016-0111-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Motion analysis for strains with different genomic separations. a When the relative orientation of two particles is maintained as they proceed along their trajectories (indicated as lines) their motion is considered isotropic (bottom), when orientation changes over time motion is anisotropic (top). A statistic D was calculated as a measure of anisotropy (Materials and Methods; Fig. 4). Larger D values correspond to greater anisotropy. D is observed to generally increase with genomic separation (b). In c the mean D is plotted against the mean distance (R2 = 0.43). d To assess the correlation of the direction of motion of the two operators flanking a locus, comparisons of the vectors describing the motion of each operator were made at each time point. An RV coefficient was calculated for this purpose. The general trend observed was a decrease of motion correlation as the genomic separation between the operators increased. However, for the 64 kb and 42 kb strains which adopt a more compact configuration, the trend was not observed
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Fig4: Motion analysis for strains with different genomic separations. a When the relative orientation of two particles is maintained as they proceed along their trajectories (indicated as lines) their motion is considered isotropic (bottom), when orientation changes over time motion is anisotropic (top). A statistic D was calculated as a measure of anisotropy (Materials and Methods; Fig. 4). Larger D values correspond to greater anisotropy. D is observed to generally increase with genomic separation (b). In c the mean D is plotted against the mean distance (R2 = 0.43). d To assess the correlation of the direction of motion of the two operators flanking a locus, comparisons of the vectors describing the motion of each operator were made at each time point. An RV coefficient was calculated for this purpose. The general trend observed was a decrease of motion correlation as the genomic separation between the operators increased. However, for the 64 kb and 42 kb strains which adopt a more compact configuration, the trend was not observed

Mentions: During imaging, it was noticeable that the relative orientations of the tagged loci in a subset of cells were markedly constrained. To analyse this quantitatively we developed a test which assigns a statistic, D (see Materials and Methods), which quantifies the anisotropy of the spatial orientation of the two-spot system. The higher D, the greater the degree of anisotropy. Lower D values were generally observed for genomic separations below 70 kb (Fig. 4b) and anisotropy is correlated with the mean distance (Fig. 4c). This could be an indication that for small physical distances that relative motion of foci is less constrained. As physical distance increases, the nuclear environment acts as a constraint restricting the extent of relative motion.Fig. 4


High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division
Motion analysis for strains with different genomic separations. a When the relative orientation of two particles is maintained as they proceed along their trajectories (indicated as lines) their motion is considered isotropic (bottom), when orientation changes over time motion is anisotropic (top). A statistic D was calculated as a measure of anisotropy (Materials and Methods; Fig. 4). Larger D values correspond to greater anisotropy. D is observed to generally increase with genomic separation (b). In c the mean D is plotted against the mean distance (R2 = 0.43). d To assess the correlation of the direction of motion of the two operators flanking a locus, comparisons of the vectors describing the motion of each operator were made at each time point. An RV coefficient was calculated for this purpose. The general trend observed was a decrease of motion correlation as the genomic separation between the operators increased. However, for the 64 kb and 42 kb strains which adopt a more compact configuration, the trend was not observed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016949&req=5

Fig4: Motion analysis for strains with different genomic separations. a When the relative orientation of two particles is maintained as they proceed along their trajectories (indicated as lines) their motion is considered isotropic (bottom), when orientation changes over time motion is anisotropic (top). A statistic D was calculated as a measure of anisotropy (Materials and Methods; Fig. 4). Larger D values correspond to greater anisotropy. D is observed to generally increase with genomic separation (b). In c the mean D is plotted against the mean distance (R2 = 0.43). d To assess the correlation of the direction of motion of the two operators flanking a locus, comparisons of the vectors describing the motion of each operator were made at each time point. An RV coefficient was calculated for this purpose. The general trend observed was a decrease of motion correlation as the genomic separation between the operators increased. However, for the 64 kb and 42 kb strains which adopt a more compact configuration, the trend was not observed
Mentions: During imaging, it was noticeable that the relative orientations of the tagged loci in a subset of cells were markedly constrained. To analyse this quantitatively we developed a test which assigns a statistic, D (see Materials and Methods), which quantifies the anisotropy of the spatial orientation of the two-spot system. The higher D, the greater the degree of anisotropy. Lower D values were generally observed for genomic separations below 70 kb (Fig. 4b) and anisotropy is correlated with the mean distance (Fig. 4c). This could be an indication that for small physical distances that relative motion of foci is less constrained. As physical distance increases, the nuclear environment acts as a constraint restricting the extent of relative motion.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Genomes 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.

Results: We 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.

Conclusions: The 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 material: The online version of this article (doi:10.1186/s12860-016-0111-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus