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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

Chromatin conformation is not inherited. Mother and daughter cells can readily be identified in Saccharomyces cerevisiae cultures as physically attached pairs (a). Each mother-daughter pair shares the same parent providing an opportunity to assess whether chromatin configuration is conserved through cell division. Movies obtained in individual cells from the 70 kb strain are plotted with mother-daughter pairs in the same colour in (b), in each case with data from the mother to the left. Unpaired cells are in grey. In some cases it is clear that mothers and daughters have differing spot distances. The distance box plots for all mothers and daughters of each strain are plotted in (c). In order to compare changes in distance between a large sample of related and unrelated mother and daughter cell pairs, changes in compaction were calculated using movies from each genomic separation (d). Compaction is calculated by dividing genomic separation by the measured distance between operators. The change in compaction between different populations was then calculated as a ratio indicated on the y-axis as ‘Log2 of compaction ratio’. This comparison could be made between related m/d pairs and unrelated m/d pairs selected at random. In both cases, the distributions of the differences in compaction are similar and not statistically significant (Mann-Whitney p-value = 0.181). This indicates that the separation of the reporter loci studied here is not inherited. Little change in separation is observed when the comparison is made between unrelated mother/mother and daughter/daughter pairs. This suggests that the difference in the volume of mother and daughter cell nuclei has little effect on the separation of these operator tagged loci
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Fig6: Chromatin conformation is not inherited. Mother and daughter cells can readily be identified in Saccharomyces cerevisiae cultures as physically attached pairs (a). Each mother-daughter pair shares the same parent providing an opportunity to assess whether chromatin configuration is conserved through cell division. Movies obtained in individual cells from the 70 kb strain are plotted with mother-daughter pairs in the same colour in (b), in each case with data from the mother to the left. Unpaired cells are in grey. In some cases it is clear that mothers and daughters have differing spot distances. The distance box plots for all mothers and daughters of each strain are plotted in (c). In order to compare changes in distance between a large sample of related and unrelated mother and daughter cell pairs, changes in compaction were calculated using movies from each genomic separation (d). Compaction is calculated by dividing genomic separation by the measured distance between operators. The change in compaction between different populations was then calculated as a ratio indicated on the y-axis as ‘Log2 of compaction ratio’. This comparison could be made between related m/d pairs and unrelated m/d pairs selected at random. In both cases, the distributions of the differences in compaction are similar and not statistically significant (Mann-Whitney p-value = 0.181). This indicates that the separation of the reporter loci studied here is not inherited. Little change in separation is observed when the comparison is made between unrelated mother/mother and daughter/daughter pairs. This suggests that the difference in the volume of mother and daughter cell nuclei has little effect on the separation of these operator tagged loci

Mentions: Within cultures of Saccharomyces cerevisiae, following cell division, mother and daughter cells remain associated and can readily be identified as larger mother cells associated with a smaller daughter cell (Fig. 6a). The ease with which related siblings can be identified cytologically provides an opportunity to investigate whether chromatin state is inherited through cell divisions.Fig. 6


High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division
Chromatin conformation is not inherited. Mother and daughter cells can readily be identified in Saccharomyces cerevisiae cultures as physically attached pairs (a). Each mother-daughter pair shares the same parent providing an opportunity to assess whether chromatin configuration is conserved through cell division. Movies obtained in individual cells from the 70 kb strain are plotted with mother-daughter pairs in the same colour in (b), in each case with data from the mother to the left. Unpaired cells are in grey. In some cases it is clear that mothers and daughters have differing spot distances. The distance box plots for all mothers and daughters of each strain are plotted in (c). In order to compare changes in distance between a large sample of related and unrelated mother and daughter cell pairs, changes in compaction were calculated using movies from each genomic separation (d). Compaction is calculated by dividing genomic separation by the measured distance between operators. The change in compaction between different populations was then calculated as a ratio indicated on the y-axis as ‘Log2 of compaction ratio’. This comparison could be made between related m/d pairs and unrelated m/d pairs selected at random. In both cases, the distributions of the differences in compaction are similar and not statistically significant (Mann-Whitney p-value = 0.181). This indicates that the separation of the reporter loci studied here is not inherited. Little change in separation is observed when the comparison is made between unrelated mother/mother and daughter/daughter pairs. This suggests that the difference in the volume of mother and daughter cell nuclei has little effect on the separation of these operator tagged loci
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Chromatin conformation is not inherited. Mother and daughter cells can readily be identified in Saccharomyces cerevisiae cultures as physically attached pairs (a). Each mother-daughter pair shares the same parent providing an opportunity to assess whether chromatin configuration is conserved through cell division. Movies obtained in individual cells from the 70 kb strain are plotted with mother-daughter pairs in the same colour in (b), in each case with data from the mother to the left. Unpaired cells are in grey. In some cases it is clear that mothers and daughters have differing spot distances. The distance box plots for all mothers and daughters of each strain are plotted in (c). In order to compare changes in distance between a large sample of related and unrelated mother and daughter cell pairs, changes in compaction were calculated using movies from each genomic separation (d). Compaction is calculated by dividing genomic separation by the measured distance between operators. The change in compaction between different populations was then calculated as a ratio indicated on the y-axis as ‘Log2 of compaction ratio’. This comparison could be made between related m/d pairs and unrelated m/d pairs selected at random. In both cases, the distributions of the differences in compaction are similar and not statistically significant (Mann-Whitney p-value = 0.181). This indicates that the separation of the reporter loci studied here is not inherited. Little change in separation is observed when the comparison is made between unrelated mother/mother and daughter/daughter pairs. This suggests that the difference in the volume of mother and daughter cell nuclei has little effect on the separation of these operator tagged loci
Mentions: Within cultures of Saccharomyces cerevisiae, following cell division, mother and daughter cells remain associated and can readily be identified as larger mother cells associated with a smaller daughter cell (Fig. 6a). The ease with which related siblings can be identified cytologically provides an opportunity to investigate whether chromatin state is inherited through cell divisions.Fig. 6

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