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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

Establishment of a work-flow for live cell 4D imaging. a Seven sample strains were generated which introduced lac ×256 and tet ×224 operator arrays flanking regions on Chrs XIV, IV and V. b Operator arrays were detected using fluorescent tagged repressor proteins as indicated. A naming convention was adopted that includes the endogenous genomic distance (a) as well as half the distance of each operator array (0.5xp, 0.5xq). Not shown: An additional strain was generated with a single tetO ×224 array on Chr XIV which expressed both tetR-GFP and tetR-mCherry to produce colocalising green and red spots. This strain was used for channel alignment and mean measurement error estimation purposes. c Summary of the work flow for image processing. d Two stage channel alignment was found to improve standard error from 154 nm to 63 nm
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Fig1: Establishment of a work-flow for live cell 4D imaging. a Seven sample strains were generated which introduced lac ×256 and tet ×224 operator arrays flanking regions on Chrs XIV, IV and V. b Operator arrays were detected using fluorescent tagged repressor proteins as indicated. A naming convention was adopted that includes the endogenous genomic distance (a) as well as half the distance of each operator array (0.5xp, 0.5xq). Not shown: An additional strain was generated with a single tetO ×224 array on Chr XIV which expressed both tetR-GFP and tetR-mCherry to produce colocalising green and red spots. This strain was used for channel alignment and mean measurement error estimation purposes. c Summary of the work flow for image processing. d Two stage channel alignment was found to improve standard error from 154 nm to 63 nm

Mentions: In order to assay chromatin organization in vivo, we generated seven strains with fluorescently tagged chromosomal loci flanking various lengths of genomic DNA (Fig. 1a). The fluorescent repressor operator system (FROS) we adopted involves flanking different sides of reporter loci with arrays of 224 tet operators and 256 lac operators. These were then visualised though their interaction with mCherry TetR and GFP LacI (Fig. 1b). In order to mitigate the potential for arrays of repressor-bound operators to generate heterochromatin low concentrations of tetracycline were included in media to reduce tetR binding and a lacI point mutant was used [52].Fig. 1


High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division
Establishment of a work-flow for live cell 4D imaging. a Seven sample strains were generated which introduced lac ×256 and tet ×224 operator arrays flanking regions on Chrs XIV, IV and V. b Operator arrays were detected using fluorescent tagged repressor proteins as indicated. A naming convention was adopted that includes the endogenous genomic distance (a) as well as half the distance of each operator array (0.5xp, 0.5xq). Not shown: An additional strain was generated with a single tetO ×224 array on Chr XIV which expressed both tetR-GFP and tetR-mCherry to produce colocalising green and red spots. This strain was used for channel alignment and mean measurement error estimation purposes. c Summary of the work flow for image processing. d Two stage channel alignment was found to improve standard error from 154 nm to 63 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Establishment of a work-flow for live cell 4D imaging. a Seven sample strains were generated which introduced lac ×256 and tet ×224 operator arrays flanking regions on Chrs XIV, IV and V. b Operator arrays were detected using fluorescent tagged repressor proteins as indicated. A naming convention was adopted that includes the endogenous genomic distance (a) as well as half the distance of each operator array (0.5xp, 0.5xq). Not shown: An additional strain was generated with a single tetO ×224 array on Chr XIV which expressed both tetR-GFP and tetR-mCherry to produce colocalising green and red spots. This strain was used for channel alignment and mean measurement error estimation purposes. c Summary of the work flow for image processing. d Two stage channel alignment was found to improve standard error from 154 nm to 63 nm
Mentions: In order to assay chromatin organization in vivo, we generated seven strains with fluorescently tagged chromosomal loci flanking various lengths of genomic DNA (Fig. 1a). The fluorescent repressor operator system (FROS) we adopted involves flanking different sides of reporter loci with arrays of 224 tet operators and 256 lac operators. These were then visualised though their interaction with mCherry TetR and GFP LacI (Fig. 1b). In order to mitigate the potential for arrays of repressor-bound operators to generate heterochromatin low concentrations of tetracycline were included in media to reduce tetR binding and a lacI point mutant was used [52].Fig. 1

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