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High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

Matzke AJ, Watanabe K, van der Winden J, Naumann U, Matzke M - Plant Methods (2010)

Bottom Line: First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence.The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle.These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.

ABSTRACT

Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.

Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B.

Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

No MeSH data available.


Related in: MedlinePlus

Visualization of fluorescent tagged sites on mitotic chromosomes. In the division zone of the root, chromosomes are dual labelled with H2B-mRed and EGFP-LacI (both genes are under control of the RPS5 promoter). (A) Mitotic chromosomes at the metaphase plate are viewed as bright red bars (white arrow; shown enlarged in B) with EGFP-tagged sites visible as two clustered pairs of green fluorescent dots (C; merged image in D). (E) Mitotic chromosomes at anaphase with two pairs of EGFP-tagged sites (F; merged image in G). Interphase nuclei are dull red ovals (A, E, G). In the merged image (G), three neighbouring interphase nuclei with superimposed green fluorescent dots can be seen. All bars indicate 5 μm. Homozygous tagged line 112 was used for A-D; homozygous tagged line 26 was used for E-G. In both cases, the EGFP-LacI fusion protein was supplied in trans.
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Figure 6: Visualization of fluorescent tagged sites on mitotic chromosomes. In the division zone of the root, chromosomes are dual labelled with H2B-mRed and EGFP-LacI (both genes are under control of the RPS5 promoter). (A) Mitotic chromosomes at the metaphase plate are viewed as bright red bars (white arrow; shown enlarged in B) with EGFP-tagged sites visible as two clustered pairs of green fluorescent dots (C; merged image in D). (E) Mitotic chromosomes at anaphase with two pairs of EGFP-tagged sites (F; merged image in G). Interphase nuclei are dull red ovals (A, E, G). In the merged image (G), three neighbouring interphase nuclei with superimposed green fluorescent dots can be seen. All bars indicate 5 μm. Homozygous tagged line 112 was used for A-D; homozygous tagged line 26 was used for E-G. In both cases, the EGFP-LacI fusion protein was supplied in trans.

Mentions: Whereas fluorescence-tagged sites appear to be rather static in plant interphase nuclei, the situation is different in mitotic cells. Observations of the fluorescence-tagged sites in these cells should be possible provided the RP-FP fusion protein can bind to operator repeats on highly condensed mitotic chromosomes. To test this possibility, we introduced into the tagged lines a second transgene construct encoding EGFP-LacI fusion protein and histone H2B fused with red fluorescent protein (Fig. 1C, construct 5), which uniformly stains Arabidopsis chromatin [28]. When the RPS5 promoter is used to drive high frequency expression of both of these genes in cells of the root division zone, green fluorescent dots can be seen superimposed on red fluorescent mitotic chromosomes (Fig. 6A-G). Dual fluorescence labelling allows tracking of tagged sites on mitotic chromosomes as they separate and decondense in daughter cells (Additional files, 2, 3, 4). In principle, it should be possible to follow complete mitotic cycles and analyze whether fluorescent tagged sites are repositioned in daughter cell nuclei, a process that may provide opportunities for alterations in gene expression [1].


High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

Matzke AJ, Watanabe K, van der Winden J, Naumann U, Matzke M - Plant Methods (2010)

Visualization of fluorescent tagged sites on mitotic chromosomes. In the division zone of the root, chromosomes are dual labelled with H2B-mRed and EGFP-LacI (both genes are under control of the RPS5 promoter). (A) Mitotic chromosomes at the metaphase plate are viewed as bright red bars (white arrow; shown enlarged in B) with EGFP-tagged sites visible as two clustered pairs of green fluorescent dots (C; merged image in D). (E) Mitotic chromosomes at anaphase with two pairs of EGFP-tagged sites (F; merged image in G). Interphase nuclei are dull red ovals (A, E, G). In the merged image (G), three neighbouring interphase nuclei with superimposed green fluorescent dots can be seen. All bars indicate 5 μm. Homozygous tagged line 112 was used for A-D; homozygous tagged line 26 was used for E-G. In both cases, the EGFP-LacI fusion protein was supplied in trans.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820019&req=5

Figure 6: Visualization of fluorescent tagged sites on mitotic chromosomes. In the division zone of the root, chromosomes are dual labelled with H2B-mRed and EGFP-LacI (both genes are under control of the RPS5 promoter). (A) Mitotic chromosomes at the metaphase plate are viewed as bright red bars (white arrow; shown enlarged in B) with EGFP-tagged sites visible as two clustered pairs of green fluorescent dots (C; merged image in D). (E) Mitotic chromosomes at anaphase with two pairs of EGFP-tagged sites (F; merged image in G). Interphase nuclei are dull red ovals (A, E, G). In the merged image (G), three neighbouring interphase nuclei with superimposed green fluorescent dots can be seen. All bars indicate 5 μm. Homozygous tagged line 112 was used for A-D; homozygous tagged line 26 was used for E-G. In both cases, the EGFP-LacI fusion protein was supplied in trans.
Mentions: Whereas fluorescence-tagged sites appear to be rather static in plant interphase nuclei, the situation is different in mitotic cells. Observations of the fluorescence-tagged sites in these cells should be possible provided the RP-FP fusion protein can bind to operator repeats on highly condensed mitotic chromosomes. To test this possibility, we introduced into the tagged lines a second transgene construct encoding EGFP-LacI fusion protein and histone H2B fused with red fluorescent protein (Fig. 1C, construct 5), which uniformly stains Arabidopsis chromatin [28]. When the RPS5 promoter is used to drive high frequency expression of both of these genes in cells of the root division zone, green fluorescent dots can be seen superimposed on red fluorescent mitotic chromosomes (Fig. 6A-G). Dual fluorescence labelling allows tracking of tagged sites on mitotic chromosomes as they separate and decondense in daughter cells (Additional files, 2, 3, 4). In principle, it should be possible to follow complete mitotic cycles and analyze whether fluorescent tagged sites are repositioned in daughter cell nuclei, a process that may provide opportunities for alterations in gene expression [1].

Bottom Line: First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence.The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle.These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.

ABSTRACT

Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.

Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B.

Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

No MeSH data available.


Related in: MedlinePlus