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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

Release of silencing of the DsRed-LacI gene and loss of DNA methylation from lac operator repeats in a ddm1 mutant. A. DsRed fluorescence in roots of seedlings doubly homozygous for a tagged locus (101, 107, 112) and an epigenetic mutation: drd1 (dr), rdr6 (r), wild type (wt), ddm1 (dd), mom1 (m). Increased fluorescence is observed only in the ddm1 mutant. The bar indicates 2 mm. B. DNA methylation analysis of lac operator repeats. The lac operator repeat array is ~9.2 kb (256 copies of a 24-bp lacO monomer plus a 12-bp linker sequence) [8]. The lac operator repeat arrays can potentially be cut into 315 bp fragments by the restriction enzymes EcoRI (E) and NarI (N) [15]. Whereas the former is insensitive to cytosine methylation, NarI will not cleave if the CG in its recognition site (GGCGCC) is methylated (compare N versus E lanes in the three WT panels). Retention of the higher band (mom1, rdr6 and drd1 lanes) after NarI digestion signifies wild type levels of CG methylation; production of a ladder of smaller bands (ddm1 lanes) indicates loss of CG methylation. Size markers are indicated to the left. Data are shown for three tagged lines (101, 107, 112) into which we successfully introduced all four mutations (drd1, ddm1, sgs2, mom1). For the other two tagged lines (26, 79), we were unable to generate homozygous mutants for all four of the epigenetic factors. However, we did successfully introgress the ddm1 mutation into these lines, which resulted in loss of CG methylation from the lac operator repeats in ddm1 (Additional file 1).
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Figure 2: Release of silencing of the DsRed-LacI gene and loss of DNA methylation from lac operator repeats in a ddm1 mutant. A. DsRed fluorescence in roots of seedlings doubly homozygous for a tagged locus (101, 107, 112) and an epigenetic mutation: drd1 (dr), rdr6 (r), wild type (wt), ddm1 (dd), mom1 (m). Increased fluorescence is observed only in the ddm1 mutant. The bar indicates 2 mm. B. DNA methylation analysis of lac operator repeats. The lac operator repeat array is ~9.2 kb (256 copies of a 24-bp lacO monomer plus a 12-bp linker sequence) [8]. The lac operator repeat arrays can potentially be cut into 315 bp fragments by the restriction enzymes EcoRI (E) and NarI (N) [15]. Whereas the former is insensitive to cytosine methylation, NarI will not cleave if the CG in its recognition site (GGCGCC) is methylated (compare N versus E lanes in the three WT panels). Retention of the higher band (mom1, rdr6 and drd1 lanes) after NarI digestion signifies wild type levels of CG methylation; production of a ladder of smaller bands (ddm1 lanes) indicates loss of CG methylation. Size markers are indicated to the left. Data are shown for three tagged lines (101, 107, 112) into which we successfully introduced all four mutations (drd1, ddm1, sgs2, mom1). For the other two tagged lines (26, 79), we were unable to generate homozygous mutants for all four of the epigenetic factors. However, we did successfully introgress the ddm1 mutation into these lines, which resulted in loss of CG methylation from the lac operator repeats in ddm1 (Additional file 1).

Mentions: Plants that are homozygous for the respective mutations were crossed with the homozygous tagged lines and the resulting F1 progeny were allowed to self-fertilize to produce a segregating F2 population. F2 seedlings were genotyped to identify ones that are doubly homozygous for the tagged locus and the mutation in the desired epigenetic factor. Doubly homozygous F2 seedlings were screened for fluorescence in root cells at low magnification. Of the four mutations tested, only ddm1 improved expression of the RP-FP fusion protein, which was easily visualized under a fluorescence microscope in extended roots of doubly homozygous F2 seedlings (Fig. 2A, Additional file 1). The release of silencing in the ddm1 mutant was accompanied by a substantial loss of DNA methylation from the operator repeats (Fig. 2B, Additional file 1). No improvement of fluorescence or loss of methylation was observed in rdr6, drd1 or mom1 mutants (Fig. 2A, B; Additional file 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)

Release of silencing of the DsRed-LacI gene and loss of DNA methylation from lac operator repeats in a ddm1 mutant. A. DsRed fluorescence in roots of seedlings doubly homozygous for a tagged locus (101, 107, 112) and an epigenetic mutation: drd1 (dr), rdr6 (r), wild type (wt), ddm1 (dd), mom1 (m). Increased fluorescence is observed only in the ddm1 mutant. The bar indicates 2 mm. B. DNA methylation analysis of lac operator repeats. The lac operator repeat array is ~9.2 kb (256 copies of a 24-bp lacO monomer plus a 12-bp linker sequence) [8]. The lac operator repeat arrays can potentially be cut into 315 bp fragments by the restriction enzymes EcoRI (E) and NarI (N) [15]. Whereas the former is insensitive to cytosine methylation, NarI will not cleave if the CG in its recognition site (GGCGCC) is methylated (compare N versus E lanes in the three WT panels). Retention of the higher band (mom1, rdr6 and drd1 lanes) after NarI digestion signifies wild type levels of CG methylation; production of a ladder of smaller bands (ddm1 lanes) indicates loss of CG methylation. Size markers are indicated to the left. Data are shown for three tagged lines (101, 107, 112) into which we successfully introduced all four mutations (drd1, ddm1, sgs2, mom1). For the other two tagged lines (26, 79), we were unable to generate homozygous mutants for all four of the epigenetic factors. However, we did successfully introgress the ddm1 mutation into these lines, which resulted in loss of CG methylation from the lac operator repeats in ddm1 (Additional file 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Release of silencing of the DsRed-LacI gene and loss of DNA methylation from lac operator repeats in a ddm1 mutant. A. DsRed fluorescence in roots of seedlings doubly homozygous for a tagged locus (101, 107, 112) and an epigenetic mutation: drd1 (dr), rdr6 (r), wild type (wt), ddm1 (dd), mom1 (m). Increased fluorescence is observed only in the ddm1 mutant. The bar indicates 2 mm. B. DNA methylation analysis of lac operator repeats. The lac operator repeat array is ~9.2 kb (256 copies of a 24-bp lacO monomer plus a 12-bp linker sequence) [8]. The lac operator repeat arrays can potentially be cut into 315 bp fragments by the restriction enzymes EcoRI (E) and NarI (N) [15]. Whereas the former is insensitive to cytosine methylation, NarI will not cleave if the CG in its recognition site (GGCGCC) is methylated (compare N versus E lanes in the three WT panels). Retention of the higher band (mom1, rdr6 and drd1 lanes) after NarI digestion signifies wild type levels of CG methylation; production of a ladder of smaller bands (ddm1 lanes) indicates loss of CG methylation. Size markers are indicated to the left. Data are shown for three tagged lines (101, 107, 112) into which we successfully introduced all four mutations (drd1, ddm1, sgs2, mom1). For the other two tagged lines (26, 79), we were unable to generate homozygous mutants for all four of the epigenetic factors. However, we did successfully introgress the ddm1 mutation into these lines, which resulted in loss of CG methylation from the lac operator repeats in ddm1 (Additional file 1).
Mentions: Plants that are homozygous for the respective mutations were crossed with the homozygous tagged lines and the resulting F1 progeny were allowed to self-fertilize to produce a segregating F2 population. F2 seedlings were genotyped to identify ones that are doubly homozygous for the tagged locus and the mutation in the desired epigenetic factor. Doubly homozygous F2 seedlings were screened for fluorescence in root cells at low magnification. Of the four mutations tested, only ddm1 improved expression of the RP-FP fusion protein, which was easily visualized under a fluorescence microscope in extended roots of doubly homozygous F2 seedlings (Fig. 2A, Additional file 1). The release of silencing in the ddm1 mutant was accompanied by a substantial loss of DNA methylation from the operator repeats (Fig. 2B, Additional file 1). No improvement of fluorescence or loss of methylation was observed in rdr6, drd1 or mom1 mutants (Fig. 2A, B; Additional file 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