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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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 <it>trans </it>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.</p

Use of Two-Color Fluorescence-Tagged Transgenes to Study Interphase Chromosomes in Living Plants

Sixteen distinct sites distributed on all five Arabidopsis (Arabidopsis thaliana) chromosomes have been tagged using different fluorescent proteins and one of two different bacterial operator-repressor systems: (1) a yellow fluorescent protein-Tet repressor fusion protein bound to tet operator sequences, or (2) a green or red fluorescent protein-Lac repressor fusion protein bound to lac operator sequences. Individual homozygous lines and progeny of intercrosses between lines have been used to study various aspects of interphase chromosome organization in root cells of living, untreated seedlings. Features reported here include distances between transgene alleles, distances between transgene inserts on different chromosomes, distances between transgene inserts on the same chromatin fiber, alignment of homologous chromosomes, and chromatin movement. The overall findings are consistent with a random and largely static arrangement of interphase chromosomes in nuclei of root cells. These transgenic lines provide tools for in-depth analyses of interphase chromosome organization, expression, and dynamics in living plants

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter–neomycinphosphotransferase II (NOSpro– NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification