Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana

Abstract

Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions.

Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C)

“…FISH can for instance be used to observe chromosome abnormalities, or to map molecular markers. When probing two regions in the genome, the FISH signals can be used to study the frequency of co-localization of two chromosomal regions 3,4,9 . FISH has the advantage that cell-to-cell variation can easily be investigated.…”

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

“…Using a fluorescence microscope equipped with a motorized z axis and image-processing software, it is possible to make optical sections through nuclei and reconstruct them in three dimensions to determine spatial relationships among fluorescencetagged loci. This technique has been employed in yeast, Drosophila, and mammalian cells to analyze interphase chromosome organization and dynamics (Gasser, 2002;Spector, 2003), but so far has been used to study only a limited number of genomic insertion sites in plant cells Lam, 2001, 2003;Esch et al, 2003;Matzke et al, 2003;Pecinka et al, 2005).…”

Engineered plant minichromosomes

“…Successful application of the LacO/LacI system for GFP tagging of distinct chromosomal loci in living A. thaliana has already been described (Kato and Lam, 2001;Pecinka et al, 2005;Watanabe et al, 2005;Jovtchev et al, 2008Jovtchev et al, , 2011. Indeed, preliminary data suggest that the LacO/LacI system can be exploited also for ectopic plant kinetochore assembly, providing dicentric chromosomes for subsequent splitting into artificial minichromosomes (Teo et al, 2013).…”

“…Also in A. thaliana transgenerational inheritance of minichromosomes may vary between accessions (Murata et al, 2006). In maize, minichromosomes generated by transgene-mediated telomere seeding in A chromosomes were transmitted through meiosis to 33% of the progeny obtained by self-pollination or to 12% to 39% of progeny via male gametes (Yu et al, 2007), a rate similar to minichromosomes generated by breakage-fusion-bridge cycles (Kato et al, 2005). Transmission of truncated chromosomes was also below the rates expected from the Mendelian rules in progenies obtained by self-pollination in other plant species, accounting for 52% to 72% for tetraploid A. thaliana (Teo et al, 2011) and 54% for tetraploid barley (Kapusi et al, 2012).…”

“…Whereas the former approach still restricts the size of the transferred genes, the latter is suitable for transfer of large genes, gene complexes, and/or multiple genes together with regulatory elements for safe, controlled, and persistent expression. Furthermore, minichromosome vectors avoid rearrangements that are often linked with transgene insertion into native chromosomes (Aufsatz et al, 2002;Pecinka et al, 2005;Kinoshita et al, 2010). Additionally, engineered chromosomes could be used to address basic questions concerning the function of specific chromosomal domains, such as centromeres (Nakano et al, 2008).…”