Abstract
BackgroundGene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated.ResultsWe have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells.ConclusionTranscriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery.
Highlights
Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing
Visualizing GAL locus position and movement in living cells To study the chromosome dynamics associated with transcription, we tagged the GAL locus in a haploid strain of Saccharomyces cerevisiae by integrating an array containing 256 lac operator sites into the intergenic region between the GAL7 and GAL10 genes, and co-expressing a green fluorescent protein-lac repressor fusion (GFP-lacI); (Figure 1a) [18,19,20,21,22]
We found that when the GAL locus was scored at the nuclear periphery for cells grown in galactose, the correlation between the two channels shows a consistent positive correlation
Summary
Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Actively transcribed genes have been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. They become associated with the nuclear pore complex (NPC) at the nuclear envelope. The relationship of transcriptional activity with the intranuclear positioning of genes has been well documented, relative to the periphery of the nucleus In the budding yeast Saccharomyces cerevisiae, silenced telomeres are localized to the nuclear periphery via the nuclear pore complex (NPC) [8,9,10,11], and repression of the silent mating type loci is associated with NPC components and the nuclear periphery [8,9,12]
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