Title

Reverse Recruitment: Activation of Yeast Genes at the Nuclear Periphery

Date of Award

2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

George Santangelo

Advisor Department

Biological Sciences

Abstract

The regulation of genes at the nuclear periphery is an evolutionarily conserved phenomenon in eukaryotes. The reverse recruitment model of transcriptional activation postulates that genes are activated by moving to and contacting transcription machinery located at subnuclear structures. In Saccharomyces cerevisiae it has been reported that this platform for gene regulation may reside at the nuclear periphery. To test this hypothesis, I utilized a GFP-gene tagging technique, which uses LacI-GFP to visualize a tandem array of its DNA-binding sequence, to monitor localization of SUC2 and GAL1 . I found that both genes preferentially localized to the nuclear periphery when transcriptionally active. By developing an in vivo single cell reporter assay, I simultaneously monitored gene location and expression of a GFP-Ras2 reporter and found that, when induced, cells with perinuclear GAL genes activated transcription 10 minutes before cells with genes localized to the nucleoplasm. Thus, interaction with the nuclear periphery correlates with more rapid initiation. Further, the GAL1 gene can anchor in response to galactose, even when transcription is blocked, suggesting that genes move to the nuclear periphery prior to transcriptional initiation. I also show that gene localization to the nuclear periphery correlates with defects in regulation caused by the removal of SUC2 and GAL1 regulatory factors. Strikingly, these factors can be biochemically purified with the perinuclear compartment. Further, I report here that components of the transcriptional pre-initiation complex are localized to the nuclear periphery in the presence or absence of transcription. This suggests that there is a highly organized subnuclear architecture that facilitates gene regulation at the nuclear periphery. Interestingly, I saw that the archetypal transcriptional activator Gal4 exhibits a quantifiable difference in its subnuclear mobility that correlates with its function. For example, induced Gal4 shifts to a more slowly mobile form. I hypothesize that this shift may be the result of interaction with perinuclear transcription factories and that the well characterized dimerization and activation domains of Gal4 play a role in this tethering. Finally, I propose a model by which chromatin structure can influence gene movement to the nuclear periphery through the dynamic conversion between heterochromatin and euchromatin.