Skatebro:research prop

Project Overview

There is much evidence to support that, under standard cellular conditions, Sus1 plays a key role in regulating SAGA activated genes by directing/confining them to the nuclear periphery for their preferential processing and export. We propose a 3-part study to look at changes in SAGA-dependent gene motility and transcription in wild-type and sus1Δ yeast strains under different cellular conditions, such as high temperature combined with galactose induction.

• Part 1: use dynamic 3D tracking in live yeast cells to visualize the motility of SAGA-dependent genes HXT1, GAL1, and INO1
• Part 2: use RNA-fluorescence in situ hybridization (FISH) to look at HXT1, GAL1, INO1 transcription levels
• Part 3: use DNA microarray to look at changes in genome-wide transcription levels in sus1Δ yeast.

Background Information

1. SAGA complex dominates transcriptional activation at a minumum of 10% of the meausurable yeast genome, and these genes tend to be stress induced.
2. Genes GAL1, HXT1, and INO1 have been previously studied and confirmed to be SAGA-dependent.
3. Sus1 interacts with both the SAGA complex and the Sac3-Thp1-Cdc31 complex, which binds to Nuclear Pore Complexes (NPC) at the nuclear periphery.
4. Under galactose induction, while Sus1 was found to be dispensable for GAL1 activation, Sus1 was found to be critical for gene motility and confinement of activated GAL loci to the nuclear periphery.
5. Sus1 is hypothesized to be likely involved in transcription coupled mRNA export in a manner similar to the following schematic:

   

6. Our Module 3 research suggested that, on galactose media and under increasing heat stress, sus1Δ yeast strains can grow more robustly than wild-type yeast strains.
7. Intrigued by this unpredicted finding, we propose a research project aimed at studying Sus1's role in gene transcription and motility under different cellular conditions, for example high temperature combined with galactose induction.

Research Problem and Goals

1. Specific problem: to determine if the role of Sus1 in gene regulation is altered by different cellular conditions.
   • Does Sus1 somehow inhibit transcription of SAGA-dependent genes at high temperatures? inhibit their expression/export?
2. General goal: Expand previous research on the relation between peripheral gene recruitment and gene expression by investigating Sus1's role in the mechanism of peripheral gene recruitment in greater detail.

Project Details and Methods

1. Controls to do:

Predicted Outcomes

1. If all goes well:

References

1. Cabal G., Genovesio, A., Rodriguez-Navarro S., Zimmer C., Gadal O., Lesne A., Buc H., Feuerbach-Fournier F., Olivo-Martin J., Hurt E., Nehrbass U. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature. 2006;441(8):770-773