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 HXK1, GAL1, and INO1
• Part 2: use RNA-fluorescence in situ hybridization (FISH) to look at HXK1, 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, HXK1, 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 interesting and 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 either the transcription or export of SAGA-dependent genes at high temperatures?
2. General goal: Expand previous research into the relation between the spatial organization of the genome and gene expression by investigating Sus1's role in the mechanism of peripheral gene recruitment in greater detail.

Project Details and Methods

1. We will be working with FY2068 and the sus1 strain we made in the previous module. Differences between these strains will give us insight into the role SUS1 in overall gene regulation (extending our previous goal of analyzing SUS1 in SAGA).
2. Methods:
   • 3D live tracking of GAL1, INO1, and HXK1 upon transcriptional activation in FY2068 and sus1 at 30 C and 37 C.
   • RNA-fluorescence in situ hybridization (FISH) to measure transcription levels of GAL1, INO1, and HXK1 in FY2068 and sus1 at 30 C and 37 C.
   • DNA microarray of FY2068 and sus1 at different temperatures and the presence of different transcriptional inducers.
   • Spot test to monitor viability of yeast under different conditions.
   • Chromatin Immunoprecipitation

Predicted Outcomes

If all goes well:

1. GAL1 has been shown to be confined to the nuclear periphery upon intranuclear activation in wild-type yeast, while in a sus1Δ mutant, GAL1 does not undergo this confinement (Cabal et al., 2006). These studies have also found that GAL1 transcription levels are unaffected by the presence or absence of SUS1. Our recent experiments suggest that the confinement of GAL genes to the nuclear periphery at higher temperatures is harmful to the yeast. We hope to confirm these findings and thus show that freer gene motility at higher temperatures allows certain genes to be better expressed.
2. INO1 has been shown to be recruited to the nuclear periphery for transcriptional activation in wild-type yeast (Brown et al., 2007). Our recent studies identified INO1 as one of the genes with most significant transcription decrease in sus1Δ yeast. Studying the movement of INO1 in the nucleus in both wild-type and sus1Δ yeast will allow us to further characterize the importance of SUS1 in recruitment to the nuclear periphery and the importance of freer gene motility for gene expression at higher temperatures. We suspect that higher temperatures will not have as great of an effect on INO1 expression since INO1 is known to be activated by Scs2 at the nuclear periphery.
3. We hope to find unaffected transcription levels for GAL1 and affected transcription levels for INO1 in sus1Δ yeast. Also, we expect both genes to have affected gene motility and gene expression in sus1Δ yeast, and thus altered cell viability (not neccesarily worse). Also, we expect to find that high temperature affects one/all of the following: gene transcription level, gene motility, and gene expression in wild-type and sus1Δ yeast.

References

1. Huasinga K., Pugh B., A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Molecular Cell. 2004;13(4):573-85
2. 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
3. Zanton S., Pugh F., Changes in genomewide occupancy of core transcriptional regulators during heat stress. PNAS:101(48);16843-16848
4. Brown CR, Silver PA (2007) Transcription regulation at the nuclear pore complex. Current Opinion in Genetics & Development 17 (2): 100-106.