Whi5 is a protein that regulates the G1/S transition in the yeast cell cycle. It forms a complex with the cell cycle transcription factor SBF and actively represses transcription of G1-specific genes. An analogous metazoan protein, retinoblastoma protein (Rb), similarly regulates the G1/S transition by interacting with transcription factor E2F. Detection of Rb mutations in several different kinds of cancer implies that Rb is a significant factor involved in tumorigenesis. As with the Whi5, Rb also actively represses the transcription of cell cycle genes. The goal of my research is to explore the mechanism by which Whi5 represses transcription, to further examine conservation between cell cycle control mechanisms in yeast and metazoans.

Active repression of gene expression by Rb appears to depend, at least in part, on the recruitment of members of the histone deacetylase (HDAC) family. I hypothesized that Whi5 gene repression may similarly depend on yeast HDACs.  Using a batch affinity chromatography assay, I detected specific interactions between Whi5 and three of the yeast HDACs Hos3, Rpd3 and to a lesser extent, Hos1. These results suggest that, like Rb, Whi5-dependent repression may involve histone deacetylases and that at least two HDACs are involved in the Whi5-mediated repression.

Only the hypophosphorylated forms of Whi5 and Rb associate with SBF and E2F. Hyperphosphorylation of Whi5 by cyclin-dependant kinases (CDKs) disrupts the Whi5-SBF interaction, allowing the transcription of G1 genes.  We hypothesize that Whi5-HDAC interaction may also be sensitive to phosphoryalation status. Where cyclins, the catalytic subunits of CDKs, are deleted we expect to see increased interaction between Whi5-HDAC while the overexpression of cyclins will decrease this interaction.

Given that Whi5 interacts with HDACs in vivo it is possible that Whi5 is responsible for the recruitment of HDACs to promoters. To test this hypothesis I will use a chromatin immunoprecipitation (ChIP) assay to detect the recruitment of Rpd3 and Hos3 to promoters of G1 genes. If Whi5 is responsible for targetting HDACs, in the absence of Whi5 I will not be able to detect either Rpd3 or Hos3 at the promoter.

Much is known about the order of events involved in transcriptional initiation but the mechanism of repression and the order of events involved in relieving this repression remain elusive. Once it has been established that Whi5 represses transcription by recruiting HDACs I plan to explore the sequence of events that follows recruitment. We have seen that two HDACs are associated with Whi5 but some questions remain regarding: (1) whether Whi5, HDACs, or both are phosphorylated; (2) whether HDACs dissociate simultaneously or sequentially; (3) whether other factors, such as chromatin remodeling proteins, involved in Rb-mediated transcriptional repression, also influence Whi5-mediated transcriptional repression.

The Rb protein is a common factor that is critical in the onset of cancer.  A great deal has already been revealed regarding cell cycle regulation by Rb and Rb-associated complexes.  However, there are also a number of questions that are difficult to resolve due to the technical limitations of experimentation with mammalian cells. By establishing an analogy between Whi5 and Rb we hope to develop a simple model system in yeast to better understand cancer regulation by Rb in humans.