Whi5 is a critical cell cycle regulator that links CDK activity in G1 phase to the broad transcriptional program that accompanies commitment to cell division. We provide substantial evidence that the multifunctional Pho85 CDK is an important regulator of Whi5 activity and G1 phase-specific transcription including: (1) Whi5 is phosphorylated and antagonized by Pho85 and is the first reported substrate for the G1-specific CDK complex, Pcl9-Pho85; (2) the activity of an SBF-dependent promoter is influenced by PHO85; (3) the Pcl9 cyclin binds to SBF-regulated promoters; (4) the repressor function of Whi5 is mediated through the HDACs Hos3 and Rpd3; and (5) HDAC-Whi5 association is regulated by G1-specific forms of both the Pho85 and Cdc28 CDKs. We therefore conclude that timely and efficient release from Whi5 inhibition and subsequent G1/S cell cycle progression requires the concerted activity of both Cdc28 and Pho85.
Several lines of evidence point to common roles for Pho85 and Cdc28. For example, a burst of both G1-specific Cdc28 and Pho85 activity is essential for cellular morphogenesis. A strain lacking the G1-specific cyclins, CLN1
, and PCL2
, undergoes a catastrophic morphogenic change and fails to establish polarized cell growth and cytokinesis 
. Consistent with these observations, a chemical genomic analysis demonstrated that expression of genes involved in polarized cell growth was sensitive to simultaneous inhibition of both kinases, but not either single kinase 
. A functional connection between Pho85 and Cdc28 is further supported by independent genetic and biochemical analyses that identify common targets phosphorylated by both kinases 
Despite the clear functional overlap for G1-specific forms of Cdc28 and Pho85 in controlling morphogenesis, up to now, a direct role for Pho85 in cell cycle commitment and G1 phase-specific transcription has remained unclear. We discovered that, like Cdc28, Pho85 activates G1 transcription through inhibition of the Whi5 repressor. While the two kinases collaborate to control certain facets of Whi5 regulation, they are also specialized to modulate Whi5 function by distinct mechanisms. We have defined a novel HDAC-dependent mechanism that impinges on Whi5 function and implicates both Pho85 and Cdc28 as regulators of this process.
On the basis of these and other observations, we propose that Whi5 functional regulation involves perturbation of specific HDAC-Whi5 interactions and requires the concerted activity of both Cdc28 and Pho85 (summarized in ). Interestingly, our genetic observations support a model whereby Pcl-Pho85 preferentially targets the Hos3-Whi5 interaction illustrating a functional distinction between the two CDKs. While Pho85 associates with several cyclin subunits, only Pcl9 exhibits temporal expression and localization patterns compatible with such a function. PCL9
is expressed at the M/G1 phase transition and encodes a short-lived protein localized exclusively to the nucleus in early G1 phase 
. Cln3 is also present in early G1 cells, but shows a complex localization pattern, with significant retention to the ER in early G1 cells, followed by chaperone-mediated release into the nucleus in late G1 phase 
. How the specific features of Pcl9 and Cln3 localization might influence the timing of HDAC inhibition remains to be explored.
Model for CDK-dependent regulation of Whi5 activity and G1/S-specific transcription.
The second component of Whi5 regulation is predicated on previous studies indicating that G1/S gene expression is preceded by Whi5-SBF complex dissociation and subsequent nuclear export of Whi5 () 
. Unlike early regulatory events, Cdc28 activity is both necessary and sufficient to drive these events since neither SBF binding to Whi5 nor nuclear localization of Whi5 was adversely affected in a pho85
Δ mutant (). Also, we are able to detect binding of SBF in vivo to CLN2
promoters when PHO85
is deleted (). However, both purified Cln3-Cdc28 and Pcl9-Pho85 failed to affect Whi5-SBF stability in vitro, while complex disruption was effectively achieved in the presence of Cln2-Cdc28 kinases (). Cln3-Cdc28 and Pcl9-Pho85 may have a more pronounced effect on the Whi5-SBF complex in vivo. Alternatively, Cln3- and Pcl9-CDKs may act primarily as agonists of HDAC interactions while physical interactions with SBF and nuclear export are optimally mediated by the late G1 CDKs, Cln1- and Cln2-Cdc28. Indeed, recent work reveals activation of CLN2
expression while Whi5 remains bound to the promoter (H. Wang, L.B. Carey, Y. Cai, H. Wijnen, and B. Futcher, personal communication). Such a mechanism may serve to sharpen the onset, as opposed to the timing, of G1/S gene expression thus ensuring a sustained transcriptional burst and irreversible commitment to cell division 
. Consistent with this idea, recent analysis of cyclin gene expression using a single cell assay affirms that positive feedback involving the Cln1 and Cln2 cyclins induces the G1/S regulon, and that this regulatory feedback is important for maintaining coherence of gene expression at Start 
SBF promoter recruitment depends on a series of well-organized chromatin remodeling events 
. SBF, in turn, regulates the recruitment of the general transcription machinery via a two-step process beginning with the mediator complex followed by CDK-dependent recruitment of RNA PolII, TFIIB, and TFIIH 
. Previous studies suggested that this CDK requirement stems from Whi5, which in its unphosphorylated state, remains bound to SBF and occludes the basal transcription machinery from binding specific promoters 
. We have extended this model to include a role for HDAC activity. We predict that Hos3 and Rpd3 contribute to Whi5 repression by preventing holoenzyme access to chromatin. During states of high CDK activity, Cdc28 and Pho85 abrogate Whi5-HDAC and Whi5-SBF interactions and initiate transcription. Consistent with our model, Pcl9 and Cln3 cyclins localize to G1 promoters and Whi5 remains associated with G1-specific promoters in the absence of HDAC-promoter interactions (; H. Wang, L.B. Carey, Y. Cai, H. Wijnen and B. Futcher, personal communication). However, Whi5 may also repress transcription by additional mechanisms since its activity is partially retained in hos3
Δ mutants ().
Rpd3 is a well-characterized HDAC that accomplishes most of its functions as part of a large protein complex 
. The Rpd3-Sin3 deacetylase complex has long been implicated as a cell cycle regulator required for silencing HO
gene expression to prevent mating type switching in newly budded cells 
. Our observations that Whi5 associates with Rpd3 and our genetic data linking G1 Cdks, Whi5, and Rpd3 reveal a more general role for Rpd3 in G1/S-phase specific transcription. These data are consistent with observations from Futcher and colleagues that the Rpd3 protein can be detected at the CLN2
promoter and that the amount of Rpd3 at the promoter is decreased when CLN3
is induced (H. Wang, L.B. Carey, Y. Cai, H. Wijnen and B. Futcher, personal communication). The Rpd3-Sin3 HDAC has also been connected to G1 transcription factors through the interaction of Sin3 with Stb1, a Swi6-binding protein 
. Both Stb1 and Sin3 are required for repression of G1 transcription early in G1 phase 
. Unlike Rpd3, Hos3 is largely uncharacterized, although a recent study suggests a role for Hos3 in yeast apoptosis upon exposure to oxidative radicals 
. We have uncovered an additional role for Hos3 in Whi5-mediated transcriptional repression.
A question that arises from our observations is what advantage does combinatorial kinase regulation impart on specific biological processes such as G1/S cell cycle progression? Contributions from multiple CDKs may provide the precision and accuracy necessary for rapid definitive decisions that irreversibly affect cellular fate. Indeed, distributive multisite phosphorylation mechanisms exhibit ultrasensitivity with respect to kinase concentration, thereby creating a “switch-like” behavior in biological circuits 
. Since cell cycle transitions typically display switch-like attributes, multisite phosphorylation by various kinase combinations may prove to be a rule rather than the exception amongst CDK targets, including key cell cycle regulators such as Whi5. In fact, a recent computational analysis showed enrichment of multiple closely spaced consensus sites for Cdc28 substrates in yeast, a pattern that proved predictive of likely CDK targets 
Although kinase combinations are likely necessary for cell cycle regulation, the contribution of each individual kinase may vary depending on specific signals and environmental stimuli. In certain environments, Pcl-Pho85 may have more dramatic, condition-specific effects on Whi5 function than Cdc28 analogous, perhaps, to the regulation of Rb that is required for quiescence and prevention of apoptosis 
. Previous studies indicate that Whi5 localizes to nuclei in stationary phase cells suggesting that Whi5 may also play a role in G0 
. Interestingly, Pho85 is required for survival in starvation conditions and plays an important role during stationary phase 
. Furthermore, CDK5, the mammalian Pho85 homolog, induces apoptosis in neuronal cells via Rb phosphorylation 
. Whether Whi5 activity is more prominently affected by Pcl-Pho85 in response to stationary or stress conditions requires additional investigation.
Similarities between metazoan and yeast cell cycle regulation are increasingly evident as we continue to characterize Whi5 function. For example, similar to proposed Pcl9/Cln3 “early” phase regulation (), cyclinD-CDK4/6 phosphorylates Rb to promote HDAC dissociation and E2F transcriptional activation. E2F activation then leads to cyclin E expression, which, similar to Cln1/2 “late” phase regulation (), may establish a positive feedback loop whereby cyclinE-CDK2 activity disrupts Rb-promoter interactions and stimulates G1-transcription further 
. Despite these similarities, the importance of multiple regulatory components in both yeast and mammalian systems remains poorly understood and may be most fruitfully dissected using the yeast model.