Ume6p destruction is required for induction of EMGs and execution of meiosis and spore formation. This proteolysis is accomplished by a two-step process that is controlled by both endogenous and exogenous signals (Mallory et al., 2007
; for review see Cooper and Strich, 2011
). The first step, which results in ~50% reduction in Ume6p levels and partial derepression of EMG, occurs when vegetative cultures are switched from dextrose to a nonfermentable carbon-based medium. The second step, resulting in the complete elimination of Ume6p and full EMG induction, requires the withdrawal of a nitrogen source and the direct association of the meiotic protein Ime1p. In the present study, we find that the initial step in this degradation program is regulated by Ume6p acetylation and deacetylation by the Gcn5p-dependent acetyltransferase complex SAGA and the HDAC Rpd3p, respectively. Specifically, acetylation at a cluster of three lysines is required for step 1 Ume6p destruction and partial EMG derepression. In addition, substitution mutations that either prevent or mimic acetylation retard or accelerate step 2 meiotic Ume6p destruction, respectively. These results suggest that these two steps are connected. Of interest, either hastening or delaying Ume6p destruction reduced the efficiency of meiosis I and II execution. These findings indicate that the fine-tuning of Ume6p destruction by acetylation is important for normal meiotic progression.
Acetyltransferases induce transcription by enhancing the activity of transcriptional activators and opening chromatin domains. The present study defines a third avenue by which gene activation is accomplished by acetylation, through the targeted destruction of the Ume6p repressor. Lysine acetylation is usually associated with protecting proteins from proteolysis by serving as a block to ubiquitylation (Sadoul et al., 2008
). A potential exception to this rule was the report that acetylation of the hypoxic transcription factor HIF1-α by the N-terminal acetyltransferase ARD1 stimulated its proteolysis (Jeong et al., 2002
). This result was surprising, given the mostly cytoplasmic localization of ARD1 and its function as an N-terminal α-acetyltransferase rather than as targeting the ε-amino group of lysine like ubiquitin ligases or Gcn5p (reviewed in Kuo and Hung, 2010
). Moreover, two subsequent reports found no connection between ARD1 function and HIF1-α stability (Arnesen et al., 2005
; Bilton et al., 2005
), bringing the initial result into question. The combination of in vitro and in vivo studies presented in the present study provides solid evidence for the regulated acetylation of a transcriptional repressor and the role of this modification in promoting APC/C-dependent destruction.
Gcn5p-dependent acetyltransferase activity stimulates partial Ume6p degradation when the cells are switched from dextrose to acetate media. In many differentiation pathways, transcriptional programs provide a series of gates that allows flexibility in cell fate decisions. For example, in hematopoietic stem cell differentiation, changes in the levels of the transcription factor PU.1 help to refine the gene expression profile to push the cell toward macrophage differentiation (Laslo et al., 2006
). A similar scenario may be occurring when yeast cells switch from a fermentable to a nonfermentable carbon source. Yeast will ferment various sugars, producing ethanol as a byproduct. When the sugar is exhausted, yeast will use the ethanol as a carbon source through mitochondrial-dependent respiration. Partial reduction in Ume6p levels induced by acetylation results in low-level derepression of EMG and rapid execution of meiosis upon further stimulation. A similar fate is observed for the yeast C-type cyclin. Along with its kinase Cdk8p, cyclin C also represses EMG transcription (Strich et al., 1989
) through its interaction with the mediator complex (Cooper and Strich, 1999
). Similar to Ume6p, cyclin C levels are reduced to approximately half when cultures are shifted from dextrose to ethanol- or acetate-based medium (Cooper et al., 1999
). Final destruction of cyclin C, which is also required for full EMG induction, occurs upon meiotic entry (Cooper et al., 1997
). These findings argue that a general down-regulation of EMG transcriptional repressors in acetate medium represents a “potentiated state” of the cell that allows rapid transition from mitosis to meiosis when nitrogen sources are exhausted.
The transition from mitotic to meiotic cell division requires Ime1p, a protein necessary for the complete Ume6p destruction (Mallory et al., 2007
). Although still a possibility, it seems unlikely that acetylation recruits Ime1p, as the Ume6p-Ime1p association domain (Rubin-Bejerano et al., 1996
) and acetylated lysines are on opposite ends of the protein (). In addition, in vitro studies revealed no difference between the association of Ime1p to Ume6p or to Ume6pKallQ
(unpublished results). A previous study suggested that one potential function of Ime1p is to disrupt the association of Ume6p to the Sin3p-Ume1p-Rpd3p HDAC complex (Pnueli et al., 2004
). The removal of Rpd3p from EMG promoters could serve two purposes. First, it would allow further acetylation of Ume6p, which would stimulate its destruction. Second, the surrounding histones would also become more likely acetylated due to the loss of Rpd3p from the promoter. Under step 1 destruction conditions (acetate-grown vegetative cells), Ime1p is not expressed, thus retaining Rpd3p at the EMG promoter and setting up a competition between this HDAC and Gcn5p activity to determine Ume6p acetylation status. This model is supported by our analysis of cis
mutations that throw the acetylation balance in one direction or the other. For example, mimicking acetylation (KallQ) promotes Ume6p step 1 destruction in the absence of Gcn5p. Similarly, deleting RPD3
enhances Ume6p destruction in acetate cultures. This constant interplay between these two activities is illustrated by our finding that although Ume6p is acetylated in acetate cultures, it is only partially destroyed compared with the KallQ mutant, which the cell may recognize as always acetylated. Taken together, these findings suggest that the interplay between Rpd3p and Gcn5p activities controls the acetylation status and stability of Ume6p, which in turn regulates meiotic entry and progression.