Although typically thought of as a site for transcription of tandem arrays of rDNA genes, rRNA processing, and ribosome assembly, the nucleolus plays other critical roles in cellular regulation. The anchoring of the conserved eukaryotic phosphatase Cdc14 in the nucleolus is a prominent example of such a function. Despite the physical association of Cdc14 and its sequestration machinery with nucleolar chromatin, the impact of chromatin modifications on this mechanism has not been reported previously.
In this study, we provide evidence for a role for the conserved ubiquitin ligase Bre1 and monoubiquitination of histone H2B in the early phase of mitotic exit. First, in cells lacking the MEN component Lte1, we observed that deletion of BRE1 or mutation of the codon encoding the target lysine in both H2B genes results in cellular inviability or severe growth defects, respectively. Such a phenotype is characteristic of mutants in components of the FEAR network responsible for the early anaphase release of Cdc14. Second, we found that Bre1 and ubH2B-K123 are indeed required for full Cdc14 release during early anaphase when the MEN is inactivated by the cdc15-2 mutant.
Furthermore, we established that this requirement for ubH2B-K123 in Cdc14 release is dependent on the downstream effects of ubH2B-K123 on the methylation of H3K4 and H3K79, as strains unable to be methylated on either residue are also defective for early anaphase Cdc14 release. We also observed Cdc14 release defects for a mutant unable to be methylated by Set2 on H3K36, which was surprising since H3K36 methylation does not require ubH2B-K123 
. Nonetheless, these data demonstrate that all three of the known methylation marks on histone H3 in S. cerevisiae
play a role in this process. These data may also help to explain the observation that a haploid H3K4R,36R,79R strain mutated for all three H3 methylation sites displays an increased population of cells with 2C DNA content 
The requirement for histone modifications in mitotic exit is most simply explained by the direct modification of nucleolar chromatin (). We demonstrated that the Cdc14 release defects we observe are not correlated with changes in expression levels of known Cdc14 release factors, as the transcript and protein levels of regulators are unchanged or increased in bre1Δ
mutants. The sole exception is the increase in levels of Cfi1/Net1, which could potentially cause an increase in Cdc14 sequestration. However, this increase does not change the levels of Cfi1/Net1 or Fob1 associated with chromatin at the rDNA locus as measured by ChIP. The presence of both H3K4 and K79 methylation has been reported at rDNA chromatin by ChIP, consistent with a direct role for these modifications in affecting rDNA function 
. Dot1, the H3K79 methylase, has also been reported to be present in the nucleolus by microscopy of cycling cells and spread mitotic nuclei 
. Lastly, deletion of Ubp10, a deubiquitinating enzyme for ubH2B-K123, increases the levels of both meH3K4 and K79 at the NTS1 region, suggesting that ubH2B-K123 is also present at the site of Cdc14 sequestration and is able to promote the methylation of H3K4 and K79 at this locus 
. These modifications may depend on the transcription of this region by RNA polymerase II (Pol II), since there is a reported transcript produced from a promoter in the NTS1 region and Pol II has been detected in this region by ChIP 
Nonredundant roles for histone modifications in promoting early anaphase release of Cdc14.
In addition to showing that histone H2B ubiquitination and histone H3 methylation are required for efficient Cdc14 release, we verified the reported genetic interaction of rpd3Δ
and lte1Δ 
and demonstrated defects in Cdc14 release of rpd3Δ
mutants when MEN activity was inhibited. Unlike histone methylation, however, the requirement for histone deacetylation in Cdc14 release was specific to deacetylation by Rpd3 and not shared by other HDACs such as Hda1 and Sir2. These results are in agreement with expression microarray data showing no significant overlap between sir2Δ
mutant expression profiles and the overrepresentation of cell cycle related transcripts in rpd3Δ
that is not observed in hda1Δ
. The transcriptional changes of cell cycle genes in rpd3Δ
mutants suggest a possible indirect transcriptional mechanism for the requirement for Rpd3 in Cdc14 release but given the many reports linking Rpd3 to rDNA functions a direct role at rDNA cannot be excluded 
Although our studies point to a role for histone modifications in controlling the early anaphase release of Cdc14, understanding the underlying biochemical mechanisms involved will require further studies. An appealing possibility is that histone modifications may serve to maintain a permissive state for mitotic exit regulators to access the chromatin associated factors Cfi1/Net1 and Fob1 at the onset of anaphase and to enact the release of Cdc14 (). Surprisingly, our genetic experiments indicate that this requirement is independent of the action of the Cdc14-associated histone deacetylase Sir2 and removal of Sir2 did not bypass the requirement for histone modifications in Cdc14 release. Potential candidates whose access to Cfi1/Net1 could be affected by this alteration of chromatin function include Cdc5 and Clb-Cdk, the kinases responsible for Cfi/Net1 phosphorylation, and PP2ACdc55
, the phosphatase complex that counteracts this phosphorylation. Recently, Zds1 and Zds2, previously identified to associate with the chromatin silencing machinery 
, have been shown to play a role in early anaphase release of Cdc14 and to interact with PP2ACdc55 
, suggesting another potential mediator for the histone modification requirement for efficient mitotic exit.
As all of the histone modifications studied here are conserved from yeast to mammals, it seems possible that their role in controlling localization of Cdc14 is also conserved. A hint that this might be the case comes from studies of HUB1, the homolog of Bre1 in Arabidopsis thaliana
. Cells from hub1-1
mutants exhibit an increased population with 4C DNA content, consistent with a delay in progression through G2 or mitosis 
. In addition, while Cdc14 is not essential for mitotic exit in animal cells, its cell cycle-regulated association with the nucleolus is remarkably conserved from yeast to humans 
. Recently, it was reported that mammalian cells have adapted the Cdc14 sequestration mechanism to regulate the G2 DNA-damage checkpoint 
. Given the emerging role of meH3K79 and other histone modifications in the DNA damage response 
, it is possible that the Cdc14 regulation by meH3K79 described here is relevant to the function in metazoans of this conserved histone modification in DNA damage checkpoint signaling.
More generally, our studies point to a role for histone modifications in a process involving chromatin but not directly impinging on transcription or DNA repair on which most functional studies have been focused. The nonredundant role for all three known histone methylations in mitotic exit suggests they do not function in this context as part of a combinatorial code but instead may affect chromatin in a similar way to promote the programmed release of a chromatin bound factor.