The conserved Cdc14 (cell division cycle 14)-family of phosphatases regulates several key events during late mitosis, most notably they promote reversal of Cdk1-dependent phosphorylation and thus mitotic exit 2,3,5
. In budding yeast, Cdc14 localises dynamically to different cellular structures in a cell-cycle dependent manner. During interphase Cdc14 is bound to the nucleolus 6
while at anaphase it is released throughout the cell 7
. Two regulatory networks activate Cdc14 during mitosis, FEAR (Cdc Fourteen Early Anaphase Release) and MEN (Mitotic Exit Network) 3
. The FEAR activation occurs in early anaphase and is important to coordinate many anaphase events, while MEN operates in late anaphase 3
. Cdc14 activation by FEAR is crucial for the faithful execution of many anaphase processes, including timely chromosome segregation. This role was identified through the observation that segregation errors are present in Cdc14 mutants but not MEN mutants 4,8,9
. Interestingly, such defects occur at specific genome regions, namely the repetitive ribosomal gene array (rDNA) and telomeres 4,8,9
. The failure to segregate ribosomal repeats in cdc14
mutants is caused by lack of RNAP-I transcription inhibition 10-12
, a process required for the loading of Condensin complex to these repeats during anaphase. In contrast, the role of Cdc14 in telomere segregation is unknown. Importantly, Cdc14 inactivation in cells tricked to transcribe ribosomal genes with RNAP-II, instead of RNAP-I, also causes segregation failure of the ribosomal repeats 11
. Therefore, one hypothesis would be that Cdc14 promotes transcription repression of RNAP-II genes at sub-telomeric sites. This possibility is particularly appealing because nucleolar Cdc14 was originally discovered as a subunit of the silencing RENT (regulator of nucleolar silencing and telophase exit) complex 5,6
, which inhibits transcription by RNAP-II at the rDNA intergenic spacers (IGS). Besides Cdc14, RENT contains Sir2 and Cfi1/Net1 5,6
. Roles for Sir2 and Net1 in rDNA silencing have been described extensively 13-15
, however a role for Cdc14 in RNAP-II silencing has not been carefully assessed.
Growth assays are available for the examination of transcription silencing within the silenced loci in the yeast genome, however the contribution of essential genes, like Cdc14, to silencing cannot be analysed using these methods. To circumvent this limitation, we used RT-qPCR to measure levels of endogenous transcripts of the intergenic sequence (IGS) regions within yeast ribosomal repeats on chromosome XII () in the conditional mutant cdc14-1 at permissive (25°C) and non-permissive (37°C) temperatures. An increase in IGS transcripts was observed in cdc14-1 mutants at 37°C (relative to 25°C), but not in wild type cells (). To test whether Cdc14 effect on IGS transcription is independent from its role in mitotic exit during anaphase, we measured total IGS transcription in cdc14-1 and wild type cells blocked in G1 and G2/M at 37°C. An increase in IGS transcripts, particularly at IGS2, was observed for cdc14-1 cells in these arrests (), therefore Cdc14 plays a role in rDNA silencing independent of its main cell cycle function during anaphase.
Cdc14 is required for rDNA silencing.
mutants cells, exit from mitosis is prevented under non-permissive conditions leading to cell cycle arrest in telophase. To ensure that the increased IGS transcription observed () is not due to the cell cycle arrest, we compared cdc14-1
mutants, which also arrest in telophase. Importantly, Cdc14 is bound to IGS regions in the final telophase arrest by cdc15-2
, which provides an ideal control for a telophase arrest with Cdc14 bound to IGS regions. An similar increase in IGS transcripts was observed in the cdc14-1
anaphase arrest relative to cdc15-2
(). These results confirm that, like other RENT members, Cdc14 contributes to rDNA silencing.
Sir2 is an NAD-dependent deacetylase that targets histone H3 and H4 to promote rDNA silencing 16
. It is possible that the role of Cdc14 in rDNA silencing () is fully mediated by Sir2 function. To test this, we first investigated whether Sir2 localises to IGS regions in the absence of Cdc14. We found that Cdc14 inactivation reduced Sir2 binding across IGS (). Next, we investigated whether Cdc14 inactivation affected rDNA silencing in the absence of Sir2. We found increased IGS transcription in double mutant cells (sir2
) compared to single mutants (), demonstrating that in the absence of Sir2, Cdc14 supports silencing at IGS regions.
Cdc14 is an RNAP-II CTD phosphatase.
In addition to silencing, transcription of non-coding RNA within the IGS regions is also subject to degradation, a process mediated by the TRAMP complex and by the exosome 17
. Next we investigated whether increased IGS transcription in cdc14-1
cells is caused by defects of this degradation pathway. To this aim, we deleted Trf4, the poly(A) polymerase component of TRAMP4, in cdc14-1
cells and investigated IGS transcription in the presence and absence of Cdc14 (). Double mutant trf4
cells showed an increase in IGS transcription compared to single mutants (), which demonstrates that elevated IGS transcription in cdc14-1
cells is not caused by defects in TRAMP-mediated degradation of ncRNA.
The transcription activity of RNAP-II is regulated through phosphorylation of the carboxyl-terminal domain (CTD) of the core subunit Rpb1 18
. CTD contains tandem repeats of the heptapeptide, Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1
. RNAP-II recruitment to promoters requires CTD to be hypo-phosphorylated, however transcription initiation involves phosphorylation on the serine-5 residues (S5
P) of CTD, and elongation on serine-2 (S2
. Although RNAP-II has been shown to bind to IGS regions, it does not elongate 19
, and thus the regions are silenced 13-15
Since Cdc14 is a proline-directed phosphatase and both S2
residues on the CTD are followed by proline, we explored whether these CTD residues are a substrate of Cdc14. We incubated purified Cdc14 (GST-Cdc14), expressed in bacterial cells, with Rpb1 extracted from yeast. In the absence of Cdc14, Rpb1 phosphorylation was detected on both S2
(). Phosphorylation at both S2
was rapidly and completely lost after incubation with Cdc14 (). To confirm Cdc14 specificity in the in vitro
phosphatase assay, several controls were also performed. Rpb1 incubation with a phosphatase-inactive Cdc14 (GST-Cdc14CR) 20
, the PP2A catalytic subunit Pph21 21
or the dual-specificity protein phosphatase (DSP) Msg5 22
did not affect S5
phosphorylation (). In contrast, incubation with the CTD S5
phosphatase Ssu72 23
led to loss of S5
but not S2
phosphorylation (). In vivo
, Cdc14 activity as a CTD phosphatase was consistent with an observed increase in CTD S5
phosphorylation on the IGS region upon Cdc14 inactivation ().
Our findings demonstrate that Cdc14 binding to IGS regions in interphase contributes to their silencing (). Although Cdc14 is confined to rDNA during interphase, it is released from the nucleolus during anaphase through the sequential activation of the FEAR (Cdc Fourteen Early Anaphase Release) and MEN (Mitotic Exit Network) regulatory networks 3
. Next, we sought to determine whether the release of Cdc14 during anaphase affects transcription of RNA Pol-II genes outside rDNA. To investigate this, we compared transcription profiles on genome-wide strand-specific tiling arrays 24
arrested cells. Comparison of cdc14-1
transcription in the arrays across the IGS regions confirmed the increase in IGS transcription previously observed for cdc14-1
(Supl. Fig. 1
). Next, we looked for regions that showed an increase in transcription in cdc14-1
relative to cdc15-2
. We found no significant changes in the profiles of cdc14-1
arrests genome-wide (data not shown), with the exception of sub-telomeric regions that contained Y’ elements. Strikingly, all telomeres containing Y’ elements showed an increase in transcription in cdc14-1
relative to cdc15-2
( and Supl. Fig. 2
). In contrast, transcription profiles of telomeres lacking Y’ elements were identical in cdc14-1
(). We observed these Cdc14-dependent transcription changes in Y’ elements to occur specifically during anaphase (). Supporting this data, we found that Cdc14 is enriched on the Y’ element of the right arm of chromosome IV (TEL4R-Y’
) () and that CTD phosphoryation increased in the absence of Cdc14 activity in this region ().
Cdc14 represses transcription of sub-telomeric Y’elements.
In budding yeast, Cdc14 is required for the segregation of repetitive regions, mainly the ribosomal gene array (rDNA) 4,8,9
and telomeres 4
. The rDNA non-disjunction phenotype observed in cdc14-1
cells stems from a defect in repressing transcription of ribosomal genes 10-12
, which is a requirement for Condensin recruitment to rDNA 4,25
and anaphase disjunction of the locus 26
. Next, we sought to determine whether Condensin binds to the sub-telomeric Y’ element on TEL4R
in a Cdc14-dependent manner during anaphase. To this aim, we compared binding of Condensin subunit Smc2 in cdc14-1
on the rDNA IGS and TEL4R-Y’
regions (). Consistent with previous reports, we observed that Condensin binding to rDNA is reduced in the absence of Cdc14 4,25
(). We note that the Smc2 binding profile in G1 arrested cells showed accumulation on IGS1
but not IGS2
(). We presently do not know the exact reason for this, however, Condensin has been recently shown to form a ring that topologically entraps DNA 27
, therefore it is possible that like in the case of Cohesin 28
, the transcription machinery can slide Condensin through the template. Next, we investigated the effect of Cdc14 on Condensin binding to telomeric regions during anaphase. We found that while Smc2 binds to both telomeric regions of chromosome IV (TEL4L
, lacking Y’ elements, and TEL4R-Y’
) in cdc15-2
arrests (), it binds to TEL4L
but not TEL4R-Y’
in cdc14-1 arrested cells (). This result demonstrates that Smc2 binds to sub-telomeric Y’ elements in a Cdc14-dependent manner during anaphase. Unlike Condensin, Sir2 binding on TEL4R-Y’
was unaffected by Cdc14 inactivation (). Therefore the Cdc14 function at telomeric Y’ elements is independent of Sir2. Recent reports demonstrated that the related rDNA structural complex Cohibin has a Sir2-dependent role telomere silencing and stability 29
. This may point to division of labour in genome maintenance between closely related complexes.
Cdc14 recruits Condensin to telomeres.
The lack of Condensin binding to TEL4R-Y’
but not TEL4L
arrests prompted us to investigate the segregation of chromosome IV in these arrests. We introduced arrays of tet
operators (tetO) at different positions on this chromosome; at centromere proximal regions (CEN4
-dot; 150kb away from CEN4
), the left telomere (TEL4L
-dot; 30kb away from TEL4L
), the right telomere (TEL4R
-dot; 30kb away from TEL4R
) and two interstitial sites on the right arm (ARM1
-dot and ARM2
-dot) (). We found that TEL4L
-dots and CEN4
-dots were fully segregated in the cdc14-1
arrest while TEL4R
-dots did not separate despite evident segregation of nuclear masses (). An intermediate degree of segregation was observed for the ARM
-dots (). We note that the TEL4R
-dot segregation failure was characterised by the presence of a single GFP dot in one of the segregated nuclei (). There are two potential interpretations for this phenotype. One possibility is that replication of telomeric regions containing Y’ elements is incomplete, since Cdc14 has been recently implicated in replication termination at the rDNA 30
. The second possibility, and more likely, is that lack of Condensin loading at these regions () prevents resolution of intertwines between sister chromatids 31,32
Segregation of telomeres with Y’-element requires Cdc14.
To test whether the telomeric segregation defects in cdc14-1
cells was caused by transcription of Y’ elements, we used α-amanitin, a RNAP-II specific inhibitor 33
, to block RNAP-II transcription. Addition of the drug improved significantly the degree of separation of the ARM
-dots (). Therefore, telomere segregation defects in cdc14-1
mutants correlate with the presence of Y’ elements in sub-telomeric regions and active RNAP-II transcription.
Cdc14 is a highly conserved phosphatase. In the human genome two Cdc14 homologues have been identified, hCDC14A and hCDC14B 14. CDC14A brings about mitotic CDK inactivation 34
, and its depletion by RNAi leads to centrosome duplication, mitotic and cytokinesis defects 35
, suggesting a role in chromosome partitioning and genome stability. It is differentially expressed in human cancer cells, interacts with Cdk1/cyclin B complex and p53 36
, and exhibits cytoplasmic localization during interphase 37
. In contrast, CDC14B, like yeast Cdc14, localizes to the nucleolus in interphase but not during mitosis 37
. Recent studies have linked CDC14B to the DNA damage checkpoint response during the G2 stage of the cell cycle 38
. The role of Cdc14 as an RNAP-II phosphatase in yeast prompted us to investigate whether this is an evolutionarily conserved function. To this end, we expressed and purified the human Cdc14 homologue hCDC14A from bacterial cells and tested its phosphatase activity in vitro
on human hRPB1 purified from HEK293T cells. Incubation of hRPB1 with purified hCDC14A led to dissapearance of S2
In summary, our study identifies a novel and conserved role for the mitotic exit phosphatase Cdc14 in the repression of RNA polymerase II transcription in repetitive regions of the yeast genome. Moreover, Cdc14 function links RNAP-II and chromosome segregation through the evidence that transcriptional repression of sub-telomeric regions containing Y’ elements by Cdc14 facilitates Condensin loading to and segregation of these telomeres. We propose that mitotic RNAP-II transcription inhibition facilitates loading of factors that mediate structural functions on mitotic chromosomes.