We initiated this study to better understand the mechanism by which Ches1 suppresses checkpoint phenotypes in S. cerevisiae checkpoint mutants. We found that the Ches1 protein interacts with Sin3, a member of the S. cerevisiae Sin3/Rpd3 HDAC complex. We further demonstrated that Ches1 does not suppress the DNA damage response in sin3 mutant strains, and overexpression of SIN3 blocks Ches1-mediated G2/M arrest after DNA damage. Thus, Ches1 appears to function by inhibition of Sin3 function. This inhibition may be the result of competition for binding between the high-level expression of the heterologous human Ches1 protein and endogenous yeast proteins.
These findings also led us to determine the role of Sin3 and its associated HDAC, Rpd3, in the response to DNA damage. Loss of Sin3/Rpd3 activity by deletion of either the SIN3 or RPD3 gene in checkpoint mutant strains increased survival in response to DNA damage and replication blocks and restored an appropriate cell cycle delay.
Prior work has demonstrated that chromatin status influences the S. cerevisiae
cell cycle. In particular, acetylation of H3 and H4 histone tails is necessary for transit through the G2
/M cell cycle phase in the absence of damage (16
). Alteration of centromeric structure by mutation of NPS1
, a component of the RSC chromatin-remodeling complex, also results in a permanent G2
/M arrest that is dependent on the spindle checkpoint (44
). In contrast, we observed no significant G2
/M delay in Sin3/Rpd3-deficient strains in the absence of damage. Our data indicate that disrupting the Sin3/Rpd3 deacetylase restores a G2
/M cell cycle arrest, but this response occurs only after the cells are exposed to DNA damage.
There are several potential models to explain how the sin3
Δ- or rpd3
/M delay is activated in response to damage. It is likely that chromatin must be remodeled in response to DNA damage in order for the damage signal to be initiated or for the damage to be processed. Much evidence suggests that checkpoint proteins must load onto damaged DNA to initiate and carry out checkpoint activities. A recent study (54
) reported the association of the human replication factor C-related Rad17 protein with the PCNA-related Rad1-Rad9-Hus1 protein complex at sites of DNA damage in vivo. Additionally, two reports (22
) demonstrated that components of the S. cerevisiae
DNA damage checkpoint complex, including Mec1, Ddc1, and Ddc2, localize to a region proximal to a single double-stranded break. An interaction between the S. cerevisiae
chromatin assembly factor, Asf1, and Rad53 has also recently been described (18
). Rad53 localizes to chromatin in a manner dependent on the DNA helicase Sgs1, which functions in the S-phase checkpoint pathway (11
). In human cells, HDAC1 interacts with the checkpoint proteins hRad9 and hHus1, possibly to regulate the G2
/M checkpoint (7
). Thus, depletion of Sin3/Rpd3 activity may directly promote a more accessible chromatin state and may partially obviate the need for Mec1 and Rad9 in response to DNA damage.
An alternative but not exclusive explanation for these data is that depleting HDAC activity could result in the specific misregulation of genes affecting G2
/M progression and DNA damage checkpoint activity. Microarray analysis of strains with SIN3
deleted showed a greater-than-twofold up- and down-regulation of approximately 170 and 265 transcripts, respectively, many of which function during meiosis (3
). Inhibition of the HDAC complex permits the expression of many meiotic genes in mitotic cells (4
). However, analysis of the sin3
mutant microarray data sets revealed no significant change in expression levels of those genes playing a primary role in the spindle checkpoint, including MAD1-3
, and MPS1
The DNA damage-induced arrest exhibited in the SIN3
mutant strains is dependent on the Mad1-containing branch of the spindle checkpoint pathway. The spindle checkpoint elicits preanaphase arrest through inhibition of the anaphase-promoting complex in cells whose replicated chromosomes have failed to form proper attachments to the mitotic spindle (2
). Three recent studies have reported potential functions of the spindle checkpoint pathways in response to DNA damage or replication blocks. Maringele and Lydall (26
) demonstrated that the cell cycle arrest exhibited by yku70
Δ mutants (which accumulate telomeric single-stranded DNA) is dependent on the DNA damage checkpoint genes CHK1
, and RAD9
, as well as the spindle checkpoint gene MAD2
. A second study also found a contribution of the spindle checkpoint to cell cycle arrest in response to MMS and HU when assayed in strains deficient for the DNA damage and replication checkpoints (12
). In this latter study, spindle checkpoint mutants containing an intact DNA damage checkpoint exhibited a wild-type response to HU and MMS; therefore, the spindle pathway may not play a major role in the response to DNA damage when the MEC1
-dependent pathway is active.
Most recently, a role for the spindle checkpoint in responding to DNA damage in some human cells has been reported (29
). Extensive chromosomal damage revealed a mitotic arrest independent of p53, and this delay was present when cells were treated with inhibitors of the ATM kinase. Immunofluorescence microscopy indicated the presence of Mad2 at kinetochores, suggesting activation of the spindle checkpoint, and inhibition of Mad2 by microinjection of a dominant-negative form of Mad2 cells alleviated the damage-induced arrest, thus allowing cells to enter anaphase. Those authors propose that the arrest exhibited by these cells is likely due to the spindle checkpoint's response to damaged kinetochores, which when disrupted can no longer attach to the mitotic spindle.
In the present work we demonstrate a similar response of S. cerevisiae
to DNA damage. Using specific mutants of the ATM-related Mec1 kinase, we demonstrate that the delay is independent of this pathway. Furthermore, the deletion of SIN3
potentiates the DNA damage response, resulting in substantial resistance to DNA damage and a wild-type cell cycle arrest even at relatively low doses of UV irradiation. Involvement of the spindle checkpoint suggests that the damage response may be acting through damaged kinetochores or, alternatively, there may be decreased tension elicited by damaged chromosomes with altered chromatin structure. Silverstein et al. (40
) recently uncovered a direct role for the Schizosaccharomyces pombe
Sin3 homolog, PstI, in regulating centromere acetylation levels through association with the Clr6 HDAC. Mutants lacking PstI exhibit reduced levels of gene silencing near the centromere and display a variety of phenotypes consistent with altered centromere and kinetochore structure, including increased sensitivity to the microtubule-destabilizing agent thiabendazole and defective centromeric sister chromatid cohesion.
Many reports implicate HDAC activity as being involved in cell cycle progression and cancer development (9
). The complicated nature of the Rb/E2F pathway provides many ways in which genetic defects can impede Rb-dependent repression, and this pathway is targeted in many tumor types (39
). Not all the transformed mammalian cell lines tested by Mikhailov exhibited a DNA damage-dependent anaphase arrest, and these differences may reflect whether they contain dysregulation of histone deacetylation. Studies using HDAC inhibitors further support the possibility that checkpoint pathways monitor acetylation status in human cells. For example, certain HDAC inhibitors cause cell cycle arrest in some tumor cell lines (31
). One study (34
) reported that the HDAC inhibitor azelaic bishydroxamic acid (ABHA) activates a novel G2
cell cycle arrest in normal human cells, and this checkpoint is absent in some ABHA-sensitive tumor cell lines.
HDAC inhibitors are being assessed for anticancer activity in clinical trials. One confounding factor in studies using pharmacologic means to inhibit HDACs is that the resulting phenotype may be secondary to other cellular targets of these agents. Our genetic studies in yeast directly demonstrate that deletion of an HDAC complex restores a DNA damage-induced G2/M arrest in checkpoint-deficient strains that is dependent on an intact spindle checkpoint. These data suggest that HDAC inhibitors may cause a paradoxical increase in survival to genotoxic agents when used in cancer cells containing mutations in genes performing functions analogous to RAD9 and MEC1, including BRCA1 and ATM, respectively.