We have found that an evolutionarily conserved SCB within RE plays an important role in donor preference and that the SBF complex, which binds this consensus sequence, is important for RE activity. We also described two independent pathways that govern donor preference: one depends on FKH1, and the other depends on SBF, CHL1, and YKU80.
Although two important transcription factors are involved in MATa
donor preference, RE activation does not depend on transcription. Around region E, Szeto et al. (55
) did find a weak transcript that does not appear to encode a protein. However, RE activity does not depend on these sequences because they are absent in the 270-bp minimum enhancer (65
) or when multimers of region A, D, or E were inserted in place of the 1.8-kb sequence containing RE. Therefore, the role of Fkh1 and SBF in donor preference is very different from their involvement as transcription factors in the G2
/M and G1
/S transition, respectively.
It is also possible that the binding of SBF and Fkh1 to RE provokes a change in the chromatin structure of the left arm leading in some way to a greater accessibility of the resident donor; however, a recent study of the global chromatin structure of 45 kb of the left arm of chromosome III covering HML
and RE did not shown any differences between MATa
α cells except for the RE region (15
). We propose that the binding of Fkh1 and SBF to RE could reorganize the architecture and the nuclear localization of the left arm of chromosome III. In mammalian cells, the position of a locus can move relative to a heterochromatin domain, in response to a change in its transcriptional state (for review, see references 52
). Also, massive decondensation can occur in the absence of transcription per se (5
). We suggest that binding of SBF and Fkh1 to RE could control the nuclear organization of the left arm of chromosome III in a similar way. SBF and Fkh1 could counteract a compact organization of the left arm, making HML
more mobile in the nucleus.
Recently, we showed that HML
motion is strongly constrained in both MAT
α and RE-deleted MATa
strains, compared with MATa
). Additionally, the three-dimensional configuration of MAT
, and HMR
is mating-type dependent, the distance between HML
and the other cassettes being greater in MATa
). These data suggest there is constitutive tethering of HML
, which is relieved in MATa
cells through the binding of SBF and Fkh1 to the RE.
The consequences of cell cycle-dependent regulation of SBF and Fkh1 binding to RE are still unclear. It is possible that the establishment of chromosome III architecture needed for the activation of HML usage relies on two independent events, taking place in G1 through SBF binding to RE and in G2 through Fkh1 binding. Even if HO breaks the DNA at the MAT locus in G1, the absence of binding of Fkh1 in G2 would compromise the accessibility of HML, because the conformation of the left arm has not been properly established. This hypothesis is in agreement with the fact that fkh1Δ and RESCB mutants show the same HML usage defects in both G1/S- and G2/M-arrested cells. It seems that the recruitment of these factors to RE creates an epigenetic state necessary for the organization of the left arm in the nucleus in the subsequent phases of the cell cycle. The defect encountered in the G1 phase in RESCB mutants could lead to the disorganization of chromosome III structure in the subsequent S and G2 phases, explaining the defect also observed in the G2-arrested phase in this mutant. The same idea could apply to the fkh1 mutant. In contrast, arrest in both G1/S and G2/M does not affect donor preference in MATα cells.
Transcriptional activation at G1
/S promoters follows a complex ordered series of events first delineated for the developmental and cell cycle-regulated HO
promoter which depend on Swi5, SBF, and Whi5 (11
). At RE, there seems to be no involvement of either Whi5 or Swi5. In addition, the study of the association of Swi5 with intergenic regions across the genome (37
) does not show any binding to the RE-containing intergenic region. Finally, we show that the MBF complex, involved in the transcription regulation of another set of genes at the G1
/S transition is not involved in RE activation, underlining the specific role of SBF in donor preference.
We have previously suggested that the role of Mcm1 is to open the chromatin structure at RE to allow effector proteins to bind (53
). We show here that this protein binds all along the cell cycle. It is therefore possible that the role of Swi5 to recruit SBF at the HO
promoter is fulfilled by Mcm1 at RE.
Fkh1 has been shown to bind the promoters of the CLB2
cluster all along the cell cycle (35
), an observation that we confirmed. However, binding of Fkh1 to RE is restricted to the G2
/M phase of the cell cycle. This property is also observed for the binding to the 4A synthetic RE. This result could mean that the binding of Fkh1 to RE is regulated by a direct modification of the protein. However, as Fkh1 binds the CLB2
cluster promoters all along the cell cycle, it seems that this regulation does not affect directly the DNA binding properties of the protein, but rather its capacity to interact with other proteins binding RE. The cycling properties of Fkh1 binding are not directly linked to Mcm1 or SBF, since these proteins do not have a binding site in the 4A synthetic RE. It is possible that unidentified factors that are involved in Fkh1 regulation bind RE in domains A, D, and E. We are currently working on identifying these factors.
Fkh1 may be more important for RE function than in the regulation of the transcription of the CLB2
cluster. Fkh1 ChIP signals are stronger for RE than for the CLB2
promoters in G2
-arrested cells, in logarithmic phase, and in synchronized cells (Fig. ). The Fkh1 ChIP signal at RE is stronger than the one of Fkh2 or Ndd1 (53
). Reciprocally, Fkh2 and Ndd1 ChIP signals are much stronger at the BCL2
cluster promoters than Fkh1 (23
). Fkh1 activity can clearly substitute for the one of Fkh2 in the control of the CLB2
cluster expression (35
), but Fkh2 cannot replace Fkh1 for RE activation.
We also found that the mutated SCB is epistatic to chl1
Δ and to yku80
Δ, showing that SBF, CHL1
, and YKU80
act in the same pathway of RE activation. Since the deletion of RE reduces the usage of HML
cells to the level observed in MAT
α cells (66
), we know that deleting the SCB element is epistatic to both chl1
Δ and yku80
Δ. Therefore, as Chl1 does not bind RE, we suggest that SBF bound to RE needs Chl1 to perform its function. Although Chl1 is involved in the establishment of sister chromatid cohesion during S phase (40
), none of the other components that genetically interact with Chl1 were found to affect donor preference, including CTF4
, or KAR3
). It is therefore possible that the role of CHL1
in donor preference is not related to its activity in sister chromatid cohesion. It is important to keep in mind that Chl1 acts also in an SBF-independent manner, since the RESCB fkh1 chl1
triple mutant shows a slightly stronger reduction of HML
usage than the RESCB fkh1
How Yku80—primarily implicated in DNA end-joining and telomere silencing—acts at RE or in facilitating recombination at HML
is unclear, especially given that yku70
Δ deletions have little effect (48
; our data not shown). Whether it associates directly with RE or binds through Swi4/Swi6 or Fkh1 remains to be established.
Finally, we have ruled out the involvement of other a-
and α-specific genes in donor preference. Among the five a
-specific genes that have been identified (17
), all of them are involved in conjugation and both their cellular localization and their enzymatic properties do not fit with a role in donor preference (25
). We cannot fully rule out the involvement of an a
-specific sterile RNA or of the product of an unidentified small ORF (29
) to explain why HML
is slightly less used in MAT
α cells when the natural RE is replaced by 4A, for example. We cannot also rule out that HMR
usage can be slightly increased in MAT
α cells by an RE-like activity (67
We still don't know how RE works, but regulation of its activity is very complex. The involvement of SBF and Fkh1 make RE resemble a hybrid-regulated promoter, which recruits SBF in a Swi5-independent manner and Fkh1 in a G2
-specific way. Given that RE does not cause modification of chromatin around HML
, we speculate that the recruitment of transcription factors can create an epigenetic modification of RE which leads to a change in the regional conformation of the left arm of chromosome III in the nucleus, making it more accessible for recombination, possibly by blocking the tethering of the chromosome arm at several as yet unidentified sites. This hypothesis is supported by the increased mobility of the left arm in MATa
). In Schizosaccharomyces pombe
, recent studies have shown that donor preference is dictated by a Swi2-dependent recombination enhancer that controls the choice of the donor by controlling the spreading of a recombination-promoting complex on the donors in a heterochromatin-dependent manner (28
). The function of RE in S. cerevisiae
appears therefore to be radically different.