The N-terminal arm of Mcm1 has a role in regulating genes that are required for tolerance to NaCl (24
). In addition to the NaCl-sensitive phenotype previously associated with deletion of the arm, we have observed other biological phenotypes, including sensitivity to CaCl2
, high pH, and CFW. We also determined that the NT arm of Mcm1 is required for transcriptional activation of a set of genes, including cell wall biogenesis and transport genes that were not earlier known to be regulated by Mcm1. In addition to the requirement for the NT arm, mutations in the MADS box domain that are defective in the transcriptional activation of other subsets of Mcm1-regulated genes also affect the transcription of many of these newly identified Mcm1 targets. Among the genes that we identified, only the promoter region of YGP1
appeared to be bound by Mcm1 by genome-wide location analysis (17
). Four of these genes, YBR071W
, and YGP1
, are induced under various cell wall-damaging conditions (25
is an uncharacterized ORF that appears to be part of the cell wall integrity pathway (25
are components of the cell wall (12
, which has 50% overall identity to YGP1
, is a spore wall component but is also induced under stress. The increased sensitivity of the mcm1-Δ2
strain to NaCl, CaCl2
, high pH, and CFW is likely caused by decreased transcription of these and other cell wall genes that are regulated by Mcm1.
The Mcm1 protein has strong in vitro DNA-binding affinity for the promoters of a subset of cell wall- and membrane-associated genes, such as GFA1
, and PIS1
). Given that we identified other cell wall-associated genes that were sensitive to the Mcm1 NT arm deletion, it was possible that these genes were regulated by a similar mechanism. However, Northern blot analysis indicated that expression of these genes was not affected by deletion of the Mcm1 NT arm (data not shown). Thus, the Mcm1 arm-dependent genes we identified are distinct from other cell wall- and membrane-associated genes that may be regulated by Mcm1. It is likely that transcriptional regulation of the two groups of genes, although both mediated by Mcm1, occur through distinct mechanisms.
Most of the genes that we have identified showed a decrease in expression in strains containing the Mcm1 NT arm deletion. This result suggests that Mcm1 is functioning as an activator of these genes. However, two of the NT arm-dependent genes we identified, SIT1
, showed an increase in expression in the deletion mutant, suggesting that Mcm1 is functioning as a repressor of these genes. It is possible that depending on the promoter, Mcm1 can function either as an activator or a repressor of the salt induced genes. This would be similar to regulation by the Mcm1-ArgR complex, which functions to both activate arginine catabolic genes and repress anabolic genes in the presence of arginine in the medium (30
). However, we were unable to detect the direct binding of either Mcm1 or Mcm1-Δ2-17 to these promoters by ChIP analysis, suggesting that Mcm1 may indirectly regulate the expression of these genes through the Mcm1 NT arm-dependent expression of a repressor protein that targets these genes (data not shown).
The decreased transcription of YGP1
in the mcm1-Δ2
mutant was not due to a decrease in Mcm1 protein level or stability. The deletion of the Mcm1 arm also did not decrease DNA-binding affinity or change the apparent DNA-bending angle, both of which contribute to transcriptional activation by Mcm1 (1
). Based on our data, we hypothesize that the Mcm1 NT arm may be required to recruit or stabilize binding of another transcription cofactor that binds to the YGP1
promoter to activate its transcription.
Transcription of YGP1
was earlier shown to be regulated by Rlm1, a type II MADS box protein (18
). In a strain with constitutively activated Rlm1 there was a decrease in YGP1
expression, leading to the conclusion that Rlm1 was a repressor of this gene (18
). However, mutational analysis of the regulatory sites in the YGP1
promoter suggests that Rlm1 may also have a minor role in the activation of YGP1
. This model is supported by Northern analysis, which showed that deletion of RLM1
resulted in a decrease in YGP1
transcript compared to the wild-type strain (18
). We have shown here that Mcm1, a type I MADS box protein, has a direct role in regulating the expression of this gene. Interestingly, this is the first report of a gene that is jointly regulated by both type I and type II MADS box proteins in yeast. Since Rlm1 is known to regulate YGP1
transcription, there is a possibility that Rlm1 may interact with the NT arm of Mcm1. Among the 25 genes that are known to be regulated by Rlm1, 20 also have putative Mcm1-binding sites (18
; our data). While the expression of some of these transcripts appear to be affected by mutations in the Mcm1 MADS box domain that decrease Mcm1 DNA-binding affinity, we found that only YGP1
transcription are dependent on the NT arm (Fig. and data not shown). If the Mcm1 NT arm is important for interaction with its cofactor, this result suggests that Rlm1 is unlikely to be the cofactor that Mcm1 is interacting with at the YGP1
promoter. Also, since mutagenesis of the Rlm1-binding site, R1, in the YGP1
reporter showed only a minor decrease in activation (Fig. ), there is little evidence for the role of Rlm1 as the cofactor recruited by the Mcm1 NT arm.
Residues S2 and T8 in the Mcm1 NT arm are heavily phosphorylated and a mutation of S2 to alanine, which partially mimics the unphosphorylated form of these residues, had a salt-sensitive phenotype that was similar to deletion of the NT arm (24
). Since the deletion of the NT arm also causes cells to be sensitive to CFW, high pH, and CaCl2
, we expected that amino acid replacements of residues S2, T8, and T10 may have similar phenotypes on these media. However, none of the point mutations affected growth on CaCl2
and had relatively mild effects on growth on CFW and high pH in comparison to
the NT arm deletion. Interestingly, mutations of these residues to either alanine or aspartate, which partially mimic constitutive dephosphorylation or phosphorylation, respectively, showed similar sensitivity to CFW and alkaline pH (Fig. ). This suggests that the phosphorylation state of these residues may have little to do with resistance to CFW and alkaline pH. Alternatively, it is also possible that the aspartate mutation does not effectively mimic the phosphorylated form of these residues.
The T35A mutation in the MADS box domain of Mcm1 suppresses the growth defect of the NT arm deletion under high-salt conditions (23
). We found that this mutation had a similar effect on suppressing the NT arm-sensitive phenotypes on media with CFW, high pH, and CaCl2
but did not restore growth to the level of wild-type Mcm1. Residue T35 of Mcm1 contacts the phosphate backbone of the DNA and a T35A mutation decreases Mcm1 in vitro DNA-binding affinity (1
). As expected from the decreased DNA-binding affinity, the T35A mutation caused a significant decrease in YGP1
transcript level. Although the T35A mutation partially suppressed the growth defects caused by the deletion of the Mcm1 NT arm, it did not cause an increase in YGP1
expression (Fig. ). Therefore, the ability to grow in medium with CFW, high pH, and CaCl2
is not simply due to restoring expression of YGP1
. This result implies that the Mcm1 NT arm may have different mechanisms of regulating YGP1
and other genes required for resistance to various cell wall stresses. This suggests that there are other Mcm1 NT arm-dependent genes that are required for resistance to these growth conditions. It is unclear how the T35A mutation suppresses the sensitivity to cell wall stresses caused by deletion of the NT arm. It is possible the T35A mutation prevents interaction with other Mcm1 cofactors or decreases Mcm1 binding to weak sites in the genome, causing an increase in the level of Mcm1 that is available to interact with cofactors at the cell wall gene promoters.
The Mcm1 point mutations in the NT arm and the MADS box domain appear to be more sensitive to CFW than growth under high-pH conditions, while hardly affecting sensitivity to CaCl2. One model to account for this differential sensitivity would be that genes conferring resistance to high pH and CaCl2 have other activators in addition to Mcm1. For example, Mcm1 may be the primary activator for NT arm-dependent genes that confer CFW resistance, causing cells with the NT arm deletion to be very sensitive to the presence of this compound. On the other hand, genes required for growth under high-pH conditions may be regulated by other activators in addition to Mcm1 and are therefore less sensitive to particular Mcm1 mutations.
While deletion of the NT arm made cells sensitive to CaCl2, in contrast to the CFW and high pH phenotypes, growth on CaCl2 was not sensitive to any of the point mutations in the MADS box domain, suggesting that a very different mechanism may be at play in regulating genes that confer resistance to this growth condition. It is possible that another cofactor that binds strongly to these promoters and may be tethering Mcm1 to the DNA mainly via interactions with its NT arm. Thus, DNA-binding defects in Mcm1 may not have significant effects on activation of these genes as long as Mcm1 is tethered to the promoter through interactions with this cofactor.