The essential liaison of two copper proteins: the Cu-sensing transcription factor Mac1 and the Cu/Zn superoxide dismutase Sod1 in Saccharomyces cerevisiae.

Although copper is an essential trace element for cell function and viability, its excess can lead to protein oxidation, DNA cleavage, and ultimate cell damage. Cells have established a variety of regulatory mechanisms to ensure copper ion homeostasis. In Saccharomyces cerevisiae, copper sensing and response to copper deficiency are regulated by the transcription factor Mac1. Our group has previously reported that in addition to copper, several chromatin proteins modulate Mac1 functionality. In this study, based on a synthetic growth deficiency phenotype, we showed that the Cu/Zn superoxide dismutase Sod1 plays an important role in Mac1 transcriptional activity, in unchallenged nutrient-rich growth conditions. Sod1 is a multipotent cytoplasmic and mitochondrial enzyme, whose main known function is to detoxify the cell from superoxide ions. It has been previously reported that Sod1 also enters the nucleus and affects the transcription of several genes, some of which are involved in copper homeostasis under Cu-depleted (Wood and Thiele in J Biol Chem 284:404-413, 2009) or only under specific oxidative stress conditions (Dong et al. Mol Cell Biol 33:4041-4050, 2013; Tsang et al. Nar Commun 8:3446, 2014). We have shown that Sod1 physically interacts with Mac1 transcription factor and is important for the transactivation as well as its DNA-binding activities. On the other hand, a constitutively active mutant of Mac1 is not affected functionally by the Sod1 ablation, pointing out that Sod1 contributes to the maintenance of the copper-unchelated state of Mac1. In conclusion, we showed that Sod1-Mac1 interaction is vital for Mac1 functionality, regardless of copper medium deficiency, in unchallenged growth conditions, and we suggest that Sod1 enzymatic activity may modify the redox state of the cysteine-rich motifs in the Mac1 DNA-binding and transactivation domains.

Distinct associations of the Saccharomyces cerevisiae Rad9 protein link Mac1-regulated transcription to DNA repair.

While it is known that ScRad9 DNA damage checkpoint protein is recruited to damaged DNA by recognizing specific histone modifications, here we report a different way of Rad9 recruitment on chromatin under non DNA damaging conditions. We found Rad9 to bind directly with the copper-modulated transcriptional activator Mac1, suppressing both its DNA binding and transactivation functions. Rad9 was recruited to active Mac1-target promoters (CTR1, FRE1) and along CTR1 coding region following the association pattern of RNA polymerase (Pol) II. Hir1 histone chaperone also interacted directly with Rad9 and was partly required for its localization throughout CTR1 gene. Moreover, Mac1-dependent transcriptional initiation was necessary and sufficient for Rad9 recruitment to the heterologous ACT1 coding region. In addition to Rad9, Rad53 kinase also localized to CTR1 coding region in a Rad9-dependent manner. Our data provide an example of a yeast DNA-binding transcriptional activator that interacts directly with a DNA damage checkpoint protein in vivo and is functionally restrained by this protein, suggesting a new role for Rad9 in connecting factors of the transcription machinery with the DNA repair pathway under unchallenged conditions.

Synergy of Hir1, Ssn6, and Snf2 global regulators is the functional determinant of a Mac1 transcriptional switch in S. cerevisiae copper homeostasis.

To gain insights on the transcriptional switches that modulate proper copper homeostasis in yeast, we have examined in detail functional interactions of the relevant transcriptional activator Mac1. We identified Hir1 transcriptional repressor and histone chaperone as a Mac1-interacting protein. This association directly recruits Hir1 on a Mac1 target, CTR1 promoter, quantitatively under induction conditions. We also found Hir1 interacting directly with a previously unknown partner, the Ssn6 (Cyc8) co-regulator. On the non-induced CTR1 promoter, a Hir1 transcriptional activation function was revealed, in the absence of Ssn6, which was dependent on the presence of Snf2 (Swi2) nucleosome remodeler. Moreover, Ssn6 was identified as a Mac1-dependent prominent repressor of CTR1 transcription, antagonizing Snf2 occupancy. Transcriptional induction by copper depletion was effected by the quantitative recruitment of Snf2 directed mainly by Mac1 and redundantly by the quantitatively accumulated Hir1 and Ssn6 pair. Our analysis showed that the activation-effecting chromatin remodeling of CTR1 was due to Snf2 and not to the Hir1 histone chaperone activity or ability to regulate histone levels and stoichiometry. Following initiation, Hir1 and Snf2, but not Ssn6, were found to associate also with the actively transcribing CTR1 coding region, where Hir1 followed the pattern of the elongating RNA polymerase II. Therefore, we have shown that, at the CTR1 gene, in association with Mac1 DNA-binding transcriptional activator, the distinct and alternate genetic and physical collaboration of three global regulators modulates the transcriptional state of a switch involved in copper homeostasis.

Rad9 interacts with Aft1 to facilitate genome surveillance in fragile genomic sites under non-DNA damage-inducing conditions in S. cerevisiae.

DNA damage response and repair proteins are centrally involved in genome maintenance pathways. Yet, little is known about their functional role under non-DNA damage-inducing conditions. Here we show that Rad9 checkpoint protein, known to mediate the damage signal from upstream to downstream essential kinases, interacts with Aft1 transcription factor in the budding yeast. Aft1 regulates iron homeostasis and is also involved in genome integrity having additional iron-independent functions. Using genome-wide expression and chromatin immunoprecipitation approaches, we found Rad9 to be recruited to 16% of the yeast genes, often related to cellular growth and metabolism, while affecting the transcription of ∼2% of the coding genome in the absence of exogenously induced DNA damage. Importantly, Rad9 is recruited to fragile genomic regions (transcriptionally active, GC rich, centromeres, meiotic recombination hotspots and retrotransposons) non-randomly and in an Aft1-dependent manner. Further analyses revealed substantial genome-wide parallels between Rad9 binding patterns to the genome and major activating histone marks, such as H3K36me, H3K79me and H3K4me. Thus, our findings suggest that Rad9 functions together with Aft1 on DNA damage-prone chromatin to facilitate genome surveillance, thereby ensuring rapid and effective response to possible DNA damage events.

Nhp6 facilitates Aft1 binding and Ssn6 recruitment, both essential for FRE2 transcriptional activation.

We found Nhp6a/b yeast HMG-box chromatin-associated architectural factors and Ssn6 (Cyc8) corepressor to be crucial transcriptional coactivators of FRE2 gene. FRE2 encoding a plasma membrane ferric reductase is induced by the iron-responsive, DNA-binding, transcriptional activator Aft1. We have shown that Nhp6 interacts directly with the Aft1 N-half, including the DNA-binding region, to facilitate Aft1 binding at FRE2 UAS. Ssn6 also interacts directly with the Aft1 N-half and is recruited on FRE2 promoter only in the presence of both Aft1 and Nhp6. This Nhp6/Ssn6 role in Aft1-mediated transcription is FRE2 promoter context specific, and both regulators are required for activation-dependent chromatin remodeling. Our results provide the first in vivo biochemical evidence for nonsequence-specific HMG-box protein-facilitated recruitment of a yeast gene-specific transactivator to its DNA target site and for Nhp6-mediated Ssn6 promoter recruitment. Ssn6 has an explicitly coactivating role on FRE2 promoter only upon induction. Therefore, transcriptional activation in response to iron availability involves multiple protein interactions between the Aft1 iron-responsive DNA-binding factor and global regulators such as Nhp6 and Ssn6.