Abstract
Transition metals, like iron and copper, are essential for life but, in excess, they can be toxic for cells. Therefore, their uptake and intracellular distribution are tightly controlled and regulated by cells in order to achieve homeostasis. Eukaryotic organisms, from yeasts to plants and animals, have developed complex mechanisms of metal homeostasis which display a significant conservation. In the present study, we look at the iron and copper homeostasis mechanism of Saccharomyces cerevisiae, a model organism for physiological and genetic studies. We examine this mechanism at the transcriptional level, using three reference genes: FRE1, FRE2 and CTR1. FRE1 and FRE2 encode two plasma membrane metal reductases necessary for iron and copper uptake. Both genes are transcriptionally induced in the absence of iron by the iron-responsive DNA-binding activator Aft1, with FRE1 being additionally induced in the absence of copper by the copper-responsive DNA-binding activator Mac1. CTR1 encode ...
Transition metals, like iron and copper, are essential for life but, in excess, they can be toxic for cells. Therefore, their uptake and intracellular distribution are tightly controlled and regulated by cells in order to achieve homeostasis. Eukaryotic organisms, from yeasts to plants and animals, have developed complex mechanisms of metal homeostasis which display a significant conservation. In the present study, we look at the iron and copper homeostasis mechanism of Saccharomyces cerevisiae, a model organism for physiological and genetic studies. We examine this mechanism at the transcriptional level, using three reference genes: FRE1, FRE2 and CTR1. FRE1 and FRE2 encode two plasma membrane metal reductases necessary for iron and copper uptake. Both genes are transcriptionally induced in the absence of iron by the iron-responsive DNA-binding activator Aft1, with FRE1 being additionally induced in the absence of copper by the copper-responsive DNA-binding activator Mac1. CTR1 encodes the main plasma membrane copper transporter and is also transcriptionally induced in the absence of copper by Mac1. We investigated the mechanism of Aft1-mediated transcriptional activation, using FRE2 gene as a model since FRE2 transcription solely depends on Aft1. We found that Nhp6a/b yeast HMG-box chromatin-associated architectural factors and Ssn6 (Cyc8) corepressor are crucial coactivators of FRE2 transcription. 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, playing a critical explicitly positive role in transcriptional activation. The Nhp6/Ssn6 role in Aft1-mediated transcription is specific for FRE2 promoter context and both regulators are required on this promoter 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. Therefore, in Saccharomyces cerevisiae, transcriptional activation in response to iron availability involves multiple protein interactions between the Aft1 iron-responsive DNA-binding factor and global transcriptional coregulators such as Nhp6 and Ssn6. We also examined the function of Mac1 copper-responsive DNA-binding activator. Mac1 C-terminal activation region includes two functionally distinct cysteine-rich repeats, termed REPI and REPII respectively. It has been shown that REPI is responsible for the copper-dependent regulation of factor’s activity. We found that REPII modulates the efficiency of Mac1 binding to DNA. In particular, we showed in vitro that REPII negatively affects Mac1 binding on CTR1 promoter, an effect that is compromised in vivo by the fact that Mac1 binds as a dimmer on two consecutive promoter target sites. This REPII domain effect implies that Mac1 function could be altered in vivo by structural modulations affecting its binding properties and possibly resulting from independent molecular interactions between this domain and other proteins or metals. Finally, we revealed a new repressive role of Aft1 in the transcription of the Mac1-dependent CTR1 gene. This role is based on the indirect recruitment and not direct binding of Aft1 on CTR1 promoter and probably implies an Aft1-Mac1 interaction. The latter is not unlikely given the fact that in yeast a link between iron and copper metabolism exists, contributing to cellular homeostasis of the two metals.
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