Abstract
Iron and copper are redox active metals essential for life. In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level. In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization. To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene. Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex. In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation. Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression. Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins. Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an up-regulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation. Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation.
Highlights
Iron and copper are redox active metals essential for life
Targets for Aft1/Aft2 regulation include (i) genes which protein products are involved in high affinity reductive iron uptake such as the plasma membrane metalloreductases FRE1– 6 [8], the high affinity iron transport complex composed of the iron permease FTR1 [9] and the multicopper oxidase FET3 [10] and the ATX1 copper chaperone and CCC2 copper-transporting ATPase, (ii) genes encoding components of siderophore iron uptake systems, which include the transporters ARN1– 4 [11] and the cell wall manoproteins FIT1–3 [12], (iii) genes involved in the mobilization of iron from vacuolar stores, which include the Fet3-Ftr1 homologue complex formed by FET5 and FTH1 [13] and the Nramp family
To completely understand the function and regulation of the Rpd3 histone deacetylases (HDAC) in cells, it is crucial to elucidate the role of proteins that function within this large complex under different environmental conditions
Summary
Yeast Strains and Growth Conditions—Genotypes for the yeast strains used in this work are listed in Supplemental Materials Table S1. To test growth on low iron, cells were grown in synthetic media (SC) to exponential phase (A600 ϭ 1.0) and spotted in 10-fold serial dilutions starting at A600 ϭ 0.1 onto SC alone (complete) or SC containing 75–100 M BPS and 75–100 M BCS (low iron/copper), or 1 mM ferrozine (not shown). Wild type and mutant alleles of the CTI6 gene were cloned in phase into pBTM116 (pADH-LexA) plasmid, a gift from Ann Vojtek (University of Michigan), using SmaI and PstI restriction sites. DNA Microarray and RNA Blot Analysis—For microarray experiments wild type BY4741 and cti mutant cells were grown to exponential phase in liquid SC medium containing 150 M BPS and 150 M BCS. 10 g of total RNA from wild type and cti cells was labeled with Cy3 and Cy5 fluorescent dyes, respectively. For staining of yeast nuclei cells were incubated for 15 min with 10 g/ml 4Ј,6-diamidino-2-phenylindole
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