The double zinc finger domain and adjacent accessory domain from the transcription factor loss of zinc sensing 1 (loz1) are necessary for DNA binding and zinc sensing

Abstract

The Loz1 transcription factor from Schizosaccharomyces pombe plays an essential role in zinc homeostasis by repressing target gene expression in zinc-replete cells. To determine how Loz1 function is regulated by zinc, we employed a genetic screen to isolate mutants with impaired zinc-dependent gene expression and analyzed Loz1 protein truncations to map a minimal zinc-responsive domain. In the screen, we isolated 36 new loz1 alleles. 27 of these alleles contained mutations resulting in the truncation of the Loz1 protein. The remaining nine alleles contained point mutations leading to an amino acid substitution within a C-terminal double zinc finger domain. Further analysis of two of these substitutions revealed that they disrupted Loz1 DNA activity in vitro. By analyzing Loz1 protein truncations, we found that the last 96 amino acids of Loz1 was the smallest region that was able to confer partial zinc-dependent repression in vivo. This 96-amino acid region contains the double zinc finger domain and an accessory domain that enhances DNA binding. These results were further supported by the findings that MtfA, a transcription factor from Aspergillus nidulans that contains a related double zinc finger, is unable to complement loz1Δ, whereas a chimera of MtfA containing the Loz1 accessory domain is able to complement loz1Δ. Together, our studies indicate that the double zinc finger domain and adjacent accessory domain preceding zinc finger 1 are necessary for DNA binding and zinc-dependent repression.

Keywords: Metal Homeostasis; Metallosensor; Transcription Repressor; Yeast Genetics; Zinc; Zinc Finger.

FIGURE 1.

Loz1 is regulated at a post-translational level by zinc. A, wild-type cells containing the empty vector or the integrated adh4-lacZ, loz1-lacZ, or zhf1-lacZ reporter constructs were grown in ZL-EMM supplemented with the indicated amount of zinc before β-galactosidase activity was measured. B, loz1Δ cells containing the empty vector, pL-loz1GFP, or pZ-loz1GFP were grown in ZL-EMM supplemented with 0, 50, 200, or 500 μm zinc. Total RNA was extracted and subjected to RNA blot analysis. Blots were probed for zrt1, adh4, and the loading control pgk1 (phosphoglycerate kinase 1). C, immunoblot analysis of crude protein extracts prepared from loz1Δ cells containing the indicated plasmids. Cells were grown in ZL-EMM supplemented with 0, 50, or 200 μm zinc. Immunoblots were probed with antibodies raised against GFP or the loading control actin (Act1). An arrow indicates the band that is specific to Loz1-GFP. Shown are the means from three independent experiments, and the error bars indicate ± S.D.

FIGURE 2.

Abnormal zinc homeostasis in antimycin A-resistant adh1Δ cells. A, wild-type, adh1Δ, adh1Δ loz1-1, adh1Δ loz1-4, adh1Δ loz1-8, and adh1Δ loz1-9 cells were grown overnight in YES medium before cells were spotted in 10-fold serial dilutions onto YES medium without or with (+AA) a 10 μg/μl antimycin A supplement. Plates were incubated for 3 days at 31 °C before photography. B, wild-type cells expressing the empty vector (Vector) or adh4-lacZ reporter (WT) or adh1Δ cells containing the indicated loz1 allele and the adh4-lacZ reporter were grown in ZL-EMM with or without a 200 or 500 μm zinc supplement. Cells were harvested, and β-galactosidase activity was measured using standard procedures. C, wild-type cells or haploid strains containing the indicated loz1 alleles or these strains with the plasmid pL-loz1GFP were grown overnight in YES medium. Total RNA was extracted and subjected to RNA blot analysis. Error bars, S.E.

FIGURE 3.

A schematic diagram illustrating amino acid substitutions present in different Loz1 alleles. Cysteine and histidine residues predicted to coordinate zinc are shown in boldface type, and the original loz1-1 mutation is shown in boldface and italic type.

FIGURE 4.

S489F and M513I substitutions affect Loz1 DNA binding in vitro. A, DNA sequence of the wild-type and mutated oligonucleotide probes. The loz1 binding site is boxed. B, SDS-PAGE analysis of the purified Trx-histidine tag (vector) and recombinant proteins TH-loz1, TH-loz1 C470G, TH-loz1 S489F, TH-loz1 M513I, or TH-loz1 ZF. Proteins were visualized by staining with Coomassie Blue. The sizes of the molecular mass markers are shown in kDa on the left. C, representative EMSA using 40 μmol of ³²P-labeled double-stranded oligonucleotide and 200 ng of the indicated purified recombinant protein. For competition studies, 0, 50×, 200×, or 500× of the WT unlabeled oligonucleotide (Comp Inhibitor), or mutant oligonucleotide (Non-Comp Inhibitor) was added to the reactions.

FIGURE 5.

Mapping a minimal zinc-responsive domain. A, schematic diagram of pL-Loz1GFP truncations. Shown are a C-terminal GFP tag, the double zinc finger domain (numbered black boxes), regions conserved in Loz1 homologs from other Schizosaccharomyces sp. (striped boxes), and introduced nuclear localization signal. The amino acids included in each truncation are also indicated. B and C, loz1Δ cells expressing the constructs shown in A were grown in ZL-EMM with or without a 200 μm zinc supplement. Cells were harvested, and crude protein extracts were prepared for immunoblot analysis (B), or total RNA was extracted for RNA blot analysis (C). D, loz1Δ cells co-expressing the constructs shown in A and reporter gene pTN-zrt1-lacZ (zrt1-lacZ) or empty vector pTN-lacZ (vector) were grown as described in B before β-galactosidase activity was measured by standard procedures. Error bars, S.E.

FIGURE 6.

A minimal domain containing the last 96 amino acids of Loz1 is sufficient to confer zinc-dependent repression of adh4. A, schematic diagram of pZ-loz1GFP truncations. See the legend to Fig. 5A for details. B and C, loz1Δ cells expressing the constructs shown in A were grown in ZL-EMM with or without a 200 μm zinc supplement. Cells were harvested, and total RNA was extracted and subjected to RNA blot analysis (B), or crude protein extracts prepared for immunoblot analysis (C).

FIGURE 7.

pZ-MtfAGFP is regulated by zinc in loz1Δ cells. A, an alignment of the double zinc finger domains from Loz1 and MtfA. B, schematic diagram of Loz1/MtfA chimeras. Loz1 sequences are shown in light gray, and MtfA sequences are shown in dark gray. Numbers represent amino acid number in the respective proteins. C and D, loz1Δ cells expressing the constructs shown in B were grown in ZL-EMM with or without a 200 μm zinc supplement. Cells were harvested, and total RNA was extracted for RNA blot analysis (C), or crude protein extracts were prepared for immunoblot analysis (D).