Dataset Information

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster.

ABSTRACT: Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence (547)CNCTNCKCDQTKSCHGGDC(565) are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Delta strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu(4)-S(6)] cage structure, characterized by a core of trigonally S(3) coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu(4)-L(6) (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).

SUBMITTER: Chen X

PROVIDER: S-EPMC2396432 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster.

Chen Xiaohua X Hua Haiqing H Balamurugan Kuppusamy K Kong Xiangming X Zhang Limei L George Graham N GN Georgiev Oleg O Schaffner Walter W Giedroc David P DP

Nucleic acids research 20080413 9

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence (547)CNCTNCKCDQTKSCHGGDC(565) are significantly or completely impaired in their ability to protect flies f ...[more]

PMID: 18411209

Similar Datasets

Project description:MTF1 is a highly conserved metal-binding transcription factor in eukaryotes. MTF1 binds to DNA sequence motifs, termed metal response elements (MREs) to induce the expression of genes involved in metal and oxidative stress homeostasis. MTF1 is responsive to both metal excess and deprivation, and can also protect cells from oxidative and hypoxic stresses. Disruption of metal homeostasis leads to the development of several pathological states. Despite its roles in these processes , MTF1 has been shown to be required for developmental processes such as embryonic liver formation. In this study, we used multiple strategies to understand the mechanism by which MTF1 functions in skeletal muscle differentiation and to determine the role cellular copper (Cu) status plays in this process. We provide the functional relationships between MTF1, Cu, myogenic gene promoters, and MyoD, an specific component of the myogenic transcriptional machinery in differentiating primary myoblasts derived from mouse satellite cells. We found that MTF1 is induced and translocated to the nucleus upon initiation of myogenesis. Consistent with previous studies from our laboratory , addition of non-toxic concentrations of Cu promote myogenesis and enhanced MTF1 expression. CRISPR/Cas9-mediated depletion of MTF1 demonstrated that MTF1 is essential for proper development of skeletal muscle, as partial Mtf1 knockdown leads to apoptosis to differentiating myoblasts. MTF1 was also found to bind Cu at a carboxy-terminal tetra-cysteine cluster, which may contribute to the mobilization of Cu to the nucleus during myogenesis. ChIP-seq and ChIP-qPCR analyses showed that MTF1 binds at the promoter regions of myogenic genes as part of a complex with MyoD, the master transcriptional regulator of the myogenic lineage. These results have the potential to initiate a new area of research in MTF1 function and in the regulation of myogenesis. Furthermore, these studies set the basis to understand the role of Cu at the transcriptional level, affects growth and development and will contribute to the largely unexplored are of muscular phenotypes observed in human pathologies associated to Cu misbalance, such as Menkes’ and Wilson’s diseases.

Project description:Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Dataset Information

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster.

Publications

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets