Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione

Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein

Characterization of MtnE, the fifth metallothionein member in Drosophila

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missin

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural ‘linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regu­latory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manne

Toxicity of Alzheimer's disease-associated Aβ peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability

Amyloid plaques consisting of aggregated Aβ peptide are a hallmark of Alzheimer's disease. Among the different forms of Aβ, the one of 42aa length (Aβ42) is most aggregation-prone and also the most neurotoxic. We find that eye-specific expression of human Aβ42 in Drosophila results in a degeneration of eye structures that progresses with age. Dietary supplements of zinc or copper ions exacerbate eye damage. Positive effects are seen with zinc/copper chelators, or with elevated expression of MTF-1, a transcription factor with a key role in metal homeostasis and detoxification, or with human or fly transgenes encoding metallothioneins, metal scavenger proteins. These results show that a tight control of zinc and copper availability can minimize cellular damage associated with Aβ42 expressio

Transcriptional repression by methylation: cooperativity between a CpG cluster in the promoter and remote CpG-rich regions

AbstractCytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA-VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation-free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PlGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PlGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PlGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlG

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro. It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis-trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domaincontaining splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcriptio

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BACKGROUND: Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. RESULTS: Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. CONCLUSION: Our studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility