Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. This study reports the generation of a metallothionein gene family knockout in Drosophila by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, different metallothioneins were found to be expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's 'copper cells.' The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, it is concluded that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. It is concluded that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop (Egli, 2006a).
The genomic arrangement of the metallothionein genes made the generation of a quadruple knockout a difficult endeavor. All four genes are located on the same chromosome, but they are interspersed with many other genes, which precluded the removal of a large segment. Rather, the genes had to be mutated one by one by means of targeted gene disruption. Drosophila metallothioneins have an important role in copper homeostasis, as well as in the detoxification of cadmium. While cadmium toxicity is often caused by industrial pollution, copper is an essential trace element, and its homeostasis is physiologically important. The copper tolerance of wild-type flies is truly remarkable. In metallothionein mutants, a slight increase in copper levels, whether from a constant exposure or a transient copper shock, results either in larval death or in a shortened life span. With increased sensitivity to copper and cadmium and, to a lesser extent, to zinc, metallothionein mutants mirror most but not all aspects of the Drosophila MTF-1 mutant phenotype. Flies lacking MTF-1 are more sensitive to zinc, silver, and mercury than metallothionein mutants. This difference can best be explained by MTF-1-dependent activation of genes involved in metal homeostasis other than the metallothioneins. For example, MTF-1 induces the expression of a putative zinc exporter (CG3994) under conditions of zinc excess, preventing zinc overload of the cell (Yepiskoposyan, unpublished, reported in Egli, 2006). The sensitivity of MTF-1 mutants to silver might be due to low expression levels of the copper importer Ctr1B, another MTF-1 target gene. Silver exerts its toxicity by competing with copper, and competition is expected to be more severe in MTF-1 mutants, which have lower copper levels than wild-type flies (H. Yepiskoposyan and K. Balamarugan, unpublished data, reported in Egli, 2006a). Besides copper and cadmium, mercury, silver, and zinc induce metallothionein transcription, but these metals are not more toxic to metallothionein mutants than to the wild type. Apparently, metallothioneins are not able to protect against all compounds that induce their synthesis (Egli, 2006a).
Wild-type flies can develop at a copper concentration at least 200-fold higher (1 mM) than the normal copper content in food (5 µM) without survival to adulthood being affected. Indeed for most organisms, copper is a relatively benign trace element, thanks to sophisticated transport and detoxification systems. However, it can be toxic under special circumstances, such as in association with genetic defects of copper homeostasis or upon environmental accumulation, notably in vineyards where it is used as an antifungal agent. Metallothioneins protect the cells from toxic effects of copper by binding and sequestering the metal inside the protein. This metallothionein-copper complex can be conveniently observed in vivo as an orange copper luminescence. The reduction of copper cell luminescence in qMtn mutants suggests that copper is now solvent accessible and able to damage the cell, possibly via the generation of ROS and/or ectopic binding to protein sulfhydryl groups. Formation of ROS by copper is often proposed as the major mechanism of copper toxicity. The sensitivity of SOD1 mutant flies to high levels of copper is in agreement with such a scenario. In the Long-Evans cinnamon rat strain, a model system for a human copper homeostasis disorder, Wilson's disease, copper accumulation in the liver induces ROS production, which results in lipid peroxidation, impaired mitochondrial function, and increased DNA damage. However, under physiological conditions, copper also has a role in antioxidant defense as an essential component of the Cu, Zn-SOD. Low dietary copper levels were found to impair the catalytic function of Drosophila Cu,Zn-SOD, similar to what has been observed with mammals, and such flies display a dramatically shortened life span. The effect of copper concentration on life span apparently follows a U-shape curve, since both copper starvation and elevated concentrations shorten the life span. In both extremes, a likely cause of the premature death of adult flies is the accumulation of ROS-mediated damage. Under conditions of copper load, the shortened life span is particularly evident for metallothionein mutant males that normally express metallothionein mRNA at higher levels than females. Thus, it is proposed that adult males depend more on the protective effects of metallothioneins than females (Egli, 2006a).
All four Drosophila metallothioneins and even a transgene with a synthetic minipromoter composed merely of four tandem copies of MREs are expressed in a very similar tissue- and cell-type-specific pattern in the intestine, which reflects the known accumulation of copper or cadmium. For example, metallothioneins are expressed in the 'copper cells,' which are well known for their peculiar ability to accumulate large amounts of copper. Cadmium has been shown to accumulate in the anterior midgut, in the 'iron cell' region, and in the posterior midgut in a pattern highly similar to the metallothionein induction reported in this study. Since MTF-1 is the only transcription factor known to bind directly to MREs but ubiquitous overexpression of MTF-1 does not change the expression pattern of metallothioneins, it is concluded that metallothionein expression is governed by metal distribution. Thus, a metallothionein promoter driving a fluorescent protein reporter can be used as a highly sensitive and semiquantitative biomarker for cellular metal transport. This may help to investigate the function of putative metal transporters in vivo, as has been demonstrated in a study of the copper exporter ATP7 (Egli, 2006a).
Metallothioneins, together with glutathione, an antioxidant with relatively nonspecific metal binding ability, constitute the first line of defense against the toxic effects of heavy metals in both mammals and insects. As ingested metals first reach the gut cells, metallothioneins in the gut probably serve to trap toxic metals and limit their distribution throughout the body. An interesting example of such trapping of toxic metal is the zinc treatment of Wilson's disease patients, who suffer from copper accumulation in the liver. Zinc treatment induces metallothionein synthesis in the intestine; due to the metallothionein's high affinity to copper, the latter is trapped within intestinal cells and eventually excreted (Egli, 2006a).
A remarkable finding in the present study is the autoregulation of metallothionein expression. Metallothionein promoters used as a reporter of MTF-1 activity are more active in metallothionein mutants. This is not due to a higher copper content, since total body copper does not differ between wild-type and metallothionein mutants. Rather, metallothioneins can inhibit their own expression by inhibiting MTF-1 function via the binding of free metal, which otherwise would directly or indirectly activate MTF-1. The nature of the signal that activates MTF-1 in vivo is still not established. While zinc can directly bind to MTF-1, copper and cadmium interfere with DNA binding of MTF-1 in vitro. They are thus thought to activate MTF-1 indirectly and were indeed shown to do so in a cell-free model system by displacement of zinc from metallothioneins, which in turn binds to MTF-1. The present study, however, shows that metal-loaded metallothioneins are not essential in vivo for the activation of MTF-1 by any of the heavy metals tested. It is therefore likely that zinc can be displaced from other cellular pools. Furthermore, as shown with mammals, MTF-1 regulation in vivo occurs at multiple levels that include nucleocytoplasmic transport, protein phosphorylation, and a conspicuous cysteine cluster toward the C terminus of MTF-1, which might act as a metal sensor. In conclusion, the present study does not support the concept of metallothioneins playing an important role in copper uptake and intracellular distribution but firmly establishes their importance for coping with metal fluctuations, notably for copper homeostasis and cadmium detoxification (Egli, 2006a).
All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion were determined. Furthermore, the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant was analyzed. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in this study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification (Yepiskoposyan, 2006).
The imbalance of metals due to genetic disorders or environmental pollution is a worldwide problem associated with adverse health effects. This study unveils a number of genes and regulatory mechanisms involved in maintaining heavy metal homeostasis. The metals tested in this study, cadmium, copper and zinc, induce a wide range of responses, including induction of genes coding for metallothioneins, transporters, components of the glutathione-mediated detoxification pathway, antimicrobial peptides, ubiquitin-conjugating enzymes, heat shock proteins and cytochrome P450 enzymes. The universal induction of metallothioneins by all three metals and the downregulation by copper depletion is in line with their metal scavenger function and protective role against metal toxicity. However, there are differences in the strength of the response of individual metallothioneins: MtnA induction is more prominent in copper, whereas MtnB and MtnC are more strongly induced by zinc and cadmium. Indeed, a recent study suggests that individual metallothioneins preferentially defend against specific metals, with MtnA playing a major role in handling copper excess, MtnB during cadmium and zinc excess, while MtnC and MtnD play only minor roles in defending against these three metals. In absolute terms, expression of MtnA is higher than that of other metallothioneins, with an impressive basal level and consequently a lesser fold-induction, suggesting MtnA to be the major metal scavenger at Drosophila larval stage (Yepiskoposyan, 2006).
Another essential system for metal ion homeostasis is metal export and import. Although transcriptional control is only one of several regulatory processes, numerous changes could be followed in characterized, as well as putative, Drosophila metal transporters in the microarray experiment. To provide adequate intracellular copper concentrations, the transcripts of the high-affinity copper importer Ctr1B are strongly induced by copper depletion; conversely, they are downregulated at high Cu concentration in order to reduce copper uptake. Two zinc transporter genes (ZnT35C and ZnT63C), both encoding ZnT family members involved in zinc efflux, as well as two ATPase transporters (CG6263 and CG18419) presumably involved in cation and lipid transport, were upregulated by Zn and Cd treatments. Of note, several ubiquitin-conjugating enzymes (Ubc) were induced by zinc. In this context it is worth mentioning that the yeast zinc importer Zrt1p is ubiquitinated and subsequently degraded upon zinc load. The upregulation of Ubc might also help to degrade misfolded proteins which are probably formed upon metal load (Yepiskoposyan, 2006).
The results indicate that the larval transcriptome responses to zinc and cadmium share several features. Even though they exert different biological effects, the chemical properties of Zn and Cd are similar, thus part of the regulatory mechanisms to cope with excess zinc are also functional in cadmium stress. Another example of the similarity between the zinc and cadmium responses is the specific and strong induction of two members of the glutathione S-transferase Delta class genes. There are more than 40 GST genes in Drosophila grouped into six classes. Three GSTs, CG17524, CG6776 and CG1681 with homology to epsilon, omega and theta class GSTs, respectively, are preferentially upregulated by zinc. GSTs catalyze the conjugation of glutathione (GSH) to a variety of harmful compounds. The resulting glutathione conjugates (GS-X) can then be sequestered by the vacuole or excreted from the cell by ATP-dependent GS-X pumps. The details of this process vary with different compounds and in different species. In S.cerevisiae, e.g. cadmium ions are conjugated to GSH and the Cd·GS2 conjugate is transported to the vacuole by YCF1, an ATP-binding cassette transporter. It is plausible that the GSTs induced in these studies catalyze the conjugation of Cd and/or Zn to GSH, which eventually will be removed from the cytoplasm. Alternatively, or in addition to conjugating metals, they might conjugate ROS which can occur as byproducts of heavy metal metabolism. Interestingly, a specific ATP-binding cassette transporter CG10505, that shares 26% identity to YCF1, is strongly upregulated by all tested metals. It is tempting to speculate that CG10505 acts in a similar fashion to its yeast counterpart in exporting the metal-GSH conjugates. Nevertheless, the metal specificity of Drosophila CG10505 differs from that of YCF1 because the former contributes to elevated resistance to zinc and copper, rather than to cadmium. Another enzyme involved in GSH conjugate metabolism and excretion, gamma-glutamyl transferase, which is an integral part of the gamma-glutamyl cycle involving the degradation and neo-synthesis of GSH was also induced by zinc in Drosophila. Taken together, these data suggest that GSH conjugation plays a substantial role in the response of Drosophila to metal stress. Support for such a scenario comes from a recent study in the mouse showing the existence of two branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, and the other via glutathione. In agreement with this concept the data indicate that when the MTF-1-dependent response is abrogated, the glutathione-associated pathway is upregulated to compensate for this compromised genetic background (Yepiskoposyan, 2006).
The knockout of the Drosophila MTF-1 gene altered the expression of more than 50 genes in the transcriptome assay. However, there is no obvious phenotype unless the animal is subjected to metal stress: copper, zinc, cadmium and mercury load, or copper starvation. The copper importer Ctr1B and metallothionein genes are well-established targets of Drosophila MTF-1. The Ctr1B mutant is sensitive to changes in copper concentration and the KO of the Drosophila metallothionein family is sensitive to cadmium and copper load; however, these mutants hardly show any phenotype upon zinc challenge. A major role is suggested in zinc detoxification for several other genes (Yepiskoposyan, 2006).
Next to the ABC transporter CG10505, zinc exporter Zn35C contributes to zinc homeostasis in that the truncation mutant ZnT35Cmut is extremely sensitive even to a mild zinc load of 2 mM, while wild-type tolerates up to 14 mM zinc excess. Also, an overexpression of this transporter confers resistance to zinc and can partially rescue the zinc sensitivity of MTF-1 knockout flies. Given the strong zinc sensitivity phenotype of the mutant, it is proposed that ZnT35C is the major transporter of cellular zinc, similar to the mammalian zinc exporter ZnT1. It remains to be seen whether the other zinc exporter-like genes annotated in the Drosophila genome transport zinc from the cytoplasm to vesicular compartments, as do several of the mouse zinc transporters, such as ZnT2, 3, 5 and 7. Drosophila ZnT35C localizes to the plasma membrane both at normal and elevated zinc concentrations; besides, mutant flies have a high zinc content, in support of a role in zinc excretion. It is noted that the mouse zinc exporter ZnT1 was also found to be induced both by cadmium and zinc treatment in an MTF-1-dependent fashion similar to ZnT35C. Thus not only the regulation of metallothioneins but also that of zinc transporters is conserved between insects and mammals. In other cases, metal responses of Drosophila and mammals diverge: the fly orthologs of the mouse cEBP alpha, ndrg1 or klf4 were unchanged in the microarray experiments (Yepiskoposyan, 2006).
An unexpected result of the present study is the induction of ferritin heavy and light chain homologue genes by heavy metals. Ferritin is well known to detoxify, store and transport iron. It was also shown to bind other metal ions and has been speculated to function as a general metal detoxicant in mammals. The results suggest an induction of Drosophila ferritins by metals other than iron, notably zinc. The latter induction depends on MTF-1, based on the following findings: (1) the presence of multiple MREs, (2) the loss of zinc induction in the MTF-1 mutant, and (3) elevated ferritin transcripts upon MTF-1 overexpression. In contrast, iron inducibility must be regulated independently since it is not altered by the absence of MTF-1. In line with different signaling pathways responding to iron load versus excess of zinc, copper or cadmium, it is noted that the classical MTF-1 dependent MtnA gene is not inducible by iron. Yet these pathways apparently overlap in the ferritin genes (Yepiskoposyan, 2006).
Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA, MtnB, MtnC, MtnD, all of which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. This study shows, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the individual metallothionein mutants, the most prominent distinction between them was that MtnA-defective flies were the most sensitive to copper load, while MtnB-defective flies were the most sensitive to cadmium. Using various reporter gene constructs and mRNA quantification, the MtnA promoter is shown to be preferentially induced by copper, while the MtnB promoter is preferentially induced by cadmium. Such a metal preference is also observed at the protein level as the stoichiometric, spectrometric and spectroscopic features of the copper and cadmium complexes with MtnA and MtnB correlate well with a greater stability of copper-MtnA and cadmium-MtnB. Finally, MtnC and MtnD, both of which are very similar to MtnB, display lower copper and cadmium binding capabilities compared to either MtnA or MtnB. In accordance with these binding studies, Drosophila mutants of MtnC or MtnD have a near wild type level of resistance against copper or cadmium load. Furthermore, eye-specific over-expression of MtnA and MtnB, but not of MtnC or MtnD, can rescue a 'rough eye' phenotype caused by copper load in the eye. Taken together, while the exact roles of MtnC and MtnD remain to be determined, the preferential protection against copper and cadmium toxicity by MtnA and MtnB, respectively, are the result of a combination of promoter preference and metal binding (Egli, 2006b).
Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. RNA interference (RNAi) was used in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. The robust response of the metallothionein genes to heavy metal was used as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, it was found that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a 'checkpoint complex' for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, it was found that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation (Marr, 2006).
There are four known metallothionein genes in Drosophila: MtnA, MtnB, MtnC, and MtnD. Of these, the best characterized is the MtnA gene, which produces a transcript of ~600 bases in length, bearing one intron. All of the regulatory elements required for robust response to heavy metals, including copper, lie within 500 bp of the transcription start site. The gene is controlled by a single activator, metal response element-binding transcription factor 1 (MTF-1), which binds two adjacent metal response elements (MRE) 50 bp upstream of the TATA-box (Zhang, 2001). Quantitative PCR (qPCR) analysis of the endogenous gene in Drosophila S2 cells shows that the gene is highly induced (~250-fold) after a short exposure to copper. The total amount of stable MtnA mRNA approximates the level of the abundant transcript for the ribosomal subunit Rp49. Primer extension analysis confirms that transcriptional activation of the endogenous MtnA gene originates from a unique start site overlapping the core promoter. The transcript accumulates linearly for ~12 h, thus measurements in this time window likely reflect relative levels of transcription of the MtnA gene. Importantly, induction at the endogenous chromosomal locus is easily assayed in order to measure physiologically relevant transcriptional activation in the context of native chromatin. Taken together, these properties establish the endogenous MtnA gene as a useful model for studying transcriptional mechanisms governing an inducible gene (Marr, 2006).
Using chromatin immunoprecipitation (ChIP), it was found that the sequence-specific DNA-binding protein MTF-1 is specifically recruited to the MtnA promoter region in response to copper. Curiously, the ChIP of the promoter region was compared to a region 1 kb downstream, a significant amount of MTF-1 was found to be present on the promoter even in the absence of added copper. Under these conditions, little transcription is detected from this gene. As a preliminary experiment to investigate a potential functional interaction between TFIID and MED, it was first asked whether the two complexes are both recruited in a signal-dependent manner to the MtnA gene. Using ChIP, it was found that both TBP and the TAFs are efficiently recruited to the promoter region in response to copper. In addition, the MED17, MED24, MED26, and MED27 subunits of MED are all recruited to the promoter region in response to copper treatment. Consistent with the high level of induction, RNAPII occupancy at the MtnA promoter is also increased in response to heavy metal treatment. Thus, both core coactivator complexes and RNAPII are efficiently recruited to the promoter region upon induction and resultant binding of MTF-1 to the MREs (Marr, 2006).
Because the ChIP assay is limited to measuring response in a heterogeneous population of cells, a transgenic model system was extablished in Drosophila S2 cells in order to visualize the response at the single-cell level. Such an approach has proved useful in understanding transcription factor dynamics in vivo. By selecting for stably transfected MtnA firefly luciferase reporters, a concatenated transgenic locus was generated in a clonal line of S2 cells. The transgenic locus was assayed for dependence on copper using a luciferase assay. Importantly, transcription initiates a unique site that maps to the correct start site of the MtnA core promoter. With this substantial increase in gene number (~2000) at the integrated transgenic locus, it should now be possible to visualize direct recruitment of specific transcription factors to the MtnA promoter within a single cell (Marr, 2006).
As expected, in the absence of heavy metal, MTF-1 is predominantly cytoplasmic; however, in agreement with ChIP data, some MTF-1 can be detected at the transgenic cluster even in the absence of a metal stimulus. Thus, antibody labeling of MTF-1 provides a useful marker for the subnuclear location of the transgene cluster in both induced and uninduced cells. Notably, the locus is not undergoing transcription (as detected by RNA FISH) in the absence of heavy metal induction despite the presence of some MTF-1 at the transgene cluster. Upon copper induction, MTF-1 vacates the cytoplasm and accumulates selectively at the transgenic locus. Under these same conditions, TBP is also actively recruited to this cluster. Consistent with not only TBP but holo-TFIID complex recruitment, it was found that TAF2 also accumulates at the transgene. Likewise MED components recruited to the transgene were detected using antibodies against MED26. As expected, RNAPII is recruited to the cluster in a copper-dependent manner consistent with the transcriptional induction of the transgene under these conditions. In contrast, TBP-related factor 1 (TRF1), a subunit known to be a key component of the RNA polymerase III core promoter recognition complex, is not recruited to the transgene. This negative control helps rule out the possibility that the tandemly reiterated transgene is simply nonspecifically attracting transcription factors (Marr, 2006).
Having established by two independent methods that both TFIID and MED complexes are recruited to the MtnA promoter in an activator-dependent manner, their role in potentiating transcriptional activation of the endogenous MtnA gene was investigated. The efficient technique of RNAi in Drosophila S2 cells was used to knock down expression of TFIID and MED subunits. In addition, the activator MTF-1 was knocked down to ascertain the extent of the activator’s role in induction. After treatment with copper, total RNA was purified from dsRNA treated and untreated S2 cells and then they were assayed by two independent methods. First, a primer extension analysis was used on equivalent amounts of total RNA. This assay revealed that an accurate transcription is detected from one distinct core promoter start site. Next, qPCR normalized to the Rp49 mRNA was used, to confirm that there is little or no global disturbance of RNAPII transcription (Marr, 2006).
Not surprisingly, depletion of MTF-1 severely reduced transcriptional activation from the MtnA promoter, confirming the central role of this activator. RNAi directed against TBP also had a dramatic inhibitory effect. The MtnA promoter is <10% as active when TBP levels are severely depleted. Surprisingly, knockdown of multiple TAFs had little apparent effect on the ability of MTF-1 to activate MtnA. Indeed, depletion of the TAFs actually stimulated (1.5- to 2-fold) production of RNA. With the exception of TAF11, a reduction of individual TAFs resulted in a remarkably uniform response. The reason for this uniformity became apparent when the stability of the TFIID complex was examined in the RNAi-treated cells. The overall stability of the holo-TFIID complex appears to be coupled to the stability of certain individual TAFs. In the most dramatic example, RNAi-targeted reduction of TAF4 leads to the concomitant loss of TAF1, TAF5, TAF6, and TAF9, as well as a detectable reduction in TBP. Interestingly, TAF2 and TAF11 are largely unaffected by depletion of TAF4. Similar results are observed for the other TAFs as well. When the transcript levels of the TAFs were measure after RNAi treatment, it is clear that the loss of stability occurs at the protein level, since the transcript levels for nontargeted TAFs are unaffected. For example, when TAF4 is targeted, only the TAF4 transcript is depleted (Marr, 2006).
In contrast to the TAFs, RNAi reduction of MED subunits gave striking but variable effects on the ability of MTF-1 to activate transcription from the MtnA promoter. Unlike TFIID, the response is far from uniform. For example, dsRNA directed against MED23 has little effect on induction of MtnA, while loss of MED17, the Drosophila SRB4 homolog, has a strong inhibitory effect. The lack of a uniform response in the MED RNAi led to a further investigation of the potential differential response upon depletion of MED subunits at related promoters activated by MTF-1. As discussed above, Drosophila has four metallothionein genes that respond to heavy metals. Three of these—MtnA, MtnB, and MtnD—are active in S2 cells. All three of these genes are specifically activated by the same factor, MTF-1. All three Mtn genes were examined in a single experiment using qPCR. First, it was confirmed that all three promoters, MtnA, MtnB, and MtnD, require MTF-1 for induction. Remarkably, distinct differential requirements were found for MED subunits depending on the promoter. For example, loss of MED13, a subunit of the larger MED complex (ARC-L) thought to play a repressive role in transcription, is not essential for MtnA induction. In contrast, MED13 was found to be important for both MtnB and MtnD activation by MTF-1. In contrast, the opposite specificity was seen with the MED26 subunit, a component of the smaller MED complex (CRSP), thought to play predominantly a coactivator role in transcription. Interestingly, MED26 is required for full induction of the MtnA promoter but is dispensable for MTF-1 activation of the MtnB and MtnD promoters. Thus, these experiments reveal a remarkable example of differential dependence on cofactor composition even though all three promoters tested use the same activator. Apparently, the precise role of individual MED subunits depends on the promoter context and structure, despite the absence of any evidence of direct binding of DNA by the MED complex (Marr, 2006).
To help rule out nonspecific effects on transcription such as a change in the concentration of free RNA polymerase, representative targets from TFIID and MED were tested in a transient transfection assay where the effect to a second promoter can be normalized. In these experiments, TAF4 and MED17 were chosed as representative targets, since TAF4 compromises much of the TFIID complex and MED 17 is likely a component of the core MED complex. The transient transfection data are largely consistent with the data generated at the endogenous locus and at the transgene (Marr, 2006).
The data presented above suggest that activation of the MtnA gene requires specific MED subunits, and at the same time the TAFs appear to be playing a potential negative regulatory role. Because it is clear that the TAFs are specifically recruited in S2 cells to the MtnA promoter in a copper-dependent manner by MTF-1, whether TFIID recruitment can occur in the absence of the MED complex was examined. To achieve this, RNAi directed against MED17 was used, which results in an almost complete loss of MED activity. Surprisingly, TFIID is still efficiently recruited to the MtnA gene. ChIP experiments confirmed that TBP and TAF2 are still actively (and likely directly) recruited to the endogenous MtnA gene by MTF-1 even when the gene is transcriptionally inactive as measured by qPCR analysis. The MtnA luciferase transgene system was used to investigate this relationship at the single-cell level. Without any RNAi, TBP, TAF2, and RNAPII were all recruited to the transgene. In agreement with the ChIP data above, even in the absence of MED activity, after MED17 depletion, TBP and TAF2 are nevertheless efficiently recruited to the transgene. In contrast, no RNAPII can be detected at the transgene consistent with the loss of transcription activation. Apparently, TFIID is recruited to the promoter, but the promoter is not active in supporting transcription. Importantly, recruitment of this 'inactive TFIID' is dependent on the activator MTF-1. In the absence of MTF-1, no TFIID or RNAPII is recruited to the transgene (Marr, 2006).
This perplexing result of recruiting an apparently 'inactive' TFIID prompted an examination of what happens when both TAFs and MEDs were depleted. Remarkably when both the TAFs and MED complex are depleted and 'removed' from the MtnA promoter, MTF-1-dependent activation of transcription is restored to ~95% the level of untreated cells, which is well above the inhibited level observed when the MEDs alone are depleted. In humans and Drosophila, TAFs can be subunits of other complexes such as TFTC and STAGA, so it is possible that the functional interaction analyzed is not TFIID-specific. To test this, specific subunits of these other complexes were targeted to determine if they would have a similar ability to rescue the MED knockdown. Unlike the TFIID subunits, RNAi against dAda2b, dGCN5, dSPT3, and dTRA1 was unable to rescue the loss of the MED subunits. These findings taken together suggest that most likely the functional relationship revealed by these experiments with the MtnA promoter, indeed, involve some regulatory transaction between TFIID and MED (Marr, 2006).
The requirement for coactivator complexes mediating transcriptional responses to activators has been well documented. However, by using an inducible Drosophila gene as a model system, a previously unknown functional interaction has been uncovered between two coactivator complexes, TFIID and MED. In the absence of TAFs, the cell responds inappropriately to a metal stimulus. The cell synthesizes 50%–200% more mRNA from the MtnA gene than it does in the presence of the TAFs. The data suggest that at this gene, TFIID is recruited in an inactive state, a state that impedes initiation of transcription. It is believed that this sets up a checkpoint early in the initiation process to meter the RNA synthesis. The MED complex must be recruited to get past this checkpoint. It is postulated that the MED complex likely modifies TFIID, converting it to an active state. This could be accomplished either through one of the known enzymatic activities of MED, phosphorylating (cdk8) or ubiquitylating (MED8) TFIID subunits, or through some, as yet undetected, chaperone-like function that remodels TFIID into an active conformation. Not surprisingly then, in the absence of MED subunits the cell cannot mount an appropriate response to environmental signals. In fact, depletions of many of the MED subunits lead to <20% of the normal amount of mRNA. Unlike the uniform response to depletion of TAFs, the response to depletion of MEDs is much less uniform. One possibility is that the MED complex is more functionally and structurally diverse than TFIID. Indeed, alternative subcomplexes of MED have been purified biochemically, whereas no such subcomplexes of TFIID have been reported (Marr, 2006).
By analysis of three different Mtn genes, all of which are dependent on the same single activator, it was found, surprisingly, that there is a differential requirement of specific MED subunits at the three Mtn promoters. This is taken as evidence that, depending on the precise arrangement of cis elements and promoter context, the same activator can require different mediator subunits or modules to transmit its signals to the basal apparatus (Marr, 2006).
Interestingly, the kinase module of the MED complex, previously linked with repression functions, is required for efficient activation at two of the promoters. This result, combined with the finding that at the MtnA promoter the TAFs have a repressive regulatory influence on transcription initiation, underscores the difficulty in assigning black and white functions to the coactivator complexes. It is likely that both TFIID and MED interpret multiple inputs from cellular signals and act either positively or negatively depending on the signals received as well as the specific promoter context. As such, the complexes may better be viewed as coregulators since they can play either a positive or negative role in the process of modulating gene expression. For example, only when both TFIID and MED are intact do Drosophila S2 cells produce the appropriate amounts of MtnA mRNA. In contrast, when either coactivator complex is disrupted, aberrant levels of transcription are seen. However, when both coactivator complexes are depleted, a significant level of metal inducible activation is actually restored. Presumably, in this 'stripped down' system, some portion of the remaining TBP pool can mediate transcription. Curiously, in the absence of TAFs but with a full complement of MEDs, there is also an aberrant level of transcription consistent with the notion that there is some finely tuned codependence between the TBP/TAF complex and the MED complex at this promoter (Marr, 2006).
The results also reinforce the notion that the activator is the primary determinant of the transcriptional response. The MTF-1 depletion experiments were the most detrimental to mRNA induction. In the absence of MTF-1, there is no detectable activation of the Mtn genes. In contrast, there is some residual transcription of MtnA even when either the MEDs or TBP are largely depleted from the Drosophila cells. This remaining activity could be due to incomplete depletion, or it could indicate alternative mechanisms of activation that are activator-dependent but can partially bypass the requirement for the coregulator complexes (Marr, 2006).
In the course of testing the requirement for TAFs in activated transcription, the codependent stability of the TFIID complex was discovered. Particularly striking is the finding that TAF4 depletion destabilizes most of the other TAFs and, to some extent, even TBP. Therefore, the TAF depletion experiments most likely reflect a loss of holo-TFIID rather than just the loss of individual subunits. It is worth noting that metazoan organisms contain multiple variants of TAF4: TAF4b in vertebrates and No-hitter in Drosophila. Both of these have been implicated in tissue-specific gene expression. It is conceivable that substitution of this keystone TAF can provide a mechanism to change the entire coregulator profile of TFIID (Marr, 2006).
One intriguing question this work raises is: Why would an activator recruit an inactive TFIID complex to the promoter? There are several previously described cases in which TFIID occupancy at a promoter does not strictly correlate with transcriptional activity. However, in most of these cases the genes being examined were either in a repressed or an unstimulated state. In contrast, the current studies were designed to specifically measure the role of coactivator complexes such as TFIID and MED in the context of an active gene MtnA upon metal stimulation. The ability to deplete MED activity under these conditions revealed the unexpected finding that although TFIID is dynamically recruited to the MtnA promoter, TFIID is mainly held in an 'inactive' state until the second cofactor complex, MED, is recruited. Perhaps this recruitment of an 'inactive' TFIID is a more common phenomenon that can only be detected in special circumstances and may represent a previously unappreciated control mechanism in transcription activation. If the activator first recruits TFIID, then subsequently recruits MED, and there is a requirement for additional factors to potentiate the secondary recruitment of coregulator assemblies, then this provides a potential checkpoint for fine-tuning the control of gene expression. Alternatively, since the cell invests a significant amount of energy in making a high level of transcript, requirement of continued stimulation (i.e., activator bound at the promoter) for mRNA production would provide the most economical use of resources (Marr, 2006).
Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. This study characterizes the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis (Zhang, 2001).
In Drosophila, the two genes encoding MTs, Mto and Mtn, have distinct but partially overlapping expression patterns, with Mto being primarily expressed in early embryogenesis and Mtn being expressed in late embryogenesis/adulthood. In addition, Mtn is expressed in hemocytes, possibly to regulate copper supply to hemocyanin. It was postulated that Drosophila Mto is important for copper homeostasis during embryogenesis, while Mtn, in particular due to its very strong expression in the gut, Malpighian tubules, and fat body, is thought to balance the toxic effects of copper and other metals, such as cadmium and mercury. Thus, Mtn seems to play a role that in the snail is delegated to two different MTs, one active in hemocytes, the other one in the gut (Zhang, 2001 and references therein).
This study reports the isolation of Drosophila dMTF-1. The expression pattern of dMTF-1 is compatible with a role in MT gene activation/heavy metal detoxification and homeostasis. Furthermore, in transfection experiments both in mammalian cells and Drosophila S2 cells, dMTF-1 is able to confer strong cadmium induction to MT promoters of both Drosophila and mammalian origins. As in mammals, metal induction seems to involve characteristic MRE sequence motifs. In support of this notion, the promoters of both Drosophila MT genes, Mto and Mtn, contain multiple sequence motifs resembling mammalian MREs. Their role in metal response was further corroborated by a synthetic minipromoter where the four MREs from the Mto gene were juxtaposed in a tandem array and conferred strong heavy metal response to the reporter gene. In spite of some striking similarities, it should also be pointed out that significant differences exist between dMTF-1 and vertebrate MTF-1. First of all, the strongly conserved zinc finger domain notwithstanding, there is limited, albeit significant, protein sequence similarity between vertebrate MTF-1 and dMTF-1. Specifically, the three hallmark activation domains of mammalian MTF-1, namely, acidic, proline-rich, and serine/threonine-rich, do not have obvious counterparts in Drosophila. The functional equivalents of one or all of them remain to be identified. Secondly, dMTF-1 is quite forgiving towards low pH (6.0 to 6.5) while mammalian MTF-1 loses its DNA-binding capacity under these conditions. This property may explain why mammalian MTF-1 was inactive in transfected Drosophila Schneider cells, which are grown at pH 6.5. Conversely, dMTF-1 performed well in mammalian cells grown in Dulbecco modified Eagle medium at pH 7.4. A third difference may concern the tissue distribution: while mammalian MTF-1 is expressed in all tissues analyzed so far, dMTF-1 is expressed in a few tissues, notably gut and fat body, although further experiments will have to probe into this issue. This is compatible with a major role in the activation of MT and possibly other toxicity/cell stress-related genes. Finally, there is a difference between mammals and Drosophila concerning heavy metal metabolism. In mammalian cells, MTF-1 mediates MT gene transcription primarily in response to zinc and cadmium and, to a lesser extent, to copper and nickel. Surprisingly dMTF-1 was unable to induce transcription in response to zinc at concentrations which readily induce transcription in mammalian cells. However, at 2 mM zinc, a concentration that is lethal to mammalian cells, dMTF-1 activated transcription from the Mtn promoter. This finding is in agreement with earlier heavy metal studies of the fly: of all metals tested for Mto and Mtn transcription, Cd2+, Cu2+, and Hg2+ were found to be more efficient than Zn2+, which was required at concentrations of at least 1 mM. The high concentrations of zinc (and copper) tolerated by Schneider cells may reflect inefficient uptake or, more likely, particularly efficient export of these two essential heavy metals. In fact, a search of the Drosophila genome revealed six homologs of mammalian zinc transporters. Whatever the reason for the poor response to zinc at submillimolar concentrations, it cannot be attributed to a species difference between MTF-1 factors themselves, since dMTF-1, upon transfection into mouse cells, mediates zinc-responsive transcription like hMTF-1. Further evidence for functional similarity is that dMTF-1, like mammalian MTF-1, requires elevated zinc concentrations for DNA binding in vitro, while copper and cadmium interfere with zinc rather than replacing it. At first sight, cadmium and copper induction of a transcription factor that strictly requires zinc appears paradoxical. However, it is possible to induce MTF-1-dependent transcription in vitro by cadmium and copper. Activation is dependent on the presence of MT, which has a particularly high affinity for these two metals and upon binding to them releases zinc on behalf of MTF-1. Another question concerns the regulation of the dMTF-1 gene itself. In mammals, the MTF-1 promoter does not contain MREs and transcripts are marginally, if at all, elevated by heavy metal treatment. Although MTF-1 transcripts have not been quantitated in Drosophila, it is noted that the MTF-1 promoter, like the mammalian one, lacks MRE-type sequence motifs, at least within a 2-kb segment around the site of transcription initiation (Zhang, 2001).
Perhaps the best evidence for an important role of dMTF-1 in MT gene regulation is provided by studies with RNAi. Without RNAi and in the absence of any Drosophila mutation in the dMTF-1 gene, one might have argued that the observed effect in transfected cells was due to a nonphysiological stimulation by a heterologous zinc finger factor. Ectopic activation effects have been observed before, when related family members of mammalian transcription factors could replace each other in transient transfection/overexpression conditions. This scenario is ruled out by the finding that dMTF-1 dsRNA completely blocked the activation of the Mtn promoter without any cotransfected MTF-1 expression plasmid, i.e., under conditions that relied entirely on the host cell's transcription factors. Furthermore, unlike many other transcription factors, MTF-1 is a unique protein without related family members in Drosophila and, apparently, in mammals. Taken together, the RNAi experiments corroborated the important role of dMTF-1 for MT transcription and thus heavy metal homeostasis (Zhang, 2001).
It certainly will be of interest to study the effect of loss of dMTF-1 in vivo, for example, by screening deletion mutants at the MTF-1 locus or by the newly introduced techniques of inheritable RNAi or targeted gene disruption in Drosophila. As mentioned, a targeted disruption of MTF-1 in the mouse results in embryonic lethality due to liver decay on embryonic days 13 to 14. It is noted that disruption of either of two other stress-associated transcription factors, namely, c-Jun and NF-kappaB/RelA, also results in embryonic death from liver decay at about the same stage of mouse embryogenesis. In this context it is of interest that AP-1 sites, which bind jun-fos heterodimers, are present in the promoters of both Drosophila MTs, pointing to a possible interconnection of MTF-1 and AP-1 in the cellular stress response of insects. Thanks to the availability of jun and fos, mutants and the power of Drosophila genetics, these and other aspects of cellular stress response are amenable to analysis (Zhang, 2001).
The zinc finger transcription factor MTF-1 is conserved from insects to vertebrates. Its major role in both organisms is to control the transcription of genes involved in the homeostasis and detoxification of heavy metal ions such as Cu2+, Zn2+ and Cd2+. In mammals, MTF-1 serves at least two additional roles. First, targeted disruption of the MTF-1 gene results in death at embryonic day 14 due to liver degeneration, revealing a stage-specific developmental role. Second, under hypoxic-anoxic stress, MTF-1 helps to activate the transcription of the gene placental growth factor (PIGF), an angiogenic protein. dMTF-1, the Drosophila homolog of mammalian MTF-1, has been cloned and characterized. This study presents a series of studies to compare the metal response in mammals and insects, that reveal common features but also differences. A human MTF-1 transgene can restore to a large extent metal tolerance to flies lacking their own MTF-1 gene, both at low and high copper concentrations. Likewise, Drosophila MTF-1 can substitute for human MTF-1 in mammalian cell culture, although both the basal and the metal-induced transcript levels are lower. Finally, a clear difference was revealed in the response to mercury, a highly toxic heavy metal: metallothionein-type promoters respond poorly, if at all, to Hg2+ in mammalian cells but strongly in Drosophila, and this response is completely dependent on dMTF-1 (Balamurugan, 2004).
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