Basal Level Of Transcription Research Articles

Transcriptional regulation of connexin 43 expression by retinoids and carotenoids: Similarities and differences

Gap junctions, connexons, are formed by assembly of trans-membrane connexin proteins and have multiple functions including the coordination of cell responses. Most human tumors are deficient in gap junctional communication (GJC) and restoration of GJC by forced expression of connexins reduces indices of neoplasia. Expression of connexin 43 (Cx43), the most widely-expressed connexin family member, is upregulated by cancer-preventive retinoids and carotenoids in normal and preneoplastic cells; an action considered of mechanistic significance. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist Ro 41-5253 was capable of suppressing retinoid-induction Cx43 luciferase reporter construct in F9 cells, but did not suppress reporter activity induced by the non-pro-vitamin A carotenoids astaxanthin or lycopene, indicating that retinoids have separate mechanisms of gene activation than non-pro-vitamin A carotenoids. Neither class of compound required protein synthesis for induction of Cx43 mRNA, nor was the 5.0 h half-life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within -158 bp and +209 bp of the transcription start site; this contains a Sp1/Sp3 GC-box to which Sp1 and Sp3 were bound, as revealed by electrophoretic mobility shift assays (EMSA), but no retinoic acid response element (RARE). Site directed mutagenesis of this GC-box resulted in increased basal levels of transcription and loss of responsiveness to a synthetic retinoid. In this construct astaxanthin and lycopene produced marginally, but not significantly higher, reporter activity than the control.

Low Temperature Induction of Arabidopsis CBF1, 2, and 3 Is Gated by the Circadian Clock

Exposing Arabidopsis (Arabidopsis thaliana) plants to low temperature results in rapid induction of CBF1, 2, and 3 (CBF1-3; also known as DREB1B, C, and A, respectively), which encode transcriptional activators that induce expression of a battery of genes that increase plant freezing and chilling tolerance. Recently, it has been shown that basal levels of CBF3 transcripts and those of certain CBF-regulated genes exhibit circadian cycling. Here, we further explored the regulation of CBF1-3 by the circadian clock. The results indicated that the extent to which CBF1-3 transcripts accumulated in response to low temperature was dependent on the time of day that the plants were exposed to low temperature and that this was regulated by the circadian clock. The highest and lowest levels of cold-induced CBF1-3 transcript accumulation occurred at 4 and 16 h after subjective dawn, respectively. An analysis of CBF2 promoter-reporter gene fusions indicated that this control included transcriptional regulation. In addition, the cold responsiveness of RAV1 and ZAT12, genes that are cold induced in parallel with CBF1-3, was also subject to circadian regulation. However, whereas the maximum level of cold-induced RAV1 transcript accumulation occurred at the same time of day as did CBF1-3 transcripts, that of ZAT12 was in reverse phase, i.e. the highest level of cold-induced ZAT12 transcript accumulation occurred 16 h after subjective dawn. These results indicate that cold-induced expression of CBF1-3, RAV1, and ZAT12 is gated by the circadian clock and suggest that this regulation likely occurs through at least two nonidentical (though potentially overlapping) signaling pathways.

Cell type–specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor

AbstractMechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type–specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

Cancer prevention by retinoids and carotenoids: Independent action on a common target

Virtually all human tumors are deficient in gap junctional communication (GJC) and the restoration of GJC by forced expression of connexins reduces indices of neoplasia. The expression of connexin 43 (Cx43) is upregulated by cancer-preventive retinoids and carotenoids which correlates with the suppression of carcinogen-induced transformation in 10T1/2 cells. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist, Ro 41-5253, suppressed retinoid-induced Cx43 protein expression in 10T1/2 cells and the induction of a Cx43 luciferase reporter construct in F9 cells, but did not suppress protein expression or reporter activity induced by the non-pro-vitamin A carotenoid astaxanthin. In contrast, Cx43 induction by astaxanthin, but not by a RAR-specific retinoid, was inhibited by GW9662, a PPAR-gamma antagonist. Neither compound required protein synthesis for the induction of Cx43 mRNA, nor was the 5.0 h half-life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within -158 bp and +209 bp of the transcription start site. Site directed mutagenesis of a GC-box in this region increased basal levels of transcription and loss of retinoid responsiveness. Simultaneous treatment with a retinoid and beta-carotene or astaxanthin resulted in supra-additive Cx43 expression, again indicating separate mechanisms of gene regulation.

Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

Transcription regulation by the adaptor protein Fe65 and the nucleosome assembly factor SET

Fe65 protein interacts with the cytosolic domain of the amyloid precursor APP. Its possible involvement in gene regulation is suggested by numerous observations, including those demonstrating that it activates transcription. Here, we show that the Fe65 transcription activation domain overlaps with the WW domain of Fe65 and binds to the nucleosome assembly factor SET. This protein is required for the Fe65-mediated transactivation of a reporter gene. Two-step chromatin immunoprecipitation experiments demonstrate that a complex including Fe65/AICD/Tip60 and SET is associated with the KAI1 gene promoter. Suppression of SET levels by RNA interference shows that this protein is required for full levels of basal transcription of the KAI1 gene. These results further support the function of Fe65 and APP in gene regulation and show a new role for the SET factor.

Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade.

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Galpha protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Galpha protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

Combinatorial Transcription of Herpes Simplex Virus and Varicella Zoster Virus Immediate Early Genes Is Strictly Determined by the Cellular Coactivator HCF-1

The mammalian transcriptional coactivator host cell factor-1 (HCF-1) functions in concert with Oct-1 and VP16 to assemble the herpes simplex virus (HSV) immediate early (IE) transcription enhancer core complexes that mediate the high level transcription of these genes upon infection. Although this transcriptional model has been well characterized in vitro, the requirements and significance of the components have not been addressed. Oct-1 was previously determined to be critical but not essential for HSV IE gene expression. In contrast, RNA interference-mediated depletion of HCF-1 resulted in abrogation of HSV IE gene expression. The HSV IE gene enhancer domain is a model of combinatorial transcription and consists of the core enhancer and multiple binding sites for factors such as Sp1 and GA-binding protein. It was striking that HCF-1 was strictly required for VP16-mediated transcriptional induction via the core enhancer as well as for basal level transcription mediated by GA-binding protein and Sp1. HCF-1 was also found to be essential for the induction of varicella zoster virus IE gene expression by ORF10, the VZV ortholog of the HSV IE transactivator VP16, and the autostimulatory IE62 protein. The critical dependence upon HCF-1 demonstrates that this cellular component is a key factor for control of HSV and VZV IE gene expression by functioning as the common element for distinct factors cooperating at the IE gene enhancers. The requirements for this protein supports the model whereby the regulated transport of HCF-1 from the cytoplasm to the nucleus in sensory neurons may control IE gene expression and reactivation of these viruses from the latent state.

Transcriptional control of RAD51 expression in the ciliate Tetrahymena thermophila.

The expression of Rad51p, a DNA repair protein that mediates homologous recombination, is induced by DNA damage and during both meiosis and exconjugant development in the ciliate Tetrahymena thermophila. To completely investigate the transcriptional regulation of Tetrahymena RAD51 expression, reporter genes consisting of the RAD51 5' non-translated sequence (5' NTS) positioned upstream of either the firefly luciferase or green fluorescent protein coding sequences have been targeted for recombination at the macronuclear btu1-1 (K350M) locus of T. thermophila strain CU522. Expression from RAD51-luciferase reporter constructs has been directly quantified from transformant whole cell lysates. Luciferase is induced to maximum levels in transformants harboring the full-length RAD51-luciferase reporter gene following exposure to DNA damaging UV irradiation. A series of truncations, deletions, insertions, substitutions and inversions of the RAD51 5' NTS have led to the identification of three distinct transcriptional promoter elements. The first of these sequence elements is required for basal levels of transcription. The second modulates expression in the absence of DNA damage, whereas the third ensures increased RAD51 transcription in response to DNA damage and during meiosis. Tetrahymena RAD51 is tightly regulated through these transcriptional elements to produce the appropriate expression during conjugation, and in response to DNA damage.

Tobacco mosaic virus regulates the expression of its own resistance gene N.

The N gene of tobacco (Nicotiana tabacum) is a typical resistance (R) gene engendering localization of tobacco mosaic virus (TMV) infection and the elicitation of a hypersensitive necrotic response. The consensus model for R gene-derived resistance is at the level of protein:protein interactions, in which proteins of the pathogen interact with already present receptor-like proteins produced by the plant's R genes. This article demonstrates, by quantitative real-time reverse transcription-PCR analysis, that in tobacco carrying the dominant allele N, a basal level of transcription indeed occurs in noninfected plants. However, accumulation of N-mRNA in infected plants indicates that transcription is stimulated by TMV infection (up to 38-fold in locally infected leaves and up to 165-fold in upper, noninoculated leaves). Potato virus Y infection did not result in accumulation of N-mRNA, indicating a specific TMV-related phenomenon. The possible uncoupling of viral restriction from necrosis is discussed.

The high-affinity Sp1 binding site in the HTLV-1 promoter contributes to Tax-independent basal expression.

Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) requires many cellular proteins and the virally encoded transcription factor Tax. Tax binds the three viral cAMP-response elements (CREs) with ATF/CREB (activating transcription factor/cAMP-response element-binding protein) and recruits the cellular coactivators CBP/p300. HTLV-1 also utilizes other cellular transcription factors that bind to the promoter to regulate transcription. One of these factors, Sp1, has been shown to bind to the viral promoter at two elements; one located within the third viral CRE, and the second located between the second and third viral CREs. The functional significance of Sp1 binding at each of these regions of the viral promoter is not completely understood. We set out to characterize Sp1 binding and to evaluate the functional significance of Sp1, both in the absence and presence of Tax. We found that Sp1 binds preferentially to the element located between the second and third viral CREs, and modestly activates transcription in vitro and in vivo. Sp1 was detected at the integrated HTLV-1 promoter in vivo. Surprisingly, point mutagenesis of the strong Sp1 binding site rendered the HTLV-1 reporter plasmid insensitive to Sp1 activation, and dramatically reduced basal transcription in vivo. These data indicate a role for Sp1 in basal level transcription of HTLV-1.

Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced CYP1A1 transcription in zebrafish embryo

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that can be activated by a diverse synthetic and naturally-occurring chemicals, such as the halogenated aromatic hydrocarbons (HAHs) and the non-halogenated polycyclic aromatic hydrocarbons (PAHs). The liganded AHR modulates the genetic activity of a variety of xenobiotic-responsive genes, including cytochrome P4501A1 ( CYP1A1). The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. Here we showed that 0.2 mM PTU induced a basal level of CYP1A1 transcription in zebrafish embryonic integument as early as 24 h postfertilization (hpf) stage. Subsequently, PTU induced CYP1A1 transcription in blood vessels at 36 hpf. During larval stage, the liver and all pharyngeal arch vessels of PTU-treated embryos exhibited CYP1A1 transcription as well. Comparing to TCDD, PTU induces CYP1A1 transcription with much lower efficacy in zebrafish embryos. Coincubating the embryos with PTU and TCDD led to repressing TCDD-induced CYP1A1 transcription. Mechanistic studies indicated that both of PTU- and TCDD-mediated CYP1A1 transcriptions are modulated by the same AHR–ARNTsignaling pathway.

A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription.

Selenophosphate synthetase catalyzes the synthesis of selenophosphate which is a selenium donor for Sec biosynthesis. In Drosophila melanogaster, there are two types of selenophosphate synthetases designated dSPS1 and dSPS2, where dSPS2 is a selenoprotein. The mechanism of gene expression of dSPS2 as well as other selenoproteins in Drosophila has not been elucidated. Herein, we report an essential regulator system that regulates the transcription of the dSPS2 gene (dsps2). Through deletion/substitution mutagenesis, the downstream DNA replication-related element (DRE) located at +71 has been identified as an essential element for dsps2 promoter activity. Furthermore, double-stranded RNA interference (dsRNAi) experiments were performed to ablate transcription factors such as TBP, TRF1, TRF2 and DREF in Schneider cells. The dsRNAi experiments showed that dsps2 promoter activities in DREF- and TRF2-depleted cells were significantly decreased by 90% and 50%, respectively. However, the depletion of TBP or TRF1 did not affect the expression level of dsps2 even though there is a putative TATA box at -20. These results strongly suggest that the DRE/DREF system controls the basal level of transcription of dsps2 by interacting with TRF2.

Androgen receptor coregulators in prostate cancer: mechanisms and clinical implications.

The transcriptional activity of androgen receptor (AR) is modulated by coregulators that have a significant influence on a number of functional properties of AR, including the ligand specificity as well as the DNA binding capacity. Because androgens and AR have pivotal roles in the development and

Identification and characterization of the human Cdc2l2 gene promoter

The CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) protein kinases are part of the large family of p34cdc2-related kinases and have been shown to play a role in cell cycle progression, RNA processing and apoptosis. They are encoded by two genes—cell division control like 1 (Cdc2L1) and cell division control like 2 (Cdc2L2). To date, little is known about the transcription factors controlling their expression. To understand the mechanisms underlying the regulation of CDK11 gene expression, we cloned and identified the Cdc2L2 promoter and determined its transcriptional regulatory elements. By deletion analysis, a region between nucleotides −145 and +10 was identified to be critical for basal level transcription of the Cdc2L2 gene. Sequencing analysis revealed that the proximal promoter of the Cdc2L2 gene is GC rich and does not contain TATA and CAAT boxes. However, multiple consensus and near consensus transcription factor binding sites were found to be present in this region, such as two Ets-1, one cAMP-responsive element (CRE) and one TCF11/LCR-F1/Nrf1 binding sites. Site-directed mutagenesis and transfection studies revealed that all these binding sites were necessary to achieve sustained transcriptional activity. Electrophoretic mobility shift assay confirmed that transcription factors Ets-1 and CREB bind to the Cdc2L2 promoter elements, indicating their potential role in the transcriptional regulation of Cdc2L2 gene. More importantly, Ets-1, CREB and phosphorylated CREB were found binding to the endogenous Cdc2L2 promoter using chromatin immunoprecipitation (CHIP) assay. Our results provide the foundation for further studies into the regulation of Cdc2L2 gene expression in normal homeostasis and cancer.

Sp1-dependent regulation of the tissue inhibitor of metalloproteinases-1 promoter.

Extracellular matrix (ECM) remodeling is involved in many cellular properties such as division, migration, differentiation, and death. The turnover of ECM is regulated by matrix metalloproteinases (MMPs) and the MMPs are inhibited by the tissue inhibitors of metalloproteinases (TIMPs). In this study, the transcriptional regulation of the TIMP-1 promoter was investigated. The 5'-deletion assay showed that the region between -1,200 and -1,101 was responsible for the TIMP-1 promoter activity. The mutations of the two Sp1 sites in this region reduced the transcription activity. In addition, the co-transfection with antisense Sp1 oligonucleotide decreased the promoter activity, suggesting that the transcription of the TIMP-1 promoter is mediated by Sp1. Previously, it was reported that the TIMP-1 expression was enhanced under hypoxia. Therefore, the TIMP-1 promoter activity was investigated with or without cobalt ion, which elicits the same physiological effect as hypoxia. The results showed that the TIMP-1 promoter was induced in the presence of cobalt ion and that the promoter activity was regulated by Sp1 as well as HIF-1. Therefore, this study suggests that Sp1 is involved in the regulation of the TIMP-1 promoter in the presence of cobalt ion as well as in the basal level transcription.

Identification and characterization of the human Cdc2l2 gene promoter.

The CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) protein kinases are part of the large family of p34(cdc2)-related kinases and have been shown to play a role in cell cycle progression, RNA processing and apoptosis. They are encoded by two genes-cell division control like 1 (Cdc2L1) and cell division control like 2 (Cdc2L2). To date, little is known about the transcription factors controlling their expression. To understand the mechanisms underlying the regulation of CDK11 gene expression, we cloned and identified the Cdc2L2 promoter and determined its transcriptional regulatory elements. By deletion analysis, a region between nucleotides -145 and +10 was identified to be critical for basal level transcription of the Cdc2L2 gene. Sequencing analysis revealed that the proximal promoter of the Cdc2L2 gene is GC rich and does not contain TATA and CAAT boxes. However, multiple consensus and near consensus transcription factor binding sites were found to be present in this region, such as two Ets-1, one cAMP-responsive element (CRE) and one TCF11/LCR-F1/Nrf1 binding sites. Site-directed mutagenesis and transfection studies revealed that all these binding sites were necessary to achieve sustained transcriptional activity. Electrophoretic mobility shift assay confirmed that transcription factors Ets-1 and CREB bind to the Cdc2L2 promoter elements, indicating their potential role in the transcriptional regulation of Cdc2L2 gene. More importantly, Ets-1, CREB and phosphorylated CREB were found binding to the endogenous Cdc2L2 promoter using chromatin immunoprecipitation (CHIP) assay. Our results provide the foundation for further studies into the regulation of Cdc2L2 gene expression in normal homeostasis and cancer.

Repression of Promoter Activity by CNOT2, a Subunit of the Transcription Regulatory Ccr4-Not Complex

The evolutionary conserved Ccr4-Not complex controls mRNA metabolism at multiple levels in eukaryotic cells. Genetic analysis of not mutants in yeast identifies a negative role in transcription, which is dependent on core promoter structure. To obtain direct support for this we targeted individual core subunits of the human Ccr4-Not complex to promoters in transient transfections of human cells. In this experimental setup we found that the CNOT2 and CNOT9(hRcd1/hCaf40) subunits act as repressors of reporter gene activity. Interestingly, recruitment of other Ccr4-Not subunits did not affect the reporter gene. The major repression function of CNOT2 is localized in a specialized protein motif, the Not-Box. This conserved motif is present in all CNOT2 orthologs and surprisingly also in CNOT3 orthologs. Repression by the Not-Box was sensitive to treatment with the histone deacetylase inhibitor trichostatin A. In addition, mutation of a canonical TATA-box enhanced repression. Our experiments show for the first time direct regulation of promoter activity by components of the Ccr4-Not complex.

Tamoxifen induces the expression of maspin through estrogen receptor-α

Maspin ( mammary serine protease inhibitor) is a tumor suppressor gene that plays an important role in inhibiting tumor growth, invasion and metastasis. Maspin expression is down regulated at transcription level in primary and metastatic breast tumor cells. Previous studies on hormonal regulation of maspin prompt us to test whether an estrogen antagonist tamoxifen (TAM) [1] can exert its anti-tumor function by up regulating maspin gene expression. For this purpose, we first tested whether maspin promoter could be activated in normal and several breast tumor cells. We then carried out a series of promoter analysis in which estrogen receptors and TAM were reconstituted in an in vitro cell culture system. Here we report our new finding that tumor suppresser gene maspin is one of the TAM target genes. TAM induces a maspin/luciferase reporter in cell culture and this induction requires the presence of (estrogen receptor alpha) ERα but not estrogen receptor-β (ERβ). Maspin promoter deletion and mutation analysis showed that the cis element(s) within a region between −90 and+87 bp but not the HRE site (−272 bp) was involved in TAM induction of maspin expression. TAM bound ERα may directly control maspin gene expression through the interaction with cofactor (s). Analysis using several ERα mutants showed that the N-terminal A/B motif (AF-1) was critical for maspin basal level transcription activation. An ERα mutant with point mutations at DNA binding domain abolished estrogen induction of an ERE–luciferase reporter but was still active in activating maspin promoter by TAM. LBD-AF2 domain was required for ERα-dependent TAM induction. Deletion of LBD-AF2 or a point mutation in the ERα LBD-AF2 region (LBDmtL539A) completely abolished the activation of maspin promoter, suggesting that TAM induction of maspin involves the recruitment of cofactor(s) by ERα to the maspin promoter region. This finding indicates that one of the pathways for cancer prevention and tumor inhibition by TAM is mediated through the activation of tumor suppressor gene maspin in breast cancer.

A Novel Cysteine Cluster in Human Metal-responsive Transcription Factor 1 Is Required for Heavy Metal-induced Transcriptional Activation in Vivo

Metal-responsive transcription factor 1 (MTF-1) specifically binds to metal response elements (MREs) associated with a number of metal- and stress-responsive genes. Human MTF-1 contains a cysteine-rich cluster, -632Cys-Gln-Cys-Gln-Cys-Ala-Cys638-, conserved from pufferfish to humans far removed from the MRE-binding zinc finger domain and just C-terminal to a previously mapped serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys-->Ala substitutions (C632A/C634A) or a deletion in this region altogether (Delta(632-644)) are significantly impaired in their ability to induce Zn(II)- and Cd(II)-responsive transcription of a MRE-linked reporter gene in transiently transfected mouse dko7 (MTF-1-/-) cells in culture under moderate metal stress but retain the ability to drive basal levels of transcription in a MRE-dependent manner in vivo and in vitro. In addition, the mutated proteins respond to induction by Zn(II) or Cd(II) with nuclear translocation and MRE binding activities comparable with wild-type MTF-1. Attempts to rescue the Delta(632-644) deletion mutant phenotype by inserting similar Cys-rich sequences from Drosophila MTF-1 were unsuccessful, suggesting that the structure of this motif within intact human MTF-1, rather than the simple presence of multiple closely spaced Cys residues, is required for function. This cysteine cluster therefore functions at a step subsequent to nuclear translocation and MRE-binding DNA to naked promoter-containing DNA and appears to be specifically required for MTF-1 to activate transcription in the presence of inducing heavy metal ions.