Downregulation of constitutive and heavy metal-induced metallothionein-I expression by nuclear factor I

Abstract

Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFL) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trails-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-I, may be involved in this repression.

Figure 1

MT-I promoter harbors three half-sites for NFI binding. (A) Comparison of nucleotide sequence of MRE-c′ and MRE-b with NFI consensus element. Common nucleotides among the cis elements are shown in bold letters. (B) Schematic diagram of mouse MT-I promoter, depicting the cis elements. NFI half-sites are shown in bold letters and underlined.

Figure 2

Factors forming complex with MRE-c′ oligo belong to NFI family of proteins. Whole cell extract (10 μg) from HepG2 cells (lane 1) was incubated with 0.1–0.2 ng of ³²P-labeled MRE-c′ oligo at 4°C under optimum binding conditions and subjected to electrophoresis on polyacrylamide (6% acrylamide) gel. (A) To identify the proteins forming the complexes, the extract was preincubated with either of the following unlabeled oligos: (a) 100- or 200-fold molar excess of MRE-c′ (lanes 2 and 3), (b) MRE-c′ mutant (lanes 4 and 5), (c) 50- or 100-fold molar excess of NFI consensus oligo (lanes 6 and 7), or (d) NFI mutant (lanes 8 and 9) along with 0.5 μg of poly(dI-dC) as nonspecific competitor. (B) The extract was also preincubated separately with l μl of normal rabbit serum (lane 2) or anti-NFI antisera (lane 3) for 30 min on ice prior to addition of the labeled probe. The arrow indicates the position of the supershifted complex.

Figure 3

Both constitutive and heavy metal-induced activities of the MT-I promoter are downregulated by NFI family of proteins. (A) HepG2 cells were transfected with the reporter plasmid pMT-Luc (1.5 μg), the internal control pRLTK (0.15 μg), CMV control vector (1.5 μg), or CMV vectors expressing NFI-A, -C, and -X proteins (1.5 μg). Transfected cells were incubated with or without 10 μM cadmium sulfate for 6 h prior to harvest. Luciferase activity was determined by dual luciferase assay kit where renila luciferase activity (pRLTK) was used to normalize the transfection efficiency. The bars represent the mean and range of data from triplicate assays of three independent transfections. (B) HepG2 cells were transiently transfected (as stated in A) except that NFI-B and NFI-L expression vectors were cotransfected here. Transfected cells were either left untreated or treated with 100 μM zinc sulfate or 10 μM cadmium sulfate for 6 h prior to harvest.

Figure 4

Mutation of NFI binding site on the MT-I promoter that abrogates binding of NFI protein does not abolish the repressor action of NFI proteins on the promoter. HepG2 cells were transfected with either pMT-Luc, pMRE-c′mut, or pNFImut (1.5 μg), pRLTK (0.15 μg), CMV control vector (not shown), or CMV vector expressing NFI-C (1.5 μg). Reporter plasmids are described in Materials and Methods. Transfected cells were either treated with or without 10 μM cadmium sulfate 6 h before harvesting. Ratio of reporter gene activity to that of the internal control is presented in the bar diagram. This is representative of two independent experiments with triplicate assays.

Figure 5

NFI proteins can downregulate both the constitutive and heavy metal-induced activity of the MT-I promoter irrespective of the presence of cognate cis element. (A) Schematic diagram of the promoter/reporter plasmids constructed (described in Materials and Methods). The boxes represent MRE-c′ sequences and the ovals represents MRE-d sequence. (B) Transient transfection of HepG2 cells and subsequent zinc treatment of the transfected cells are as described in the previous experiment (Fig. 3). The promoter/reporter plasmids transfected in HepG2 cells are either p(MRE-c′)4, p(MRE-d)s, or p(MRE-c′/d) (1.5 μg). The data are representative of three independent experiments.

Figure 6

NFI-C truncated proteins lacking DNA binding domain can still exert its repressor action on the constitutive and heavy metal-induced MT-I promoter activity. (A) Schematic diagrams of HA-tagged NFI-C carboxy-terminal deletion constructs. (B) HepG2 cells were transfected with pMT-Luc reporter plasmid (1.5 μg), pRLTK (0.15 μg), and CMV vector expressing full-length NFI-C (pCHmNFI-C), or N-terminal 240 amino acids of NFI-C (pCHmNFI-C-240) or N-terminal 160 amino acids of NFI-C (pCHmNFI-C-160) (1.5 μg). Transfected cells were treated for 6 h with or without zinc sulfate (100 μM). The bars represent mean and range of data from three independent transfections with duplicate assays.

Figure 7

Overexpression of MTF-1 protein can recover the basal and zinc-induced MT-I promoter activity from NFI-mediated repression. HepG2 cells were transfected with pMT-Luc (1.0 μg), pRLTK (0.1 μg), and either of the following vectors (1.0 μg): (a) CMV control vector (E.V. NFI-C), (b) CMV vector expressing NFI-C, (c) control vector for MTF-1 (E.V. MTF-1), (d) MTF-1 expression vector or a combination of (b) and (c) or (b) and (d) (1.0 μg each), as indicated in the figure. Transfected cells were either left untreated or treated with 10 μM cadmium sulfate (A) or 100 μM zinc sulfate (B) 6 h prior to harvest. The data are representative of three independent experiments.

Figure 8

DNA binding activity of MTF-1 is not affected by NFI overexpression. Whole cell extract (10 μg) from HepG2 cells transfected with CMV control vector or different isoforms of NFI protein (NFI-A, -B, -C, or -X) was incubated with 0.1–0.2 ng of ₃₂P-labeled MRE-d oligo at 4°C under optimum binding conditions and subjected to electrophoresis on polyacrylamide (6% acrylamide) gel. The extracts were preincubated with or without unlabeled Sp1 oligo as indicated to disrupt the Sp1 complex, so that specific MTF-1/MRE-d complex is distinctly visible.