(Received for publication, May 15, 1995; and in revised form, June 28, 1995)
From the
Growth inhibitory factor/metallothionein III (GIF/MT-III) is
expressed specifically in brain, and neither mRNA nor protein is
detected in other organs. This tissuespecific expression might be
regulated by negative elements as well as positive elements, such as
tissue-specific enhancers. To investigate the repression mechanisms of
this gene in organs other than the brain, transfection experiments were
performed by using various deletion mutants. Interestingly, a 25
CTG repeat in the promoter region seemed to contribute to the
repression activity. Moreover, the repression activity of this 25
CTG repeat was also observed in various promoters and in a
direction and position independent manner, indicating that this element
could act as a silencer. However, no binding protein was detected by
gel-shift and footprint analyses. These results strongly suggest that
the CTG repeat functions as a negative element, and that this effect is
caused by unknown mechanisms, rather than by interactions between
specific cis-elements and specific trans-acting
factors as reported previously. It is also possible that the CTG repeat
functions as a general silencer in many genes.
Gene expression is regulated mainly at the transcription level. Many trans-acting factors, such as enhancer-binding proteins, participate in this regulation (see (1) and (2) for review). For tissue-specific expression, tissue-specific enhancers are known to be involved. Recently, it was proposed that, as well as enhancer elements and enhancer-binding proteins, negative regulators are also involved. Mechanisms include competition, quenching, direct inhibition, and squelching (see (3) and (4) for review). However, compared with enhancer functions, the mechanisms of negative regulation still remain to be investigated.
Growth
inhibitory factor (GIF) ()has been purified from human
brain(5) . Both amino acid sequence analysis and cDNA cloning
have revealed that GIF is a small protein, 68 amino acids long, and
quite similar to metallothionein (MT) with insertions of 1 amino acid
and 6 amino acids in the N-terminal and C-terminal portions,
respectively(5, 6, 7, 8) .
Therefore, GIF is also termed MT-III, since MT-I and MT-II have been
reported(6) . In addition, a new MT isoform, MT-IV was reported
recently(9) . MTs are small, cysteine-rich, and metal-binding
proteins, and MT-I and MT-II are thought contribute to detoxication of
heavy metals, such as mercury and cadmium, and homeostasis of essential
trace elements, such as zinc and copper (see (10) and (11) for review). Whereas MT-I and MT-II are expressed widely
in almost all organs and are strongly induced by heavy metals,
GIF/MT-III is expressed strictly in the brain and not induced by heavy
metals(6) . GIF/MT-III is also known to be deficient in the
brain of Alzheimer's disease (AD) patients(5) .
Therefore, in addition to
-amyloid precursor protein and
,
GIF/MT-III seems to have an important role in Alzheimer's
disease(5, 12, 13, 14) .
In this report, we characterized the promoter region of the mouse GIF/MT-III gene and found that a CTG repeat in this region functions as a silencer-like element and contributes to negative regulation.
Figure 1: Transcriptional activity of deletion mutants of the promoter in mouse GIF gene. Various deletion mutants of the mGIF gene promoter were constructed in the luciferase reporter gene and transfected into HepG2 cells. Values are relative luciferase activities compared with the mGIF(-257)-luciferase construct.
Figure 2:
Nucleotide sequence of the negative
regulatory region in the mGIF gene promoter. 25 CTG repeats are underlined.
Figure 3:
Effect of the CTG repeat on
transcriptional activity of the mGIF gene promoter. A, effect
of an internal deletion of the CTG repeat. 25 CTG repeats (75
bases) were deleted exactly as described under ``Experimental
Procedures.'' B, effect of the CTG repeat on a CTG
repeat-less mGIF gene promoter. Both are indicated as relative
luciferase activity.
Figure 4:
Effect of CTG repeat on transcriptional
activity of various gene promoters. A, effect of the CTG
repeat on various promoters, mGIF, C/EBP, SV40, glutathione S-transferase (GST) P, and human metallothionein
II
(hMT-IIA), in which the CTG repeat was joined
to the upstream region of the promoter. B, effect of the CTG
repeat on various promoters, mGIF, C/EBP
, and SV40, in which the
CTG repeat was joined to the downstream region of the promoter. Values
are represented as relative luciferase activity compared with each
CTG-less construct.
In this report, we characterized the promoter region of the mouse GIF/MT-III gene and identified its CTG repeat as a silencer. Since this gene is not expressed in organs other than the brain(5, 6) , this function is quite important for negative regulation in the mouse. Recently, we cloned the human GIF/MT-III gene, including the promoter region up to -2 kilobases, using the coding region as a probe (kind gift from Dr. R. D. Palmiter). Unfortunately, we have not yet identified a CTG repeat in this region by Southern blotting. Therefore, it is possible that the CTG repeat in the GIF/MT-III gene is species-specific. Indeed, only 200-base pair region from the cap site showed high similarity between the mouse and human genes(6) . It is unclear whether the CTG repeat contributes to brain-specific transcription of the GIF/MT-III gene, since suitable cell lines for the brain are not available now. Transfection experiments using primary culture and/or in vitro transcription assay may dissolve this question in the near future.
Amplification of CTG or CAG repeats is known in several inherited neurodegenerative diseases including Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia, dentatorynural-pallidoluysian atrophy, and myotonic dystrophy(20, 21) . In the first four diseases, CAG repeats in the N-terminal portions of their related genes encode polyglutamine residues, and the expansion of a glutamine-rich segment is related to the diseases. Although there is no evidence that an expanded CAG repeat has negative activity on transcription, except that the presence of the polyglutamine tract is inhibitory to trans-activation on the androgen receptor gene (22) , it is possible that the CAG repeat functions as a silencer under normal conditions. In the case of the myotonic dystrophy gene, in which the CTG repeat is found in the 3`-untranslated region, decreased expression is observed(23) . However, an opposite effect has also been reported(24) . In Fragile X syndrome, hypermethylation of the CGG triplet repeat in the 5`-untranslated region represses the expression of the Fragile X mental retardation-1 gene(25) . GIF/MT-III is the first example of a CTG triplet repeat located in the 5` promoter region, and that has repression activity.
We have tried to detect whether nuclear proteins bind to the CTG repeat. However, we could not observe binding, although the conditions used here could detect other transcription factors. These results strongly suggest that a specific binding protein is not necessary to this function. First, we thought that a conformational change caused by the CTG triplet repeat might cause an inhibition effect on protein-protein interaction between the promoter and basal machinery. However, using the method for the detection of DNA bending by native electrophoresis (26) in the absence of the protein, we failed to detect the conformational change. Moreover, even when the CTG repeat was in the downstream region of the luciferase gene, that is, very far from the promoter, the negative effect was also observed. Therefore, we prefer to consider the following mechanism; while a specific DNA-binding protein would not bind to the CTG repeat, the CTG repeat itself could interact with some proteins in the complexes of basal and specific transcription factors with weak affinity. If this is the case, it is also possible that the CTG repeat may function as an activator. So far as we tested, we observed only a negative effect. We also failed to detect CTG repeat binding activity in the brain nuclear extract, although we could detect the binding activity in other regions in the promoter. Therefore, it seems that the CTG repeat functions as a general silencer which is active in all organs including the brain, and some activator proteins overcome the silencer function in the brain. Further studies are required to clarify the effect of the CTG triplet repeat on gene expression.