Effect of MITF on transcription of transmembrane tryptase gene in cultured mast cells of mice.

Mouse mast cell protease (mMCP)-6, mMCP-7 and transmembrane tryptase (TMT) are all tryptases. The normal mi transcription factor (+-MITF) transactivated mMCP-6 gene by binding three consensus motifs in the promoter region, but no MITF-binding motifs were found in the mMCP-7 promoter. Instead, c-Jun transactivated mMCP-7 gene, and +-MITF cooperated with it. The mi-MITF encoded by mutant mi allele inhibited the transactivation by c-Jun and reduced the mMCP-7 promoter activity. Here, the effect of MITF on the TMT gene expression was examined. The +-MITF enhanced the TMT promoter activity by binding two consensus motifs. The mi-MITF showed the inhibitory effect on TMT gene expression. The effect of +-MITF on TMT gene was similar to the effect on mMCP-6 gene, and that of mi-MITF was similar to the effect on mMCP-7 gene. The effects of MITF on TMT gene appeared distinct from its effects on either mMCP-6 or mMCP-7 gene.

Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper transcription factor that is important for the development of mast cells. Cultured mast cells (CMCs) of mi/mi genotype express abnormal MITF (mi-MITF), but CMCs of tg/tg genotype do not express any MITFs. It was previously reported that mi/mi CMCs showed more severe abnormalities than tg/tg CMCs, indicating that mi-MITF had inhibitory function. Whereas mi-MITF contains a single amino acid deletion in the basic domain, MITF encoded by mi(ew) allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain. Here the effect of a large deletion of the basic domain was examined. In mi(ew)/mi(ew) CMCs, the expression pattern of genes whose transcription was affected by MITF was comparable to that of tg/tg CMCs rather than to that of mi/mi CMCs. This suggested that ew-MITF lacked any functions. The part of the basic domain deleted in ew-MITF appeared necessary for either transactivation or inhibition of transactivation.

Inhibitory effect of the mi transcription factor encoded by the mutant mi allele on GA binding protein-mediated transcript expression in mouse mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amounts of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. The synthesis of heparin is abnormal in the skin mast cells of mi/mi mice. Because N-deacetylase/N-sulfotransferase 2 (NDST-2) is essential for the synthesis of heparin, the amount of NDST-2 messenger RNA (mRNA) was compared among cultured mast cells (CMCs) of +/+, mi/mi, and tg/tg genotypes. The NDST-2 mRNA was detected by in situ hybridization in the skin mast cells of +/+ and tg/tg mice, but not in the skin mast cells of mi/mi mice. The amount of NDST-2 mRNA decreased significantly in CMCs derived from mi/mi mice when compared to the values of +/+ and tg/tg mice, suggesting that the defective form of MITF inhibited the expression of the NDST-2 transcript. The expression of NDST-2 transcript was mediated by the GGAA motif located in the 5'-untranslated region. GA binding protein (GABP) bound the GGAA motif and increased the amount of NDST-2 transcript. The mi-MITF appeared to inhibit the ability of GABP to express NDST-2 transcript by disturbing its nuclear localization. This is the first study to show that expression of an abnormal form of a bHLH-Zip transcription factor can dramatically alter the intracellular location of another DNA/RNA binding factor, which in turn brings about profound and unexpected consequences on transcript expression.

Molecular cause of the severe functional deficiency in osteoclasts by an arginine deletion in the basic domain of Mi transcription factor.

Severe osteopetrosis was observed in mi/mi mutant mice. However, the bone of VGA9/VGA9 mutant mice, in which Mi gene expression is undetectable, showed normal histology. No osteopetrosis was found in mi/+ mice, but was observed in VGA9/mi mice. Biochemical analysis revealed that the gene product encoded with the mi mutant allele (mi-Mi) has impaired DNA binding activity and nuclear translocation ability. Furthermore, inhibitory effects of mi-Mi were shown not only on the DNA binding activity of wild-type Mi, but also on the nuclear translocation ability of Mi, PU.1 and cFOS. The present results suggest the presence of a target gene for Mi that is essential for the proliferation/differentiation of osteoclasts.

Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mi(ce)/mi(ce) mutant mice of which MITF lacks the zipper domain.

The mi transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amount of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. Mast cells of mi/mi mice show more severe abnormalities than those of tg/tg mice, indicating that the mi-MITF possesses the inhibitory function. The MITF encoded by the mi(ce) mutant allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain using mi(ce)/mi(ce) mice. The amounts of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) messenger RNAs decreased in mast cells of mi(ce)/mi(ce) mice to levels comparable to those of tg/tg mice, and the amounts were intermediate between those of +/+ mice and those of mi/mi mice. Gr B mediates the cytotoxic activity of mast cells, and TPH is a rate-limiting enzyme for the synthesis of serotonin. The cytotoxic activity and serotonin content of mi(ce)/mi(ce) mast cells were comparable to those of tg/tg mast cells and were significantly higher than those of mi/mi mast cells. The phenotype of mi(ce)/mi(ce) mast cells was similar to that of tg/tg mast cells rather than to that of mi/mi mast cells, suggesting that the ce-MITF had no functions. The Zip domain of MITF appeared to be important for the development of mast cells. (Blood. 2001;97:2038-2044)

Inhibitory effect of the transcription factor encoded by the mutant mi microphthalmia allele on transactivation of mouse mast cell protease 7 gene.

The transcription factor encoded by the mi locus (MITF) is a transcription factor of the basic-helix-loop-helix zipper protein family. Mice of mi/mi genotype express a normal amount of abnormal MITF, whereas mice of tg/tg genotype do not express any MITFs due to the transgene insertional mutation. The effect of normal (+) and mutant (mi) MITFs on the expression of mouse mast cell protease (MMCP) 6 and 7 was examined. Both MMCP-6 and MMCP-7 are tryptases, and their coding regions with high homology are closely located on chromosome 17. Both MMCP-6 and MMCP-7 genes are expressed in normal cultured mast cells (+/+ CMCs). Although the transcription of MMCP-6 gene was severely suppressed in both mi/mi and tg/tg CMCs, that of MMCP-7 gene was severely suppressed only in mi/mi CMCs. The study identified the most significant segment for the transcription in the 5' flanking region of MMCP-7 gene. Unexpectedly, no CANNTG motifs were found that are recognized and bound by +-MITF in this segment. Instead, there was an AP-1 binding motif, and binding of c-Jun to the AP-1 motif significantly enhanced the transcription of MMCP-7 gene. The complex formation of c-Jun with either +-MITF or mi-MITF was demonstrated. The binding of +-MITF to c-Jun enhanced the transactivation of MMCP-7 gene, and that of mi-MITF suppressed the transactivation. Although the former complex was located only in the nucleus, the latter complex was predominantly found in the cytoplasm. The negative effect of mi-MITF on the transcription of MMCP-7 gene appeared to be executed through the interaction with c-Jun.