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The mitranscription 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 ofmi/migenotype express normal amounts of abnormal MITF (mi-MITF), whereas mast cells of tg/tggenotype do not express any MITFs. The synthesis of heparin is abnormal in the skin mast cells of mi/mimice. 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/tggenotypes. The NDST-2 mRNA was detected by in situ hybridization in the skin mast cells of +/+ andtg/tgmice, but not in the skin mast cells ofmi/mimice. The amount of NDST-2 mRNA decreased significantly in CMCs derived from mi/mimice when compared to the values of +/+ and tg/tgmice, 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.

The mitranscription 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 ofmi/migenotype express normal amount of abnormal MITF (mi-MITF), whereas mast cells of tg/tggenotype do not express any MITFs. Mast cells of mi/mimice show more severe abnormalities than those of tg/tgmice, indicating that the mi-MITF possesses the inhibitory function. The MITF encoded by the micemutant allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain usingmice/micemice. The amounts of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) messenger RNAs decreased in mast cells ofmice/micemice to levels comparable to those of tg/tgmice, and the amounts were intermediate between those of +/+ mice and those of mi/mimice. 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 ofmice/micemast cells were comparable to those of tg/tgmast cells and were significantly higher than those of mi/mimast cells. The phenotype of mice/micemast cells was similar to that of tg/tgmast cells rather than to that of mi/mimast cells, suggesting that thece-MITF had no functions. The Zip domain of MITF appeared to be important for the development of mast cells.

The transcription factor encoded by the milocus (MITF) is a transcription factor of the basic-helix-loop-helix zipper protein family. Mice of mi/migenotype express a normal amount of abnormal MITF, whereas mice oftg/tggenotype 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 bothmi/miand tg/tgCMCs, that of MMCP-7 gene was severely suppressed only in mi/miCMCs. 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 ormi-MITF was demonstrated. The binding of +-MITF to c-Jun enhanced the transactivation of MMCP-7 gene, and that ofmi-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 ofmi-MITF on the transcription of MMCP-7 gene appeared to be executed through the interaction with c-Jun.

The milocus of mice encodes a transcription factor of the basic-helix-loop-helix-leucine zipper protein family (MITF). The MITF encoded by the mutant miallele (mi-MITF) deletes 1 of 4 consecutive arginines in the basic domain. The mice of mi/migenotype express mi-MITF, whereas the mice of tg/tggenotype have a transgene at the 5′ flanking region of themigene and do not express any MITF. To investigate the function of mi-MITF in cultured mast cells (CMCs), we took two approaches. First, mRNA obtained from mi/miCMCs ortg/tgCMCs was subtracted from complementary (c) DNA library of normal (+/+) CMCs, and the (+/+-mi/mi) and (+/+-tg/tg) subtraction libraries were obtained. When the number of clones that hybridized more efficiently with +/+ CMC cDNA probe than with mi/mior tg/tgCMC cDNA probe was compared using Southern analysis, the number was larger in the (+/+-mi/mi) library than in the (+/+-tg/tg) library. Second, we compared mRNA expression of six genes betweenmi/miand tg/tgCMCs by Northern analysis. The transcription of three genes encoding mouse mast cell proteases was impaired in both mi/miand tg/tgCMCs. On the other hand, the transcription of three genes encoding c-kitreceptor, tryptophan hydroxylase, and granzyme B was markedly reduced inmi/miCMCs, but the reduction was significantly smaller intg/tgCMCs. These results indicated the inhibitory effect ofmi-MITF on the transactivation of particular genes in CMCs.

The milocus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). Mutant alleles of mi,Mior, and Miwhare deletion or point mutation of the basic domain by which MITF binds DNA. The basic domain also has nuclear localization potential. In the present study, we compared the mast cell abnormalities ofMior/MiorandMiwh/Miwhmice with those ofmi/mimice, of which many have been described by us. The number of mast cells in the skin of Mior/Miorsuckling mice was remarkably decreased from that observed inmi/misuckling mice, but the number was normal in the skin ofMiwh/Miwhsuckling mice. The decrease in skin mast cells was more severe in the mi/miembryos than inmi/misuckling mice, but the magnitude of the decrease was comparable between Mior/Miorembryos and Mior/Miorsuckling mice. The poor mRNA expression of granzyme B and tryptophan hydroxylase genes was observed in all cultured mast cells (CMCs) derived from the spleens ofMiwh/Miwh,Mior/Mior, and mi/mimice. However, the poor expression of mouse mast cell protease-4 (MMCP-4), MMCP-5, and MMCP-6 was observed only inMior/Miorand mi/miCMCs. MITF encoded by Miwhmutant allele (Miwh-MITF) showed deficient but demonstratable DNA binding, but mi-MITF and Mior-MITF did not show any DNA binding ability. Although Miwh-MITF and Mior-MITF showed normal nuclear localization potential, the potential was significantly impaired in mi-MITF. The rank order of mast cell abnormality (mi/mi>Mior/Mior>Miwh/Miwh) appears to be related to the functional abnormality of MITF encoded by each mutant gene.

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) ofmi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). Because attachment of mi/mi CMCs to fibroblasts is impaired, we examined the expression of integrin genes in mi/mi CMCs in the present study. Among the integrin genes examined, the expression of integrin ?4 subunit was barely detectable in mi/mi CMCs, and the ?4 protein was not detected by flow cytometry either. The specific adhesion to vascular cell adhesion molecule-1 (VCAM-1), the ligand for ?4 subunit, was observed in +/+ CMCs but not in mi/mi CMCs, indicating that the expression of integrin ?4 subunit at a functional level did not occur in mi/mi CMCs. In the promoter region of the ?4 subunit gene, there was a CACTTG motif to which normal MITF (+- MITF) bound. The coexpression of +-MITF but not of mi-MITF transactivated the promoter of the ?4 subunit gene. The deletion or mutation of the CACTTG motif abolished the transactivation by +-MITF, suggesting that +-MITF directly transactivated the gene encoding ?4 subunit of integrin. © 1998 by The American Society of Hematology.

Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/ Wsrats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the Wlocus and because the c-kitgene was mapped at the Wlocus of mice, we characterized the c-kitgene of Ws/Wsrats. The authentic sequence of the rat c-kitcDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kitcDNA of Ws/Wsand normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.© 1991 by The American Society of Hematology. 0006-4971/91 / 7808-0007$3.00/0

The Wshis a mutant allele at the W (c-kit)locus of mice, but no significant abnormalities are found at the coding region of the Wshallele. Since cultured mast cells derived from the spleen of Wsh/Wshmice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c-kitmRNA expression in the skin of Wsh/Wshmice of various ages. The number of mast cells in the skin of Wsh/Wshembryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kitmRNA was detectable in the skin of 18-day pc Wsh/Wshembryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wshmice. The number of c-kitprotein-containing cells was significantly greater in the skin of 18-day pc Wsh/Wshembryos than in the skin of 5-day-old Wsh/Wshmice. The abolishment of c-kitmRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kitmRNA was detectable by in situ hybridization in skin mast cells of 5-day-old Wsh/Wshmice. Taken together, the expression of c-kitmRNA was abolished first, then the content of c-kitprotein dropped to undetectable levels, and then the disappearance of Wsh/Wshmast cells themselves followed.