SYSTEMATIC METHOD TO OBTAIN NOVEL GENES THAT ARE REGULATED BY MI TRANSCRIPTION FACTOR - IMPAIRED EXPRESSION OF GRANZYME-B AND TRYPTOPHAN-HYDROXYLASE IN MI MI CULTURED MAST-CELLS/

The mi locus encodes a member of the basic-helix-loop-helix-leucine zi pper protein family of transcription factors (hereafter called MITF). We have reported that the expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi genotype, and demonstrated the inv olvement of MITF in the transcription of these genes. To obtain new ge nes whose transcription may be regulated by MITF, we prepared a subtra cted cDNA library using +/+ and mi/mi CMCs. We found two clones carryi ng the granzyme (Gr) B and tryptophan hydroxylase (TPH) cDNAs in the s ubtracted library. The expression of the Gr B and TPH genes decreased in mi/mi CMCs, and recovered to nearly normal level by the overexpress ion of normal (+) MITF but not of mutant (mi) MITF. The +-MITF bound t hree and one CANNTG motifs in the Gr B and TPH promoters, respectively , and transactivated these two genes, indicating the involvement of +- MITF in their expression. Because TPH is the rate-limiting enzyme for serotonin synthesis, we examined the serotonin content of +/+ and mi/m i CMCs. The serotonin content was significantly smaller in mi/mi CMCs than in +/+ CMCs. The introduction of +-MITF but not of mi-MITF normal ized the serotonin content in mi/mi CMCs, (C) 1998 by The American Soc iety of Hematology.