Abstract
The transcription factors GATA-1, GATA-2, and GATA-3 were found to be expressed in several mouse and rat mast cell lines that contain mast cell carboxypeptidase A (MC-CPA) and other proteases in their cytoplasmic granules. GATA-1 mRNA was not detected in P815 cells, an immature mouse mastocytoma-derived cell line that lacks electron-dense granules and has low levels of secretory granule proteases. Because the 5'-flanking regions of the mouse and human MC-CPA genes contained a conserved GATA-binding motif 51 base pairs upstream of their translation initiation sites, the ability of GATA-binding proteins to regulate the promoter activity of the MC-CPA gene was examined in rat basophilic leukemia cells, mouse P815 cells, and transfected mouse P815 cells that expressed GATA-1. In all three mast cell lines, the promoter activity of the MC-CPA gene depended on the GATA binding site. GATA-1, GATA-2, and GATA-3 are thus the first DNA-binding proteins identified in mast cells which regulate the promoter activity of a gene that encodes a secretory granule protease.
Highlights
From the Departments of $Pediatrics and IIMedicine, Harvard MedicalSchool, the §Divisionof Hematology-Oncology, ChildrenS Hosoital, the **Department of Rheumatolom and Immunology, Brigham and Women’s Hospital, and nnthe Howard Hughes Medical Institute, Boston, Massachuset&ts“115
CPA genes containeda conserved GATA-bindingmotif 51 base pairs upstream of their translation initiation sites, the ability of GATA-bindingproteins to regulate the promoter activity of the mast cell carboxypeptidase A (MC-CPA) gene was examined in rat basophilicleukemia cells, mouse P815 cells, and transfected mouse P815 cells that expressed GATA-1
Twoto 4-fold more hGH (Fig. 4B) was produced by the P815/ pGT6 mastocytomacell line thathad been inducedto express high levels of GATA-1 compared with the parent P815 mastocytoma cell line (Figs. 1and 2B). These findings indicated that the promoter activity of the 5”flanking region of the MC-CPA gene in P815 cells, P815/pGT6 cells, and RBLcells was dependent on theGATA-binding site
Summary
EXPERIMENTALPROCEDURES duce a point mutation at residue -45 in the promoter of the mouse. The first PCR wasperformed with the wild-type MC-CPA promoter/hGH gene construct aastemplate, oneof the 36meroligonucleotides thatcontainedthepointmutation,andan oligonucleotide that corresponds to residue-1s60 to -148 of the MCCPA gene. Poly(A)+ RNA was prepared from 2 X 10' RBL cells transfected with the construct containing the wild-t.ype MC-CPApromoterusing a singlestepextractionkit(Invitrogen, containing the thymidine kinase promoter and the neomycin resistance gene was introduced into a pXM/GATA-1 expression plasmid [27]. This new expressionplasmid(designatedpGT)containsthe adenovirus major late promoter and provides constitutive.
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