Characterization of the Promoter Region of the Human c‐kit Proto‐oncogene

Abstract

The c‐kit proto‐oncogene encodes a tyrosine kinase receptor for stem cell factor and plays a critical role in the growth and differentiation of various types of cells including hematopoietic stem cells. To investigate the mechanisms of its transcriptional regulation, we isolated the 5’flanking region of the human c‐kit gene and characterized its promoter activity in hematopoietic cells. Nucleotide sequence analysis revealed that the 1.2 kb 5’flanking region lacked a typical “TATA box,” but had a relatively high G + C content and four potential Sp1‐binding sites. Putative binding sites for AP‐2, basic helix‐loop‐helix proteins, Ets‐domain proteins, Myb and GATA‐1 were also found. Primer extension and S1 nuclease protection analyses of hematopoietic cells indicated that the major transcription start sites are 62 bp and 58 bp upstream of the translation start site. Essentially the same start sites were detected in non‐hematopoietic cells such as small cell lung carcinoma and glioblastoma: this single promoter in c‐kit is different from the multiple promoter system of c‐fms, a c‐kit‐related gene, in which at least two promoters are differently used in hematopoietic and non‐hematopoietic cells. An analysis of the c‐kit 5’flanking region using the bacterial chloramphenicol acetyltransferase gene (CAT assay) in human erythroleukemia HEL cells, which express the endogenous c‐kit mRNA at high levels, showed that a region from ‐180 to ‐22 is important for the expression of the c‐kit gene. In addition, a negative regulatory element(s) is suggested to be involved in the regulation of the c‐kit gene expression in mammals.

Myb binding sites within the N-ras promoter repress transcription

“…Based on the presence of the Myb consensus sequences in the 5'-¯anking regions, several other candidate Myb-regulated genes have been proposed, including the c-kit proto-oncogene (Yamamoto et al, 1993), the human CDC2 gene (Ku et al, 1993), the human hematopoietic stem cell antigen CD34 (Melotti et al, 1994), the CD13/APN gene (Shapiro et al, 1995), the human oxytocin receptor gene , the murine neutrophil elastase gene (Nuchprayoon et al, 1994), the mouse CD4 promoter (Siu et al, 1992), the human c-myc gene (Evans et al, 1990), and the N-ras gene (Thorn et al, 1991). So far little is known about the functional relevance of the Myb recognition sites within these promoters, and none of these potential Myb-regulated genes have been de®nitively linked to the ability of Myb proteins to transform cells or induce leukemia.…”

PLZF-mediated control on c-kit expression in CD34+ cells and early erythropoiesis

“…c-kit is a direct target gene of PLZF in leukemic cells We analysed the DNA genomic sequence of the proximal promoter region of the human c-kit receptor (prom c-kit) and found, at position (281-305 bp) (Yamamoto et al, 1993), the sequence 5 0 -GAATCA TTGTACTTTTATTTTGCTC-3 0 , related to the previously described PLZF DNA-binding site (Ivins et al, 2003).…”

“…c-kit, a target of PLZF in early erythropoiesis I Spinello et al Electrophoretic mobility shift assay Electrophoretic mobility shift assays were performed as previously reported (Labbaye et al, 2002), using the 32 Plabeled double-stranded oligonucleotide probe (kit*): 5 0 -GAA TCATTGTACTTTTATTTTGCT-3 0 (from 281 to 305 bp) from the first nucleotide in the proximal c-kit promoter sequence (Yamamoto et al, 1993). A 100-, 300-and 600-fold molar excess of unlabeled oligonucleotide probe kit (kit) or unlabeled mutated oligonucleotide probe kit (mt): 5 0 -GAATC ATTGCGCGTCTATTTTGCTC-3 0 was used for competition experiments.…”

“…A genomic region of 155 bp containing the PLZFbinding site in the c-kit promoter (Yamamoto et al, 1993) was amplified by PCR using specific primers flanking the site PLZF (sense (216/236 bp): 5 0 -CGTTCAAGGATCCACCATGAC-3 0 ; antisense (351/370 bp): 5 0 -TCTCTGCCTCTGTAACAGAC-3 0 ) and PCR conditions: 94 1C for 1 min; 40 cycles of (95 1C for 30 s; 58 1C for 30 s; 72 1C for 30 s) and 72 1C for 5 min. PCR products were analysed by Southern blot analysis and hybridized with the internal 32 P-labeled oligonucleotide kit* probe described above.…”

“…A 1200 bp DNA fragment of the proximal promoter of c-kit (Prom c-kit) (Yamamoto et al, 1993) was PCR-amplified from genomic DNA using the primers forward 5 0 -GAGGGAT GTCTTATTAGTTT-3 0 and reverse 5 0 -TAGCTGCGATGG GATCCGAG-3 0 , and cloned upstream to the luciferase gene into the pGL3Basic (pGL3Basic/Prom c-kit) reporter vector (Promega, Madison, WI, USA). By mutagenesis of the PLZF site described above into the pGL3Basic/Prom c-kit vector, using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according manufacturer's instructions, we prepared the PLZF mutated-Prom c-kit vector (pGL3Basic/mt-Prom c-kit).…”