The human receptor tyrosine kinase Axl gene--promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation.
Axl is a receptor tyrosine kinase which promotes anti-apoptosis, mitogenesis, invasion, angiogenesis and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, cis-acting elements and promoter methylation driving expression of Axl. The 2.4 kb sequence upstream of the translational start site, and sequential 5'-deletions were cloned and revealed a minimal GC-rich region (-556 to +7) to be sufficient for basal Axl promoter activity in Rko, HCT116 and HeLa cells. Within this minimal region, five Sp (specificity protein)-binding sites were identified. Two sites (Sp a and Sp b) proximal to the translation start site were indispensable for Axl promoter activity, whereas mutation of three additional upstream motifs (Sp c, Sp d and Sp e) was of additional relevance. Gel-shift assays and chromatin immunoprecipitation identified that Sp1 and Sp3 bound to all five motifs, and mutation of all motifs abolished binding. Mithramycin, which inhibits binding of Sp factors to GC-rich sites, dramatically reduced Axl promoter activity and Axl, Sp1 and Sp3 expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl promoter activity. Use of Sp1/Sp3 siRNAs (small interfering RNAs) significantly reduced Axl promoter activity and protein levels in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG sites within three Sp motifs (Sp a, Sp b and Sp c) and GC-rich flanking sequences, and demethylation by 5-aza-2'-deoxycytidine up-regulated Axl and Sp3 expression in low-Axl-expressing Colo206f/WiDr cells, but not in high-Axl-expressing Rko cells. The results of the present study suggest that Axl gene expression in cancer cells is (1) constitutively driven by Sp1/Sp3 bound to five core promoter motifs, and (2) restricted by methylation within/around Sp-binding sites. This might enhance the understanding and treatment of essential mechanisms associated with cancer and other diseases.
Department of Experimental Surgery Mannheim/Molecular Oncology of Solid Tumors, Mannheim Medical Faculty, Ruprecht-Karls-University Heidelberg, German Cancer Research Center-DKFZ-Heidelberg, 68167 Mannheim, Germany.
Giridhar
G
Mudduluru
Heike
H
Allgayer
D020816:Amino Acid Motifs; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000473:pathology; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D000077209:Decitabine; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051705:Sp3 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D000094963:Axl Receptor Tyrosine Kinase
D009363:Neoplasm Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D000077209:Decitabine; D020794:Receptor Protein-Tyrosine Kinases; D001374:Azacitidine; D000094963:Axl Receptor Tyrosine Kinase; C000722289:AXL protein, human