Cloning and characterization of EphA3 (Hek) gene promoter: DNA methylationregulates expression in hematopoietic tumor cells

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal a nd spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promote r elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the E phA3 gene promoter region. A region of 86 bp located at -348 bp to -262 bp upstream from the transcription start site was identified as the basal prom oter, This region was shown to be active in both EphA3-expressing and -none xpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive res triction enzymes and bisulfite sequencing of genomic DNA, sites of DNA meth ylation were identified in hematopoietic cell lines which correlated with t heir levels of EphA3 gene expression. We showed that EphA3 was not methylat ed in normal tissues but that a subset of clinical samples from leukemia pa tients showed extensive methylation, similar to that observed in cell lines , These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors, (C) 1999 by The Ame rican Society of Hematology.