Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' e nhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34(+) cell lines, suggestin g that this element might be responsible for stem cell-restricted expressio n of the CD34 gene. In the current work, through deletion and transient tra nsfection experiments, we delineated the core enhancer sequence. We examine d the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using D NaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensit ive sites, we detected a strong CD34(+) cell type-specific site in intron 4 . This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' e nhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In con trast, a 160 kb genomic fragment encompassing the entire CD34 gene containe d regulatory elements sufficient for high-level CD34 mRNA expression in mur ine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression. (C) 1998 Published by Elsevier Science B.V. All rights re served.