TY - JOUR
T1 - Multiple control elements are required for expression of the human CD34 gene
AU - Radomska, Hanna S.
AU - Satterthwaite, Anne B.
AU - Burn, Timothy C.
AU - Oliff, Ira A.
AU - Tenen, Daniel G.
N1 - Funding Information:
The authors are indebted to Dr Karina Yazdanbakhsh for sharing her experience in DNaseI hypersensitivity assays and Mohan Philip for providing preliminary sequence of the 3′ enhancer. Rex cells were kindly provided by Dr James Griffin, Dana-Farber Cancer Institute. Constanze Bonifer and members of the Tenen laboratory contributed to this work by numerous discussions and valuable suggestions. This work was supported by fellowship awards #DK09721 (to H.S.R.) and #DK02359 (to I.A.O.) from the National Institutes of Health, and grant DK48660 to D.G.T.
PY - 1998/11/19
Y1 - 1998/11/19
N2 - Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, 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, suggesting that this element might be responsible for stem cell- restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive 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' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.
AB - Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, 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, suggesting that this element might be responsible for stem cell- restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive 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' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.
KW - Chromatin
KW - DNA methylation
KW - DNaseI hypersensitive sites
KW - Locus control region
KW - Stable integration
KW - Stem cell-specific
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U2 - 10.1016/S0378-1119(98)00491-0
DO - 10.1016/S0378-1119(98)00491-0
M3 - Article
C2 - 9831666
AN - SCOPUS:0032548163
SN - 0378-1119
VL - 222
SP - 305
EP - 318
JO - Gene
JF - Gene
IS - 2
ER -