Abstract
The human alpha-globin gene complex includes three functional globin genes (5'-zeta2-alpha2-alpha1-3') regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human alpha-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged alpha-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human alpha-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted alpha-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation.
Highlights
Human ␣-globin genes are clustered on a single complex located in the telomeric region of the short arm of chromosome 16
Several studies have shown that the erythroidspecific transcriptional activation of all ␣-globin genes present in the locus is controlled by a single positive regulatory element, named HS-40, which corresponds to a DNase I-hypersensitive site located 40 kb upstream from the 2 gene [7, 8]
We addressed these questions by using homologous recombination and CRE recombinase-mediated cassette exchange to target the insertion of either an extra ␣-globin gene or a marker gene driven by the non-erythroid promoter immediately upstream or downstream from HS-40 into the single chromosome 16 present in hybrid mouse erythroleukemia cells
Summary
All experiments were performed in the mouse erythroleukemia (MEL) hybrid cell line LT585P3, which contains a single copy of normal human chromosome 16 [18, 22]. Plasmid Constructions pLTR-neo Targeting Plasmids—All four plasmids used to target the insertion of the LTR-neo gene are derived from a single starting plasmid, pHS-40 This pHS-40 plasmid is based on pUC18 in which 9.2 kb of isogenic genomic DNA overlapping the HS-40 regulatory region has been cloned. The pHS-neoS targeting plasmid was obtained by subcloning the LTR-neo gene (taken as a SalI-XhoI fragment) into the single SalI site of pHS-40 and in the same transcriptional orientation as that of resident ␣-globin genes. The resulting L1-CMV-hygroTK-1L cassette isolated as a XhoI-PvuII fragment was subcloned into the single SalI site of plasmid pHS-40 in the same transcriptional orientation as that of resident ␣-globin genes, leading to the targeting plasmid pCMV-hygroTK. The ␣T-globin gene cassette was taken as a 1.5-kb PstI fragment and cloned using BamHI linkers between two inverted Lox sequences L1 and 1L, generating the p␣T gene exchange plasmid
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