Abstract
A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.
Highlights
The promoters or enhancers of a variety of chicken, human, and mouse globin genes have been found to contain binding sites for a transcription factor (NF-E1) that has been shown to be essential for erythroid-specific expression [5,6,7, 14, 15, 17, 18, 23, 25]
In the case of the PBG-D promoter, the human and mouse sequences are highly conserved and contain an adjacent NF-E1-binding site and a CACCC motif, but the CACCC motif occurs as a tandem duplication in the mouse genome [1]. (The human PBG-D [hPBG-D] promoter contains a binding site for another erythroid-specific factor, NF-E2, at about -180 nucleotides [nt] [11, 12], but this site is not conserved in the mouse promoter [1].) Using as a model a minimal 114-base-pair truncated hPBG-D promoter that retains erythroid-specific activity, we have examined whether interactions between NF-E1 and an adjacent CACCC motif are essential for the erythroid-specific activity of the hPBG-D promoter
To study in more detail the role of NF-E1 in regulating the hPBG-D promoter, in particular whether it cooperates with the factor binding to the adjacent CACCC motif, the effects of mutating or altering the spacing of the NF-E1-binding site and the CACCC motif were examined
Summary
5.3 a Average of two independent transfections, with CAT activities differing only by about 10%. TCATC) reduced the hPBG-D promoter activit, almost completely (to about 12% of the wild-type activity, compared with 5% for the promoterless hGH vector alone); mutating the CACCC motif to GGGCC or deleting a 24-bp fragment containing the CACCC motif had a similar effect (Table 2). Both the NF-El-binding site and the CACCC motif are essential for promoter activity. Insertion of a 71-bp fragment of polylinker sequence into the Ball site between the NF-E1-binding site and the CACCC motif in pPBGhGH1 to give pPBGhGH1+71 (Fig. 1) was found to reduce hGH production in MEL cells eightfold. The oligonucleotides used for mutagenesis of the NF-E1-binding site were as follows: for the coding strand, 5'-AAGCTGATGGGCCTCATCTCTCTTTACCCACC-3' (containing a single point mutation at -75 bp) and 5'-AAGCTGATGGGAATTCTCTCTITACCCACC-3' (containing three mutations at -78, -77, and -74 bp); and for the CACCC-binding site (coding strand), 5'-CTGCAGGCCCGGGCCTTCCTGTGGCC-3' (containing three mutations at -105, -104, and -103 bp)
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