Nucleic Acids Research, Vol 26, Issue 2 407-414, Copyright © 1998 by Oxford University Press
C Ohtaka-Maruyama, X Wang, H Ge and AB Chepelinsky
The MIP gene, the founder of the MIP family of channel proteins, is
specifically expressed in fiber cells of the ocular lens and expression is
regulated temporally and spatially during development. We previously found
that a DNA fragment containing 253 bp of 5'-flanking sequence and 42 bp of
exon 1 of the human MIP gene contains regulatory elements responsible for
lens-specific expression of the MIP gene. In this report we have analyzed
the function of overlapping Sp1 and AP2 binding sites present in the MIP
promoter. Using DNase I footprinting analysis we found that purified Sp1
and AP2 transcription factors interact with several domains of the human
MIP promoter sequence -253/+42. Furthermore, addition of purified Sp1 to
Drosophila nuclear extracts activates in vitro transcription from the MIP
promoter -253/+42. This promoter activity is competed by oligonucleotides
containing domains footprinted with Sp1. Using promoter-reporter gene ( CAT
) constructs we found that the sequence -39/-70 contains a cis regulatory
element essential for promoter activity in transient assays in lens cells.
EMSA analysis showed that lens nuclear extracts contain factors that bind
to the MIP 5'-flanking sequence containing overlapping Sp1 and AP2 binding
domains at positions -37/-65. Supershift experiments with lens nuclear
extracts indicated that Sp3 is also able to interact with this regulatory
element, suggesting that Sp1 and Sp3 may be involved in regulation of
transcription of the MIP gene in the lens.
ARTICLES
Overlapping Sp1 and AP2 binding sites in a promoter element of the lens- specific MIP gene
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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