Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression.
Regulation of myelin proteolipid protein (PLP:) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a PLP:-lacZ fusion gene (which includes the entire sequence for PLP: intron 1 DNA) is regulated in a similar manner to endogenous PLP: gene expression. Furthermore, by deletion-transfection analyses using assorted PLP:-lacZ constructs with partial deletion of PLP: intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of over-coming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore beta-galactosidase activity when inserted into PLP:-lacZ constructs, which originally exhibited low levels of beta-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of PLP: gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.
Dobretsova
A
A
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Kokorina
N A
NA
Wight
P A
PA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D007438:Introns / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018991:Myelin Proteolipid Protein / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D018991:Myelin Proteolipid Protein; D009687:Nuclear Proteins; D004247:DNA; D001616:beta-Galactosidase