Functional Characterization of a cis‐Acting DNA Antisilencer Region that Modulates Myelin Proteolipid Protein Gene Expression

Abstract

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 overcoming 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 ␤-galactosidase activity when inserted into Plp-lacZ constructs, which originally exhibited low levels of ␤-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.

Expression of a Myelin Proteolipid Protein (Plp)-lacZ Transgene is Reduced in both the CNS and PNS of Plp jp Mice

“…The transgene contains Plp genomic DNA extending from the proximal 2.4 kb of 5′-flanking DNA, downstream to the first 37 bp of exon 2, which were used to drive expression of a lacZ reporter gene cassette. Thus the transgene contains the Plp promoter as well as a tissue-specific enhancer located in Plp intron 1 DNA [24,25]. Expression of the PLP(+)Z transgene was evaluated in affected Plp jp males, which express a mutated Plp gene (Plp jp ), and compared to that produced in wild-type male littermates containing the normal Plp gene.…”

Where, when and how much: regulation of myelin proteolipid protein gene expression

“…Originally this positive regulatory element was classified as an antisilencer since it seemed to override repression mediated by multiple negative elements located elsewhere within intron 1 DNA [76]. However, subsequent experiments showed that the positive regulatory element possessed enhancer-like properties; i. e. it functioned in an orientation-independent manner and multiple copies of the element resulted in higher levels of reporter gene expression in transfected N20.1 cells [109]. The positive regulatory element, probably better characterized as a region, is contained within mouse intron 1 DNA positions 1083 -1777 (based on consecutively numbering the intron sequence with the first bp designated as 1).…”

“…This was the only positive regulatory element to be mapped in the intron by deletiontransfection analysis. A plethora of putative transcription factor binding sites within this sequence has been noted [109]. Multiple factors appear bind to this region [76,109], possibly forming an enhanceosome.…”

“…By transfection analysis[109], the positive regulatory region in the mouse gene has been localized to intron 1 positions 1083 -1177 (based on consecutively numbering the intron with the first base designated as position 1). The sequence of the sense strand is shown for the mouse gene and has been aligned to comparable sequences present within Plp intron 1 DNA from the rat, human, pig and cow (numbers indicate positions within the intron).…”

YY1 Negatively Regulates Mouse Myelin Proteolipid Protein (PLP1) Gene Expression in Oligodendroglial Cells

“…Transfection analysis using a battery of Plp1-lacZ constructs containing partial deletion of Plp1 intron 1 DNA revealed the presence of a single positive-regulatory element within the intron that is active in N20.1 cells (Dobretsova and Wight, 1999). Besides being able to overcome (or counterbalance) the effects from negative-regulatory elements located elsewhere in the intron, the positive-regulatory element functions as an enhancer in N20.1 cells (Dobretsova et al, 2000; Meng et al, 2005). Thus, we designated the positive regulatory element ASE ( a nti- s ilencer/ e nhancer).…”