Involvement of intronic sequences in cell-specific expression of the peripherin gene

Peripherin is an intermediate filament protein expressed in restricted popu lations of neurons. Our previous study of the chromatin structure of the mo use peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be in volved in its cell specificity. In the present work, we performed an in vit ro functional analysis of the 5' flanking region of the mouse peripherin ge ne and observed that this region up to 9 kb contained both enhancer and inh ibiting activities; however, it was insufficient to achieve a complete exti nction of reporter gene expression in peripherin-negative cells. Furthermor e, analysis of the first three introns with the 5' flanking sequences of th e gene showed that intron I greatly increased specificity of the gene expre ssion. Intron I also conferred the same properties to thymidine kinase hete rologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompass es an AP-2-like binding site that interacted with both neuroblast and fibro blast nuclear factors, as well as with the recombinant: AP-2 alpha protein. However, gel shift experiments suggested that the interacting nuclear fact ors are distinct from AP-2 alpha itself and probably belong to the AP-2 fam ily. Inl B perfectly matched the consensus binding site for Sp1 and specifi cally interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I in dicated that the Inl A element alone is responsible for its enhancing prope rties, whereas a region located between +789 and +832 gives to intron I its silencer activity.