Involvement of intronic sequences in cell-specific expression of the peripherin gene.
Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse 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 involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.
Lecomte
M J
MJ
CNRS, URA 2115 CHU Pitié-Salpêtrière, Paris, France.
Basseville
M
M
Fauquet
M
M
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D017353:Gene Deletion; D017930:Genes, Reporter; D007381:Intermediate Filament Proteins / Q000235:genetics*; D007438:Introns; D008562:Membrane Glycoproteins; D051379:Mice; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009447:Neuroblastoma; D064531:Peripherins; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; C493668:Tfap2a protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; D013937:Thymidine Kinase; D003850:Deoxyribonuclease I