Functional organization of a Schwann cell enhancer.
Myelin basic protein (MBP) gene expression is conferred in oligodendrocytes and Schwann cells by different upstream enhancers. In Schwann cells, expression is controlled by a 422 bp enhancer lying -9 kb from the gene. We show here that it contains 22 mammalian conserved motifs > or =6 bp. To investigate their functional significance, different combinations of wild-type or mutated motifs were introduced into reporter constructs that were inserted in single copy at a common hypoxanthine phosphoribosyltransferase docking site in embryonic stem cells. Lines of transgenic mice were derived, and the subsequent qualitative and quantitative expression phenotypes were compared at different stages of maturation. In the enhancer core, seven contiguous motifs cooperate to confer Schwann cell specificity while different combinations of flanking motifs engage, at different stages of Schwann cell maturation, to modulate expression level. Mutation of a Krox-20 binding site reduces the level of reporter expression, whereas mutation of a potential Sox element silences reporter expression. This potential Sox motif was also found conserved in other Schwann cell enhancers, suggesting that it contributes widely to regulatory function. These results demonstrate a close relationship between phylogenetic footprints and regulatory function and suggest a general model of enhancer organization. Finally, this investigation demonstrates that in vivo functional analysis, supported by controlled transgenesis, can be a robust complement to molecular and bioinformatics approaches to regulatory mechanisms.
Denarier
Eric
E
Laboratory of Developmental Biology, Molecular Oncology Group H-5, McGill University Health Centre, Montreal, Quebec, H3A 1A1, Canada.
Forghani
Reza
R
Farhadi
Hooman F
HF
Dib
Samar
S
Dionne
Nancy
N
Friedman
Hana C
HC
Lepage
Pierre
P
Hudson
Thomas J
TJ
Drouin
Régen
R
Peterson
Alan
A
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001369:Axons / Q000502:physiology; D002645:Chickens; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D008297:Male; D051379:Mice; D008818:Mice, Neurologic Mutants; D008822:Mice, Transgenic; D004676:Myelin Basic Protein / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000502:physiology; D012583:Schwann Cells / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013045:Species Specificity
D004676:Myelin Basic Protein; D007041:Hypoxanthine Phosphoribosyltransferase