Functional Organization of a Schwann Cell Enhancer

Abstract

Myelin basic protein (MBP) gene expression is conferred in oligodendrocytes and Schwann cells by different upstream enhancers. InSchwann cells, expression is controlled by a 422 bp enhancer lying Ϫ9 kb from the gene. We show here that it contains 22 mammalian conserved motifs Ն6 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.

Separate Proteolipid Protein/DM20 Enhancers Serve Different Lineages and Stages of Development

“…Moreover, the factor repertoire appears to change as development proceeds, e.g., wmN2 demonstrates activity in oligodendrocyte progenitors and myelin elaborating oligodendrocytes but downregulates in mature oligodendrocytes. Similar stage-specific activities were observed for multiple derivatives of the SCE1 enhancer of the myelin basic protein locus (Denarier et al, 2005). This may accommodate the disparity in regulatory output detected using in vivo and in vitro preparations.…”

“…Although the transgenesis strategy used here docks the reporter gene in single copy at a common insertion site and thus overcomes the variability associated with randomly inserted transgenes, the insertion site is adjacent to the ubiquitously expressed HPRT locus. We have observed expression in heart, large blood vessels, and several neuronal populations, including the hippocampus, with numerous HPRT docked constructs, including an hspeGFPlacZ construct to which no additional regulatory sequences were added (Denarier et al, 2005). Notably, when this same hspeGFPlacZ construct carries with it robust enhancers such as wmN1, wmN2, and pk211, these HPRT-directed expression programs are attenuated or extinguished (supplemental Fig.…”

“…So far, high-mobility group (Sox8, Sox10), basic helix-loophelix (Olig1, Olig2), and homeodomain (Nkx6.2, Nkx2.2) factors have been linked to myelin gene enhancer activity (Awatramani et al, 1997;Lu et al, 2000;Zhou et al, 2001;Stolt et al, 2004;Denarier et al, 2005;Ghislain and Charnay, 2006;Liu et al, 2007). As enhancers from the coordinately regulated myelin gene family emerge, characterization of their element content and configuration should reveal shared components and common features of the code controlling myelin gene transcription (Farhadi et al, 2003;Denarier et al, 2005;Dionne, 2006).…”

“…As enhancers from the coordinately regulated myelin gene family emerge, characterization of their element content and configuration should reveal shared components and common features of the code controlling myelin gene transcription (Farhadi et al, 2003;Denarier et al, 2005;Dionne, 2006). Also, because wmN2 is active in neural crest, its derivatives, and in oligodendrocyte and Schwann cell progenitors, its further investigation may reveal transcription factors that participate in the transition to committed glial progenitors (Deneen et al, 2006).…”

“…PCR products were digested and subcloned into a multiple cloning site upstream of the heat shock protein (hsp) promoter in an hspeGFPLacZ Entry vector, pHEL, modified from Denarier et al (2005). BD InFusion cloning was used according to the instructions of the manufacturer.…”