Figure 3.

In a chromatin complex, enhancer activity is dependent on both the CME and the Nctc1 Promoter. (A) Reporter constructs were generated by cloning Nctc1 sequences into the multiple cloning site of plasmid pβgal-promoter as described in ‘Materials and Methods’ section. The reporter carriers a minimal SV40 promoter fused to the lacZ reporter so that high levels of expression depend on enhancer activity from the Nctc1 insertion fragments. Depicted are key Nctc1 inserts with Nctc1 exons 1 and 2 (grey rectangles) and the CME (black circle). (B) The CME is not sufficient for enhancer function in a chromosomal context. Primary myocytes isolated from wild-type mice were transfected with the constructs depicted in panel A. Expression of lacZ in transiently (grey bars) and in stably (black bars) transfected cells is normalized as described in ‘Materials and Methods’ section and reported relative to the expression observed using Construct II. Essentially identical results were obtained using mouse C2C12 myoblasts. (C) Reporter gene activation in stably transfected cells correlates with Nctc1 promoter activity. Primary myocytes isolated from ΔME/ΔME mice were transfected with constructs depicted in (A). Expression of Nctc1 and of lacZ is normalized to that seen in cells transfected with construct II carrying the full Nctc1 gene. Nctc1 expression is quantitated using primers specific for spliced message. The ΔME deletion removes the entire Nctc1 coding region (see Figure 1A) so that no Nctc1 RNA can be generated by the endogenous locus. For panels B and C, results are reported as average values with standard deviations calculated using at least three independent samples. In panel C, N/D means not determined.