Downstream utrophin enhancer is required for expression of utrophin in skeletal muscle

Duchenne muscular dystrophy is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of utrophin is expected to compensate for the dystrophin deficit. We previously reported that the 5.4-kb 5'-flanking region of the utrophin gene containing the A-utrophin core promoter did not drive transgene expression in heart and skeletal muscle. To clarify the regulatory mechanism of utrophin expression, we generated a nuclear localization signal-tagged LacZ transgenic (Tg) mouse, in which the LacZ gene was driven by the 129-bp downstream utrophin enhancer (DUE) and the 5.4-kb 5'-flanking region of the utrophin promoter.

Two Tg lines were established. The levels of transgene mRNA expression in several tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Cryosections of several tissues were stained with haematoxylin and eosin and X-gal.

The transgene expression patterns were consistent with endogenous utrophin in several tissues including heart and skeletal muscle. Transgene expression was also up-regulated more in regenerating muscle than in nonregenerating muscle. Moreover, utrophin expression was augmented in the skeletal muscle of DUE Tg/dystrophin-deficient mdx mice through cross-breeding experiments. We finally established cultures of primary myogenic cells from this Tg mouse and found that utrophin up-regulation during muscle differentiation depends on the DUE motif.

Our results showed that DUE is indispensable for utrophin expression in skeletal muscle and heart, and primary myogenic cells from this Tg mice provide a high through-put screening system for drugs that up-regulate utrophin expression.