A compact unc45b-promoter drives muscle-specific expression in zebrafish and mouse

Parts used

Key findings

• The 195-bp unc45b promoter fragment unc45bmin (nucleotides −505 to −310 of the zebrafish unc45b promoter) acts as a promoter without the heterologous gata2 minimal promoter, restricting reporter expression to skeletal muscle cells and cardiomyocytes in zebrafish. Injecting unc45bmin:tfp into one-cell-stage embryos gives strong teal fluorescent protein (TFP) expression in skeletal muscle and heart at 72 hpf, and stable Tg(unc45bmin:gfp) embryos show GFP only in skeletal muscle and cardiomyocytes at 48 and 72 hpf, matching the pattern of the full-length zebrafish unc45b promoter.
• At 195 bp, the zebrafish unc45bmin promoter fragment is smaller than the 509-bp dMTK, 720-bp tMCK and 770-bp MHCK7 muscle promoters and far smaller than the 6.5-kb muscle creatine kinase (MCK) promoter. Adeno-associated virus (AAV) vectors have a packaging capacity of about 4.5 kb, so unc45bmin leaves more room for a therapeutic transgene than these promoters.
• The unc45bmin promoter fragment is heat-shock inducible. Incubating 48-hpf Tg(unc45bmin:gfp) zebrafish embryos for 1 h at 39°C significantly increases GFP fluorescence at 72 hpf compared with untreated siblings, and the same 1 h heat shock increases fluorescence in Tg(unc45bmin:smyd1b-gfp) embryos, so unc45bmin-driven transgene levels can be titrated in vivo.
• A Smyd1b-GFP fusion driven by the unc45bmin promoter in Tg(unc45bmin:smyd1b-gfp) zebrafish is expressed specifically in cardiomyocytes and skeletal muscle cells at 72 hpf. The fusion protein localizes to the sarcomere in a pattern alternating with the Z-disk marker α-actinin, consistent with the sarcomeric M-band localization of endogenous Smyd1b.
• Expressing zebrafish smyd1b-gfp from the 195-bp unc45bmin promoter rescues the paralysis of smyd1b-deficient flatline (fla−/−) mutant zebrafish. At 24 hpf, 80.7 ± 4.6% of fla−/− embryos carrying Tg(unc45bmin:smyd1b-gfp) move spontaneously (n = 32), compared with 80 ± 3.6% of fla siblings (n = 111) and 0% of non-transgenic fla−/− mutants (n = 35), which are completely paralyzed. At 72 hpf, 88.2% of transgenic fla−/− embryos (n = 26) show a touch-evoked flight response, compared with 86.6% of transgenic fla siblings (n = 61), and fla−/− fish carrying the transgene can be raised to adulthood without an overt muscle phenotype.
• Expressing smyd1b-gfp from the unc45bmin promoter restores sarcomere structure in smyd1b-deficient flatline (fla−/−) zebrafish embryos, which otherwise lack sarcomeric organization and birefringence. Transgenic fla−/− embryos carrying Tg(unc45bmin:smyd1b-gfp) show bright birefringence and regular α-actinin-labeled sarcomeres, and Smyd1b-GFP localizes to the sarcomeric M-band, a complete structural rescue of the skeletal muscle defect.
• The zebrafish unc45bmin promoter fragment drives reporter expression in mouse skeletal muscle. Ten days after electroporation into mouse tibialis anterior muscle, unc45bmin:tfp gives fluorescent reporter expression in muscle fibers with similar efficiency to the full-length −1.8-kb unc45b promoter, while the 570-bp −369+195(unc45b):tfp fragment, which lacks the 195-bp unc45bmin sequence, gives no detectable expression.

Abstract

Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off-target effects the therapeutic gene should be driven by a tissue-specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue-specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle-specific expression but presents heat-shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b-deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle-specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431-438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.