Abstract
The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.
Highlights
The interactions of trans-acting factors with their rer-espective cis-acting sequences is presumably required to dispective cis-acting elements in5’tuhpestream region of rect high levels of tissue-specific gene expression in the adult. the p myosin heavy chain gene(MyHC)regulate its tis- It is unclear whether the factors which are active during the sue- and developmental stage-specific expression
Promoter,fusedtothechloramphenicolacetyltransferase reporter gene, and used to generate transgenic encoded isoformis found in slow skeletal muscles but not in the cardiac compartment, must be actively maintained
Tation of any two tohfe cis-acting elemenaltso does not We previously initiated an investigation of the p MyHC proaffect transgene expression
Summary
The amount of protein used and incubation times are described in the figure legends. Generation of Mutation Constructs-Site-directed mutagenesis of the from the non-acetylated products by thin layer chromatography. The. MCAT, C-rich, and pe regionsby overlap extension using thepolym- products were visualizedby autoradiography. Erase chain reaction (PCR) was performed as described [37]. For the MCAT and pe mutations, fourPCR primers were usedfor each muta-
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