Cooper, Brian; (1999) Regulation of two muscle-specific genes during Xenopus heart development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Xenopus cardiac myosin light chain 2 gene (XMLC2) encodes a contractile protein localised exclusively to the heart of the embryo. To identify the regulatory sequences responsible for cardiac muscle-specific expression of XMLC2,1 investigated the sequences immediately upstream of the gene. Characterisation of a 3kb XMLC2 promoter fragment reveals the presence of four MEF2 and four GATA transcription factor binding sites. Transgenic frog technology indicates that the 3kb 5' proximal promoter fragment can direct cardiac muscle-specific reporter expression during Xenopus embryogenesis, reproducing the normal expression pattern of the endogenous gene. Moreover, a 3kb XMLC2 transgene directs heart restricted expression during murine embryogenesis, and is active in cultured cardiomyocytes, but not in a skeletal muscle cell line. Deletion mapping of the 3kb XMLC2 promoter by Xenopus transgenesis defines the most proximal 708bp as the minimal promoter fragment sufficient to confer high level reporter expression in the embryonic heart. Further 5' or 3' truncations attenuate the transcriptional activity of the XMLC2 promoter, whilst sequences contained between -217bp and -86bp are critical for myocardial-specific activity. Mutation of the MEF2 elements indicate that they might play redundant roles in XMLC2 regulation. Gel shift experiments reveal that the four consensus MEF2 sites in the 5' flanking region of the XMLC2 gene bind Xenopus MEF2D protein. The ability of MEF2 factors to activate transcription from the XMLC2 promoter is assayed using microinjection of oocytes. Expression of a 3kb XMLC2 promoter-CAT reporter gene introduced into the oocyte germinal vesicle is only detected in oocytes previously injected with MEF2D RNA, but not MEF2A. This suggests that XMLC2 is a direct target for activation by MEF2D and indicates that functional differences exist between MEF2 family members to activate the XMLC2 gene. The cardiac α-actin gene is a contractile protein expressed concomitantly in the somitic and cardiac muscle during Xenopus embryogenesis. Using transgenic frog technology, I demonstrate that a 580bp cardiac α-actin promoter fragment drives high level expression of a GFP reporter in the heart and skeletal muscle of Xenopus embryos. An SRF binding site (CArG box 1) in the 580bp cardiac α-actin promoter is necessary but not sufficient for high level muscle-specific expression. In contrast, deletion of the E-box elements from the 580bp cardiac α-actin promoter had no effect on transcription of the transgene. Mutation of an Sbp sequence just downstream of CArG box 1, results in the abolition of reporter expression in the heart, yet somitic expression is unaffected. This Sbp sequence does not conform to any known transcription factor binding site involved in contractile isoform regulation, therefore, might represent a novel control element required for cardiac muscle-specific activation of the cardiac α-actin gene.

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