Sequence elements required for transcriptional activity of the human myoglobin promoter in intact myocardium.

Abstract

To define sequence elements required for myoglobin gene transcription in the intact heart, we examined the expression of a reporter gene under the control of a 380-bp upstream segment (-373 to +7) from the human myoglobin gene in transgenic mouse embryos and after gene transfer into left ventricular myocardium of adult rats. This proximal upstream region was sufficient to direct expression of luciferase selectively in cardiac and skeletal muscle of mouse embryos and to recapitulate the pattern of expression of the endogenous mouse myoglobin gene. This same upstream region was transcriptionally active after injection of plasmid DNA into the left ventricular wall of adult rats. Point mutations within two evolutionarily conserved sequence elements--a cytosine-rich (CCAC-box) motif and an A+T-rich (A/T) motif--severely impaired transcription within the intact heart. Nuclear extracts from neonatal cardiomyocytes contain protein factors that bind to each of these elements in a sequence-specific manner. We conclude that combinatorial interactions between the cognate DNA binding factors that recognize these motifs are necessary for transcriptional activity of the myoglobin upstream region in cardiac muscle.

A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type

“…A myoglobin promoter segment truncated to nucleotides −373 to +7 (Mb380) was sufficient for muscle-specific expression in prior experiments (Devlin et al 1989;Bassel-Duby et al 1993;Grayson et al 1995) and was responsive to calcineurin stimulation in our current studies (Fig. 3A).…”

“…1). Previously, we defined two conserved upstream response elements within the myoglobin promoter, both of which are required for transcriptional activity in skeletal myotubes or cardiac myocytes (Devlin et al 1989;Bassel-Duby et al 1993;Grayson et al 1995Grayson et al , 1998. These CCAC and A/T elements represent binding sites for Sp1 and MEF2 proteins, respectively, and function synergistically in muscle-specific gene regulation (Grayson et al 1995(Grayson et al , 1998.…”

Regulatory Elements Governing Transcription in Specialized Myofiber Subtypes

“…Plasmid DNA (50 g) containing segments of the human myoglobin gene promoter (19) was introduced into the soleus or extensor digitorum longus (EDL) muscle 3 days after injury. Muscles were harvested 10 days after bupivacaine injection and assayed for reporter activity.…”

“…Transcriptional Activity of the Human Myoglobin Gene Promoter in Regenerating Muscles of the Rat-Previous studies of the human myoglobin gene promoter in cultured cells (14,18) and following somatic cell gene transfer into the hearts of intact rats (19) suggested that important transcriptional regulatory elements reside in the proximal 5Ј flanking region. Here we determined that a segment of the human myoglobin gene extending from Ϫ373 to ϩ7 base pairs relative to the transcriptional initiation site is sufficient to direct muscle-specific expression of a luciferase reporter gene in a pattern that mirrors that of the endogenous myoglobin gene.…”

“…These sites were chosen for mutational analysis on the basis of their conservation in myoglobin genes of other mammalian species (Fig. 5A) and because of previous data from cell culture models (14,15,19). The Ϫ357 to ϩ55 segment of the murine myoglobin gene was sufficient to direct high levels of luciferase expression in myoglobin-rich soleus muscles as compared with myoglobin-deficient white vastus lateralis muscles in each of 10 independent transgenic founder mice carrying this transgene (Fig.…”