Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene.
To examine the relationship between the cardiac and skeletal muscle gene programs, the current study employs the regulatory (phosphorylatable) myosin light chain (MLC-2) as a model system. Northern blotting, primer extension, and RNase protection studies documented the high level expression of the cardiac MLC-2 mRNA in both mouse cardiac and slow skeletal muscle (soleus). Transgenic mouse lines harboring a 2100- or a 250-base pair rat cardiac MLC-2 promoter/luciferase fusion gene were generated, demonstrating high levels of luciferase activity in cardiac muscle, and only background luminescence in slow skeletal muscle and non-muscle tissues. As assessed by in situ hybridization, immunofluorescence, and luminescence assays of luciferase reporter activity in various regions of the heart, both the endogenous MLC-2 gene and the MLC-2 luciferase fusion gene were expressed exclusively in the ventricular compartment, with expression in the atrium at background levels. Point mutations within the conserved regulatory sites HF-1a and HF-1b significantly cripple ventricular muscle specificity, while mutation of the single E-box site was without effect, suggesting that ventricular muscle-specific expression occurs through an E-box-independent pathway. This study provides direct evidence that the cis regulatory sequences in the cardiac/slow twitch MLC-2 gene which confer cardiac and skeletal muscle-specific expression can be clearly segregated, suggesting that distinct regulatory programs may have evolved to control the tissue-specific expression of this single contractile protein gene in cardiac and skeletal muscle.
Lee
K J
KJ
Department of Medicine, University of California, San Diego, School of Medicine, La Jolla 92093.
Ross
R S
RS
Rockman
H A
HA
Harris
A N
AN
O'Brien
T X
TX
van Bilsen
M
M
Shubeita
H E
HE
Kandolf
R
R
Brem
G
G
Price
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003285:Contractile Proteins / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006321:Heart / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology*; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D003285:Contractile Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; D008156:Luciferases; D009218:Myosins