Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.
The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892-2730, USA.
R
R
Gopal-Srivastava
J I
JI
Haynes
J
J
Piatigorsky
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003459:Crystallins / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D016762:Genes, fos; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D007908:Lens, Crystalline / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection
D003459:Crystallins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase