The murine alpha B-crystallin/small heat shock protein enhancer: identification of alpha BE-1, alpha BE-2, alpha BE-3, and MRF control elements.
The murine alpha B-crystallin gene (a member of the small heat shock protein family) is expressed constitutively at high levels in the lens and at lower levels in many other tissues, including skeletal muscle. We have previously used the herpes simplex virus thymidine kinase promoter fused to the human growth hormone gene to identify an alpha B-crystallin enhancer at positions -427 to -259 that has high activity in muscle and low activity in lens cell lines. In the study reported here, we performed DNase I footprinting, transfection, mutagenesis, and electrophoretic mobility shift experiments using the murine C2C12 muscle and alpha TN4-1 lens cell lines and the rabbit N/N1003A lens cell line to identify sequences responsible for activity of this enhancer. Enhancer activity in both the muscle and lens cells was dependent on novel elements called alpha BE-1 (-407 to -397), alpha BE-2 (-360 to -327), and alpha BE-3 (-317 to -306). These elements were also weakly occupied by nuclear proteins in L929 cells, which appear to express the alpha B-crystallin gene at a very low level (detectable only by the polymerase chain reaction). A fourth element containing a consensus muscle regulatory factor-binding site called MRF (-300 to -288) was occupied and used only by the C2C12 muscle cells. Cotransfection in NIH 3T3 cells and antibody-gel shift experiments using C2C12 nuclear extracts indicated that MyoD, myogen, or a similar member of this family can activate the alpha B-crystallin enhancer by interaction with the MRF site. Taken together, we conclude that the alpha BE-1, alpha BE-2, and alpha BE-3 elements are shared by both lens and muscle cells, but the MRF element is used only in muscle cells, providing the first example of a muscle-specific control element in a crystallin gene.
Gopal-Srivastava
R
R
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892.
Piatigorsky
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003459:Crystallins / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D003459:Crystallins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D004247:DNA; D013937:Thymidine Kinase; D003850:Deoxyribonuclease I