Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells.
The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B-cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4-1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.
Haynes
J I
JI
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Gopal-Srivastava
R
R
Frederikse
P H
PH
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D011061:Poly A; D012313:RNA / Q000032:analysis
D003459:Crystallins; D018076:DNA, Complementary; D011061:Poly A; D012313:RNA