MULTIPLE REGULATORY ELEMENTS CONTRIBUTE DIFFERENTIALLY TO MUSCLE CREATINE-KINASE ENHANCER ACTIVITY IN SKELETAL AND CARDIAC-MUSCLE

被引:141
作者
AMACHER, SL [1 ]
BUSKIN, JN [1 ]
HAUSCHKA, SD [1 ]
机构
[1] UNIV WASHINGTON,DEPT BIOCHEM SJ70,SEATTLE,WA 98195
关键词
D O I
10.1128/MCB.13.5.2753
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.
引用
收藏
页码:2753 / 2764
页数:12
相关论文
共 98 条
[1]   IMMUNOCHEMICAL ANALYSIS OF MYOSIN HEAVY-CHAIN DURING AVIAN MYOGENESIS INVIVO AND INVITRO [J].
BADER, D ;
MASAKI, T ;
FISCHMAN, DA .
JOURNAL OF CELL BIOLOGY, 1982, 95 (03) :763-770
[2]   DIFFERENCES AND SIMILARITIES IN DNA-BINDING PREFERENCES OF MYOD AND E2A PROTEIN COMPLEXES REVEALED BY BINDING-SITE SELECTION [J].
BLACKWELL, TK ;
WEINTRAUB, H .
SCIENCE, 1990, 250 (4984) :1104-1110
[3]   THE MUSCLE REGULATORY GENE, MYF-6, HAS A BIPHASIC PATTERN OF EXPRESSION DURING EARLY MOUSE DEVELOPMENT [J].
BOBER, E ;
LYONS, GE ;
BRAUN, T ;
COSSU, G ;
BUCKINGHAM, M ;
ARNOLD, HH .
JOURNAL OF CELL BIOLOGY, 1991, 113 (06) :1255-1265
[4]   THE SARCOMERIC ACTIN CARG-BINDING FACTOR IS INDISTINGUISHABLE FROM THE C-FOS SERUM RESPONSE FACTOR [J].
BOXER, LM ;
PRYWES, R ;
ROEDER, RG ;
KEDES, L .
MOLECULAR AND CELLULAR BIOLOGY, 1989, 9 (02) :515-522
[5]   PROMOTER UPSTREAM ELEMENTS OF THE CHICKEN CARDIAC MYOSIN LIGHT-CHAIN 2-A GENE INTERACT WITH TRANS-ACTING REGULATORY FACTORS FOR MUSCLE-SPECIFIC TRANSCRIPTION [J].
BRAUN, T ;
TANNICH, E ;
BUSCHHAUSENDENKER, G ;
ARNOLD, HH .
MOLECULAR AND CELLULAR BIOLOGY, 1989, 9 (06) :2513-2525
[6]   TRANSCRIPTIONAL ACTIVATION DOMAIN OF THE MUSCLE-SPECIFIC GENE-REGULATORY PROTEIN MYF5 [J].
BRAUN, T ;
WINTER, B ;
BOBER, E ;
ARNOLD, HH .
NATURE, 1990, 346 (6285) :663-665
[7]   MYF-6, A NEW MEMBER OF THE HUMAN GENE FAMILY OF MYOGENIC DETERMINATION FACTORS - EVIDENCE FOR A GENE-CLUSTER ON CHROMOSOME-12 [J].
BRAUN, T ;
BOBER, E ;
WINTER, B ;
ROSENTHAL, N ;
ARNOLD, HH .
EMBO JOURNAL, 1990, 9 (03) :821-831
[8]   DIFFERENTIAL EXPRESSION OF MYOGENIC DETERMINATION GENES IN MUSCLE-CELLS - POSSIBLE AUTOACTIVATION BY THE MYF GENE-PRODUCTS [J].
BRAUN, T ;
BOBER, E ;
BUSCHHAUSENDENKER, G ;
KOTZ, S ;
GRZESCHIK, KH ;
ARNOLD, HH .
EMBO JOURNAL, 1989, 8 (12) :3617-3625
[9]   MYOGENIN RESIDES IN THE NUCLEUS AND ACQUIRES HIGH-AFFINITY FOR A CONSERVED ENHANCER ELEMENT ON HETERODIMERIZATION [J].
BRENNAN, TJ ;
OLSON, EN .
GENES & DEVELOPMENT, 1990, 4 (04) :582-595
[10]   IDENTIFICATION OF A MYOCYTE NUCLEAR FACTOR THAT BINDS TO THE MUSCLE-SPECIFIC ENHANCER OF THE MOUSE MUSCLE CREATINE-KINASE GENE [J].
BUSKIN, JN ;
HAUSCHKA, SD .
MOLECULAR AND CELLULAR BIOLOGY, 1989, 9 (06) :2627-2640