Multiple Regulatory Elements Contribute Differentially to Muscle Creatine Kinase Enhancer Activity in Skeletal and Cardiac Muscle

Abstract

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.

ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation

“…In support of this possibility, we found that a number of muscle‐restricted genes contain ZEB consensus binding sequences [i.e. the bHLH MRF genes (Edmondson et al ., 1992; Tapscott et al ., 1992; Black et al ., 1995), muscle creatine kinase (MCK) (Amacher et al ., 1993) and the acetylcholine receptor δ subunit (Simon and Burden, 1993)]. Indeed, ZEB has been shown to interact with the MEF‐1 site in the MCK gene (Genetta et al ., 1994; Sekido et al ., 1994).…”

“…Silencer activity is dependent upon two consensus ZEB sites (CACCTG) at −361 and −399. Consensus ZEB sites from other muscle genes, such as MCK (the MEF‐1 site; Amacher et al ., 1993) and the acetylcholine receptor δ subunit (the E1 box; Simon and Burden, 1993), showed similar silencer activity (Figure 3B). In addition, non‐E box ZEB sites (Genetta et al ., 1994; Sekido et al ., 1994) were also effective repressors of −76CAT (results not shown).…”

“…PGXSV contains four Gal4 sites upstream of the SV40 enhancer and E1B TATA box (Chow et al ., 1996). The reporter p72‐4E‐5G was created by cloning the CAT gene into Eco RV and Bgl II sites of pSP72 (Promega), the E1B TATA box into the Xba I and Bam HI sites, four MEF‐1 E boxes from the MCK gene (Amacher et al ., 1993) into Xba I and Hin dIII sites and five Gal4 sites into the Xho I site (∼120 bp upstream of the E boxes). To construct LexA–ZEB (L‐ZEB), full‐length ZEB was cloned in the Bam HI and Xba I sites of PBXL‐3 (Weintraub et al ., 1995).…”

“…E361 and E399 from the α4 promoter (Rosen et al ., 1994), three copies of the MEF‐1 site from the MCK gene (Amacher et al ., 1993) or three copies of the E1 box from the acetylcholine receptor δ subunit gene (Simon and Burden, 1993) were cloned in the Kpn I and Apa I sites of the 76α4CAT construct (Rosen et al ., 1994).…”

A Hormone-Encoding Gene Identifies a Pathway for Cardiac but Not Skeletal Muscle Gene Transcription

“…This fact has led many investigators to postulate the existence of similar mechanisms of gene expression in heart and skeletal muscles. Consistent with this hypothesis, studies from several laboratories revealed that transcription of many of these muscle-specific genes in heart and skeletal muscle requires similar AT-rich regulatory elements termed MEF-2 (myocyte-specific enhancer-binding factor 2) (1,26,41,42). A family of transcription factors which interact with this element and are present in mesodermal tissues, including the heart, have recently been cloned (52,73).…”