Muscle Creatine Kinase Gene Research Articles

A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.

The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.

Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.

Factors Involved in Cardiogenesis and the Regulation of Cardiac-Specific Gene Expression

The delineation of the mechanisms that regulate cardiac gene expression is central to our understanding of cardiac growth and development. Much progress has been made toward the identification of factors involved in tissue-restricted gene expression, especially in skeletal muscle cells. However, the mechanisms regulating the expression of cardiac-specific genes remain less well understood. Certain homeodomain proteins have been implicated in commitment to the cardiac phenotype. Among the best characterized are the murine proteins Csx, Nkx-2.5, and Nkx-2.6, related to the protein tinman, which is essential for heart formation in Drosophila. The expression of these genes precedes that of cardiac-specific genes and is therefore believed to play a critical role in the development of the heart. The GATA proteins are a family of zinc finger proteins that are also expressed early in cardiac development and may act separately from, or in concert with, the homeodomain proteins as crucial regulators of heart development. The myosin heavy and light chain genes, the actin genes, the troponin genes, and the atrial natriuretic factor and muscle creatine kinase genes have served as excellent paradigms for the study of cardiac gene expression. Although differences in cis-acting elements and their behavior in binding assays have been observed between different genes, there exist similarities that are noteworthy. In this review, we will discuss the factors involved in the regulation of cardiac-specific gene expression in an attempt to provide a better understanding of the process of cardiogenesis.

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

A Novel Site in the Muscle Creatine Kinase Enhancer Is Required for Expression in Skeletal but Not Cardiac Muscle

Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5' tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues.

M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice.

The molecular pathways and regulatory molecules that underlie changes in gene transcription during mechanical overload of skeletal muscle remain obscure. To better understand this process, we have examined mouse muscle creatine kinase (MCK) gene expression in mechanically overloaded plantaris (OP) muscle of transgenic and nontransgenic mice. Northern blot analysis revealed that endogenous MCK-specific mRNA transcripts were decreased 150% in the OP muscles after 6 wk. To identify the MCK gene regions involved in the response to mechanical overload, three different mouse MCKCAT transgenes were studied by measuring chloramphenicol acetyltransferase (CAT assays) activity in OP and sham-operated (control plantaris) muscles. Mouse lines carrying (+enh206)117MCKCAT and -1256MCKCAT transgenes exhibited 30 and 40% lower CAT levels, whereas two mouse lines carrying -3300MCKCAT transgenes exhibited average decreases of 430%. Nearly identical results, including measurements of exogenous CAT mRNA, were obtained 2 days postoverload. Six weeks or 2 days of mechanical overload led to an average decrease in MM-CK isoprotein of 140%. These data provide evidence that mechanical overload induces changes in MCK gene expression that appear to be regulated by at least two portions of the MCK gene: the 206 base pair 5' enhancer and the -3,300 to -1,257 region.

Muscle Gene E-box Control Elements: EVIDENCE FOR QUANTITATIVELY DIFFERENT TRANSCRIPTIONAL ACTIVITIES AND THE BINDING OF DISTINCT REGULATORY FACTORS

The muscle creatine kinase gene enhancer contains two regulatory elements (MCK-R and MCK-L) with the consensus E-box sequence (CAnnTG). A myocyte specific protein complex, MEF1, binds the MCK-R site. MEF1 contains several basic H-L-H myogenic determination factors (MDFs), each dimerized with ubiquitous members of the bH-L-H family (e.g. E12/E47). We now demonstrate that the ubiquitous bH-L-H factor E2-2 is a major component of the endogenous MCK-R site specific complex. Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. However, we find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2-2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. The differential behavior of the MCK-L and MCK-R sites may be a general trait of E-box elements since one among several E-boxes in the MLC 1/3 enhancer also binds preferentially to the MCK-L factor. From our studies we now propose separate consensus sequences for MCK-R and MCK-L E-box types: AACAc/gc/gTGCa/t and GGa/cCANGTGGc/gNa/g. Our results suggest that while many muscle gene E-boxes are capable of binding the previously characterized spectrum of MDF/bH-L-H heterodimers in vitro, MCK-L type E-boxes probably bind qualitatively different factors in vivo.

Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice.

In extrahepatic tissues lipoprotein lipase (LPL) hydrolyzes triglycerides thereby generating FFA for tissue uptake and metabolism. To study the effects of increased FFA uptake in muscle tissue, transgenic mouse lines were generated with a human LPL minigene driven by the promoter of the muscle creatine kinase gene. In these mice human LPL was expressed in skeletal muscle and cardiac muscle, but not in other tissues. In proportion to the level of LPL overexpression, decreased plasma triglyceride levels, elevated FFA uptake by muscle tissue, weight loss, and premature death were observed in three independent transgenic mouse lines. The animals developed a severe myopathy characterized by muscle fiber degeneration, fiber atrophy, glycogen storage, and extensive proliferation of mitochondria and peroxisomes. This degree of proliferation suggests that FFA play an important role in the biogenesis of these organelles. Our experiments indicate that LPL is rate limiting for the supply of muscle tissue with triglyceride-derived FFA. Improper regulation of muscle LPL can lead to major pathological changes and may be important in the pathogenesis of some human myopathies. Muscle-specific LPL transgenic mouse lines will serve as a useful animal model for the investigation of myopathies and the biogenesis of mitochondria and peroxisomes.

Characterization of a glucocorticoid responsive element and identification of an AT-rich element that regulate the link protein gene.

The cartilage matrix is composed of characteristic components including type II collagen, aggrecan and link protein. In this paper, we report two DNA elements that regulate the link protein gene. Using transient transfection assays with link protein gene constructs in chondrocytes, chloramphenicol acetyl transferase (CAT) assays were used to measure the transcriptional activity of the link protein gene. Previously, we identified an enhancer-like activity within the first intron of the gene. In this paper, we report an active 34 bp (+1390 to +1424) fragment within this region that contains a glucocorticoid-like response element (GRE). Both deletion of, and site-specific mutations within this sequence motif reduced the dexamethasone-inducible activity. The GRE-like sequence from the rat link protein gene, or the homologous sequence from the human link protein gene were included in vectors containing the thymidine kinase promoter linked to the CAT gene (tkCAT). Both human and rat elements transferred the ability to respond to dexamethasone and hydrocortisone with a > 10-fold induction. Deletions through the promoter from -923 to -900 identified a second site required for both glucocorticoid and serum responsiveness. A four base substitution at this site resulted in a loss of serum responsiveness. This region contains an AT-rich element, similar to the AT-rich elements involved in homeotic protein regulation of the growth hormone gene and the muscle creatine kinase gene. Southwestern analysis using oligonucleotides containing the AT-rich element from the link protein gene or the muscle creatine kinase gene, identified a 32 kDa protein band from nuclear extracts of chick chondrocytes. Using these AT-rich oligonucleotides in band-shift analyses, nuclear extracts of chick sternal muscle, rat chondrosarcoma and chick sternal chondrocytes each showed formation of different complexes suggesting cell specificity. AT-rich elements have been identified as binding sites for homeodomain-containing proteins and can contribute to gene regulation by serum response factors. The identification of an AT-rich element in the link protein gene suggests similar functions for this element.

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene.

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene.

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.

Distinguishable patterns of protein-DNA interactions involving complexes of basic helix-loop-helix proteins.

Myogenic factors and TAL1 possess distinguishable DNA binding characteristics when they form a complex with basic helix-loop-helix (bHLH) proteins of class A. These characteristics were evident in electrophoretic mobility shift assays showing that complexes of myogenic factors and HTF4 displayed a relatively high affinity for the enhancer in the muscle creatine kinase gene, whereas TAL1 appeared to greatly attenuate the interaction of HTF4 with this enhancer. In addition, by forming a complex with HTF4 in solution, TAL1 could exert a negative effect on the interactions of HTF4 with elements that include E box motifs of microE2 (CAGCTG) and kappa E2/microE5 (CACCTG) type. Similarly, heterodimers containing TAL1 and the DNA binding domain of E47 exhibited a relatively weak affinity for microE2 and kappa E2/microE5 core motifs. The results of both studies invoked the hypothesis that in vivo TAL1 might act as a negative regulator of microE2 and kappa E2/microE5 sequence motifs by forming a complex with the products of the E2A and HTF4 genes. Support for this hypothesis was obtained by transient expression analyses where TAL1 was found to inhibit the activation effects produced by E2-5 and HTF4a on a reporter gene construct containing repeated microE2 and microE5 motifs, derived from the immunoglobulin gene enhancer.

Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

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.

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

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.

Muscle-specific expression of the acetylcholine receptor alpha-subunit gene requires both positive and negative interactions between myogenic factors, Sp1 and GBF factors.

The dependence of the muscle-specific enhancer of the acetylcholine receptor alpha-subunit gene on other domains of the promoter has been analysed by performing point mutagenesis and modular reconstitution of the enhancer--promoter sequences. The enhancer is inactive in the absence of the proximal region containing an Sp1 binding site and an overlapping G-C homopolymer binding factor site (referred to as GBF). The proximal region can be replaced by an Sp1 binding site from SV40 or an MEF-2 binding site from the muscle creatine kinase gene. Specific mutation of the Sp1 site markedly affects transactivation by CMD1 or myogenin. Mutation of the GBF binding site leads to higher promoter activity in primary cultures of chick myotubes or in quail fibroblasts. In addition, binding of a purified Sp1 protein prevents the binding of GBF in vitro. It is proposed that in the case of the alpha-subunit promoter, the myogenic factors activate transcription in cooperation with Sp1, and that GBF contributes to muscle-specific expression of the promoter by interfering with Sp1 binding in nonmuscle muscle cells or myoblasts.

Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.

Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.

The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation

A cellular phosphoprotein with an apparent molecular mass of 90 kd (p90) that forms a complex with both mutant and wild-type p53 protein has been characterized, purified, and identified. The protein was identified as a product of the murine double minute 2 gene ( mdm-2). The mdm-2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. To determine if mdm-2 could modulate p53 transactivation, a p53-responsive element from the muscle creatine kinase gene was employed. A wild-type p53-expressing plasmid enhanced the expression of the p53-responsive element when cotransfected into cells that contain no endogenous p53. When a cosmid expressing mdm-2 was transfected with this p53-expressing plasmid, the transactivation of the p53-responsive element was inhibited. Thus, a product of the mdm-2 oncogene forms a tight complex with the p53 protein, and the mdm-2 oncogene can inhibit p53-mediated transactivation.

Activity and enhancer binding factors for BK virus regulatory elements in differentiating embryonal carcinoma cells

We have studied cell type specificity of expression of the human papovavirus BK regulatory elements in undifferentiated and differentiated embryonal carcinoma (EC) cells as a model system. While the activity of the regulatory elements of this virus was marginal in undifferentiated cells, differentiation by retinoic acid and DMSO resulted in a dramatic increase in the activity. To correlate in vivo activity of the regulatory elements with interaction with cellular transcription factors, we performed DNase I footprinting experiments. A GC-rich region was protected in both undifferentiated and differentiated cells. An additional four protected sites were detected in retinoic acid-differentiated cells and at least one of these additional sites was weakly protected in DMSO-differentiated cells. The sequences of the differentiated cell type-specific protected regions showed homology to a nuclear factor 1 (NF-1) binding motif and to a muscle creatine kinase gene enhancer motif. The intensity, competition, and pattern of protection of these sites were different in the two differentiated cell types, suggesting the involvement of different transcription factors regulating the activity of BKV regulatory elements in the two cell types.

Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

Rabbit muscle creatine kinase: genomic cloning, sequencing, and analysis of upstream sequences important for expression in myocytes

Muscle creatine kinase (MCK) is a major enzyme of cellular energy metabolism that is expressed upon differentiation of myoblasts into myotubes. Previously we cloned and sequenced the entire rabbit enzyme cDNA which was used as a probe in these studies to obtain a genomic clone from a rabbit library. The transcription start site was identified by primer extension analysis and over 800 bp of 5' flanking DNA was sequenced. Comparison of this sequence with the published sequences from the upstream regions of the mouse MCK gene and the human MCK gene showed two conserved regions and a large intervening block of non-conserved sequence. The conserved regions are separated by about 800 bp in the mouse and by about 400 bp in the human, but are much closer (200 bp) in the rabbit. The upstream conserved region of the mouse gene encompasses a region possessing the properties of an enhancer and containing two MyoD binding sites; the downstream element is adjacent to the start of transcription. A set of of overlapping deletions of the 5' upstream DNA was fused to the CAT gene and transfected into mouse C2 myocytes, chick primary myocytes, and chick primary liver cells. Constructs which contained both conserved 5' regions were strongly expressed in C2 and chick myocytes, but were not expressed (above background) in primary liver cells. Surprisingly, while the upstream enhancer element was required for strong expression in C2 myocytes, it was less important for expression in chick myocytes. This suggests that there are important muscle-specific transcriptional signals in the proximal promoter region of mammalian MCK genes.