Muscle-specific Regulatory Regions Research Articles

Cdk9‐55: A new player in muscle regeneration

Adult skeletal muscle contains a specialized population of myogenic quiescent stem cells, termed satellite cells, which contribute to repair myofibers after injury. During muscle regeneration, satellite cells exit their normal quiescent state, proliferate, activating MyoD and Myf-5 expression, and finally differentiate and fuse to reconstitute the injured muscle architecture. We have previously reported that cdk9 is required for myogenesis in vitro by activating MyoD-dependent transcription. In myoblasts induced to differentiate, MyoD recruits cdk9 on the chromatin of muscle-specific regulatory regions. This event correlates with chromatin-modifying enzyme recruitment and phosphorylation of cdk9-specific target residues at the carboxyl-terminal domain of RNA polymerase II. Here we report that a second cdk9 isoform, termed cdk9-55, plays a fundamental role in muscle regeneration and differentiation in vivo. This alternative form is specifically induced in injured myofibers and its activity is strictly required for the completion of muscle regeneration process.

Quantitative proteomic identification of six4 as the trex-binding factor in the muscle creatine kinase enhancer.

Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of approximately 900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: identification of a novel muscle-preferred regulatory element.

To further our understanding of the role of stress proteins in development as well as in adaptation of fish to adverse environmental conditions, we undertook molecular analyses of stress protein encoding genes from the hermaphroditic teleost Rivulus marmoratus. We isolated a genomic clone containing the Hsc71 gene (rm-hsc71m) and its upstream sequences. rm-Hsc71m is not induced by external stress, but is enriched in a tissue-specific manner during early development. In adult, the strongest expression appeared in skeletal muscle, whereas lower expression was seen in the gill, eye and brain. To understand the regulatory basis of high muscle expression of rm-hsc71m, transfection of R.marmoratus muscle tissue was performed using 5' deletion fragments containing the rm-hsc71m promoter driving EGFP expression. An upstream region from -2.7 to -1.9 kb was identified as a muscle-specific regulatory region. Within this region, we identified at least three sites with the novel sequence TGTnACA interacting with a fish muscle factor having an M(r) of 32 000. Our data indicate that rm-hsc71m expression in skeletal muscle is controlled by a muscle-specific regulatory element containing this novel motif.

Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family

A novel human potassium channel gene was identified and isolated. The maximal open reading frame encodes a protein of 456 amino acids. The predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel protein Kv1.7 (Kcna7), which plays an important role in the repolarization of cell membranes. Based on the high similarity, the human gene has been classified as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identified a pore region characteristic of potassium channels and six membrane-spanning domains. Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle, heart and kidney. However, it is expressed at lower level in other tissues, including liver. A single mRNA isoform was observed, with a size of approximately 4.5 kb. Using fluorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR3000). A genomic sequence was identified in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-specific regulatory region. Initial comparison to the published murine Kcna7 cDNA suggested a different N-terminal sequence for the human protein, however, further analysis suggests that the original mouse sequence contained an error or an unusual polymorphism.

Two upstream enhancers collaborate to regulate the spatial patterning and timing of MyoD transcription during mouse development.

MyoD is a member of the basic-helix-loop-helix (bHLH) transcription factor family, which regulates muscle determination and differentiation in vertebrates. While it is now well established that the MyoD gene is regulated by Sonic hedgehog, Wnts, and other signals, it is not known how MyoD transcription is initiated and maintained in response to these signals. We have investigated the cis control of MyoD expression to identify and characterize the DNA targets that mediate MyoD transcription in embryos. By monitoring lacZ reporter gene expression in transgenic mice, we show that regulatory information contained in 24 kb of human MyoD 5' flanking sequence is sufficient to accurately control MyoD expression in embryos. Previous studies have identified two muscle-specific regulatory regions upstream of MyoD, a 4-kb region centered at -20 kb (designated fragment 3) that contains a highly conserved 258-bp core enhancer sequence, and a more proximal enhancer at -5 kb, termed the distal regulatory region (DRR), that heretofore has been identified only in mice. Here, we identify DRR-related sequences in humans and show that DRR function is conserved in humans and mice. In addition, transcriptional activity of MyoD 5' flanking sequences in somites and limb buds is largely a composite of the individual specificities of the two enhancers. Deletion of fragment 3 resulted in dramatic but temporary expression defects in the hypaxial myotome and limb buds, suggesting that this regulatory region is essential for proper temporal and spatial patterning of MyoD expression. These data indicate that regulatory sequences in fragment 3 are important targets of embryonic signaling required for the initiation of MyoD expression.

Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activated the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also showed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR were necessary and sufficient to mediate their physical interaction. Our results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression.

MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products.

The myocyte-specific enhancer-binding factor MEF-2 is a nuclear factor that interacts with a conserved element in the muscle creatine kinase and myosin light-chain 1/3 enhancers (L. A. Gossett, D. J. Kelvin, E. A. Sternberg, and E. N. Olson, Mol. Cell. Biol. 9:5022-5033, 1989). We show in this study that MEF-2 is regulated by the myogenic regulatory factor myogenin and that mitogenic signals block this regulatory interaction. Induction of MEF-2 by myogenin occurs in transfected 10T1/2 cells that have been converted to myoblasts by myogenin, as well as in CV-1 kidney cells that do not activate the myogenic program in response to myogenin. Through mutagenesis of the MEF-2 site, we further defined the binding site requirements for MEF-2 and identified potential MEF-2 sites within numerous muscle-specific regulatory regions. The MEF-2 site was also found to bind a ubiquitous nuclear factor whose binding specificity was similar to but distinct from that of MEF-2. Our results reveal that MEF-2 is controlled, either directly or indirectly, by a myogenin-dependent regulatory pathway and suggest that growth factor signals suppress MEF-2 expression through repression of myogenin expression or activity. The ability of myogenin to induce MEF-2 activity in CV-1 cells, which do not activate downstream genes associated with terminal differentiation, also demonstrates that myogenin retains limited function within cell types that are nonpermissive for myogenesis and suggests that MEF-2 is regulated independently of other muscle-specific genes.

Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products.

The myocyte-specific enhancer-binding factor MEF-2 is a nuclear factor that interacts with a conserved element in the muscle creatine kinase and myosin light-chain 1/3 enhancers (L. A. Gossett, D. J. Kelvin, E. A. Sternberg, and E. N. Olson, Mol. Cell. Biol. 9:5022-5033, 1989). We show in this study that MEF-2 is regulated by the myogenic regulatory factor myogenin and that mitogenic signals block this regulatory interaction. Induction of MEF-2 by myogenin occurs in transfected 10T1/2 cells that have been converted to myoblasts by myogenin, as well as in CV-1 kidney cells that do not activate the myogenic program in response to myogenin. Through mutagenesis of the MEF-2 site, we further defined the binding site requirements for MEF-2 and identified potential MEF-2 sites within numerous muscle-specific regulatory regions. The MEF-2 site was also found to bind a ubiquitous nuclear factor whose binding specificity was similar to but distinct from that of MEF-2. Our results reveal that MEF-2 is controlled, either directly or indirectly, by a myogenin-dependent regulatory pathway and suggest that growth factor signals suppress MEF-2 expression through repression of myogenin expression or activity. The ability of myogenin to induce MEF-2 activity in CV-1 cells, which do not activate downstream genes associated with terminal differentiation, also demonstrates that myogenin retains limited function within cell types that are nonpermissive for myogenesis and suggests that MEF-2 is regulated independently of other muscle-specific genes.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.