Bruce M. Paterson

Transfection of a DNA locus that mediates the conversion of 10T1 2 fibroblasts to myoblasts

Stable myoblast cell lines were isolated after a brief exposure of mouse fibroblasts (10T1/2 cells) to 5-azacytidine. We show that transfection of 10T1/2 cells with DNA from these azacytidine-induced myoblasts (or from mouse C2C12 myoblasts) results in myogenic conversion of approximately 1 in 15,000 transfected colonies. In contrast, transfection of 10T1/2 cells with DNA from nonmyogenic cells (parental 10T1/2 cell DNA) does not give rise to myoblast colonies. These results indicate that an azacytidine-induced structural modification (presumably demethylation) in the DNA of a single locus is sufficient to convert 10T1/2 cells into determined myoblasts.

Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures

Messenger RNA which stimilates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.

Coupling of the cell cycle and myogenesis through the cyclin D1-dependent interaction of MyoD with cdk4.

Proliferating myoblasts express the muscle determination factor, MyoD, throughout the cell cycle in the absence of differentiation. Here we show that a mitogen‐sensitive mechanism, involving the direct interaction between MyoD and cdk4, restricts myoblast differentiation to cells that have entered into the G0 phase of the cell cycle under mitogen withdrawal. Interaction between MyoD and cdk4 disrupts MyoD DNA‐binding, muscle‐specific gene activation and myogenic conversion of 10T1/2 cells independently of cyclin D1 and the CAK activation of cdk4. Forced induction of cyclin D1 in myotubes results in the cytoplasmic to nuclear translocation of cdk4. The specific MyoD–cdk4 interaction in dividing myoblasts, coupled with the cyclin D1‐dependent nuclear targeting of cdk4, suggests a mitogen‐sensitive mechanism whereby cyclin D1 can regulate MyoD function and the onset of myogenesis by controlling the cellular location of cdk4 rather than the phosphorylation status of MyoD.

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three of four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.

Regulation of MyoD function in the dividing myoblast

Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing. Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation. Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation. Co‐expression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter. Similarly, transfection of myoblasts with the cyclin‐dependent kinase (cdk) inhibitors p16 and p21 supports some muscle‐specific gene expression even in growth medium. Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks. We review evidence coupling growth status, the cell cycle and myogenesis. We describe a novel mitogen‐sensitive mechanism that involves the cyclin D1‐dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen‐sensitive manner.

Direct inhibition of G1 cdk kinase activity by MyoD promotes myoblast cell cycle withdrawal and terminal differentiation

MyoD has been proposed to facilitate terminal myoblast differentiation by binding to and inhibiting phosphorylation of the retinoblastoma protein (pRb). Here we show that MyoD can interact with cyclin‐dependent kinase 4 (cdk4) through a conserved 15 amino acid (aa) domain in the C‐terminus of MyoD. MyoD, its C‐terminus lacking the basic helix–loop–helix (bHLH) domain, or the 15 aa cdk4‐binding domain all inhibit the cdk4‐dependent phosphorylation of pRb in vitro. Cellular expression of full‐length MyoD or fusion proteins containing either the C‐terminus or just the 15 aa cdk4‐binding domain of MyoD inhibit cell growth and pRb phosphorylation in vivo. The minimal cdk4‐binding domain of MyoD fused to GFP can also induce differentiation of C2C12 muscle cells in growth medium. The defective myogenic phenotype in MyoD‐negative BC3H1 cells can be rescued completely only when MyoD contains the cdk4‐binding domain. We propose that a regulatory checkpoint in the terminal cell cycle arrest of the myoblast during differentiation involves the modulation of the cyclin D cdk‐dependent phosphorylation of pRb through the opposing effects of cyclin D1 and MyoD.

Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coli, corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function. Furthermore, D-elp1 depletion results in increased steady state levels of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast, although Dcr-2 depletion results in increased transposon RNA levels and a reduction in the corresponding endo siRNAs, there is no change in the transposon antisense RNA levels. In D-elp1 null third instar larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly with Dcr-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. These results identify an aspect of the RNAi pathway in Drosophila that suggest transposon derived endo siRNAs, critical for transposon suppression, are produced, in part, in a D-elp1 dependent step that converts transposon RNA into dsRNA that is subsequently processed by Dcr-2. The generality of this mechanism in genome defense and RNA silencing in higher eukaryotes is suggested.

COMPLETE CDNA SEQUENCE AND TISSUE LOCALIZATION OF N-RAP, A NOVEL NEBULIN-RELATED PROTEIN OF STRIATED MUSCLE

We have cloned and sequenced the full-length cDNA of N-RAP, a novel nebulin-related protein, from mouse skeletal muscle. The N-RAP message is specifically expressed in skeletal and cardiac muscle, but is not detected by Northern blot in non-muscle tissues. The full-length N-RAP cDNA contains an open reading frame of 3,525 base pairs which is predicted to encode a protein of 133 kDa. A 587 amino acid region near the C-terminus is 45% identical to the actin binding region of human nebulin, containing more than 2 complete 245 residue nebulin super repeats. The N-terminus contains the consensus sequence of a cysteine-rich LIM domain, which may function in mediating protein-protein interactions. These data suggest that the encoded protein may link actin filaments to some other proteins or structure. We expressed full-length N-RAP in Escherichia coli, as well as the nebulin-like super repeat region of N-RAP (N-RAP-SR) and the region between the LIM domain and N-RAP-SR (N-RAP-IB). An anti-N-RAP antibody raised against a 30 amino acid peptide corresponding to sequence from N-RAP-IB detected recombinant N-RAP and N-RAP-IB, but failed to detect N-RAP-SR. This antibody specifically identified a 185 kDa band as N-RAP on immunoblots of mouse skeletal and cardiac muscle proteins. In an assay of actin binding to electrophoresed and blotted proteins, we detected significant actin binding to expressed nebulin super repeats and N-RAP-SR, but only a trace amount of binding to N-RAP-IB. In immunofluorescence experiments, N-RAP was found to be localized at the myotendinous junction in mouse skeletal muscle and at the intercalated disc in cardiac muscle. Based on its domain organization, actin binding properties, and tissue localization, we propose that N-RAP plays a role in anchoring the terminal actin filaments in the myofibril to the membrane and may be important in transmitting tension from the myofibrils to the extracellular matrix.

Negative control of the helix-loop-helix family of myogenic regulators in the NFB mutant

We have characterized a nondifferentiating mouse muscle cell line, NFB, that represses the activity of the helix-loop-helix (HLH) family of myogenic regulators, yet expresses sarcomeric actins. The NFB MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts, the activity of these regulators is also repressed:myogenesis is not induced in 10T1/2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. We conclude that the myogenic HLH regulators are not required for sarcomeric actin gene activation and that myogenesis is subject to dominant-negative control.

A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis

Satellite cells are skeletal muscle stem cells that provide myogenic progeny for myofiber growth and repair. Temporal expression of muscle regulatory factors (MRFs) and the paired box transcription factor Pax7 defines characteristic phases of proliferation (Pax7+/MyoD+/myogenin−) and differentiation (Pax7−/MyoD+/myogenin+) during myogenesis of satellite cells. Here, using bromodeoxyuridine (BrdU) labeling and triple immunodetection, we analyzed expression patterns of Pax7 and the MRFs MyoD, Myf5, or myogenin within S phase myoblasts prepared from posthatch chicken muscle. Essentially, all BrdU incorporation was restricted to Pax7+ cells, of which the majority also expressed MyoD. The presence of a minor BrdU+/Pax7+/myogenin+ population in proliferation stage cultures suggests that myogenin up‐regulation is alone insufficient for terminal differentiation. Myf5 was detected strictly within Pax7+ cells and decreased during S phase while MyoD presence persisted in cycling cells. This study provides novel data in support of a unique role for Myf5 during posthatch myogenesis. Developmental Dynamics 238:1001–1009, 2009.