MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient.

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.

p27Kip1 is expressed transiently in developing myotomes and enhances myogenesis.

Vertebrate skeletal muscle development is characterized by tight coupling of muscle differentiation with cell cycle arrest in G1/G0. Key regulators of G1 progression are the G1 cyclin-dependent kinases, their positive regulators, the G1 cyclins, and their negative regulators, the cyclin-dependent kinase inhibitors (CDIs). Here we show that p27Kip1 protein, a G1 CDI, is expressed in a prominent but transient wave in the developing myotomes of the mouse embryo. We relate its expression to expression of MyoD and myogenin proteins, which are determination and differentiation class myogenic regulatory factors, respectively. Functional assays showed that ectopic p27 expression can powerfully enhance the efficiency of MyoD-initiated muscle differentiation in cell culture. When considered together with the myotomal expression patterns of p18, p21, and p57, these results suggest a model in which p27 acts as a "trigger" CDI while myoblasts are exiting the cell cycle and initiating differentiation. At later times, when p27 protein has been down-regulated, it is proposed that accumulation of p18, p21, and p57 maintain the differentiated myocytes in a postmitotic state.

Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells.

Repair and regeneration of adult skeletal muscle are mediated by satellite cells. In healthy muscle these rare mononucleate muscle precursor cells are mitotically quiescent. Upon muscle injury or degeneration, members of this self-renewing pool are activated to proliferate and then differentiate. Here we analyzed in single satellite cells the expression of a set of regulatory genes that are candidates for causal roles in satellite cell activation, maturation, and differentiation. Individual cells were identified as satellite cells and selected for analysis based on their physical association with single explanted myofibers or their position beneath the basal lamina in unperturbed muscle tissue. Using a multiplex single-cell RT-PCR assay we simultaneously monitored expression of all four MyoD family regulators of muscle determination and differentiation (MRFs) together with two candidate markers of satellite cell identity, c-met and m-cadherin. By making these measurements on large numbers of individual cells during the time course of satellite cell activation, we were able to define which expression states (possible combinations of the six genes) were represented and to specify how the representation of each state changed with time. Activated satellite cells began to express either MyoD or myf5 first among the MRFs; most cells then expressed both myf-5 and MyoD simultaneously; myogenin came on later in cells expressing both MyoD and myf5; and many cells ultimately expressed all four MRFs simultaneously. The results for fiber-associated satellite cells from either predominantly fast or slow muscles were indistinguishable from each other. The c-met receptor tyrosine kinase was also monitored because it is a candidate for mediating activation of quiescent satellite cells (Allen et al., 1995) and because it might also be a candidate molecular marker for satellite cells. A significant difficulty in studying mouse satellite cells has been the absence of molecular markers that could identify them in the quiescent state before expression of MRFs or desmin and distinguish them from fibroblasts. We show here that c-met receptor is present beneath the basal lamina on presumptive satellite cells in intact muscle and that c-met mRNA and protein are expressed by all myofiber-associated satellite cells from the time of explant through the course of activation, proliferation, and differentiation. c-met was not detected in muscle-derived fibroblasts or in other mononucleate cells from healthy muscle explants. When compared directly with m-cadherin, which has previously been suggested as a marker for quiescent satellite cells, m-cadherin mRNA was detected only in a small subset of satellite cells at early times after myofiber explant. However, at late times following activation (by 96 hr in this fiber culture system), c-met and m-cadherin were uniformly coexpressed. From the individual satellite cell expression types observed, a model of the satellite cell population at rest and during the time course of activation was generated.

Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.

Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

BRCA1 mRNA is expressed highly during meiosis and spermiogenesis but not during mitosis of male germ cells.

The 17q-linked breast and ovarian cancer susceptibility gene (BRCA1) is believed to function as a tumor suppressor gene (Miki et al., 1994). In this report BRCA1 RNA expression has been analysed in adult mouse tissues with detailed attention to its expression in prepuberal and adult testis. Measurements of BRCA1 mRNA levels in highly purified somatic cells of the testis and in staged germ cells showed that high level BRCA1 mRNA expression is limited to the germ cells. Within the germ cell lineage, the high level expression was detected in meiotic cells, specifically pachytene spermatocytes and in post-meiotic round spermatids. This is in contrast to premeiotic germ cells which were found to express little or no BRCA1 mRNA. These observations, considered together with recent data on the expression of BRCA1 in breast epithelium, argues against a function for BRACA1 in early progenitor cells in both tissues and cells attention instead to roles intimately associated with terminal differentiation or with final rounds of cell division.

Evidence for developmentally programmed transdifferentiation in mouse esophageal muscle.

Transdifferentiation is a relatively rare phenomenon in which cells of one differentiated type and function switch to a second discrete identity. In vertebrate embryos, smooth muscle and skeletal muscle are distinct tissues that arise from separate compartments of the mesoderm. The musculature of the mouse esophagus was found to undergo a conversion from smooth muscle in the fetus to skeletal muscle during early postnatal development. The switch from smooth to skeletal muscle features the transitory appearance of individual cells expressing a mixed phenotype, which suggests that this conversion is a result of programmed transdifferentiation.

Inhibition of Klenow fragment DNA polymerase on double-helical templates by oligonucleotide-directed triple-helix formation.

We have examined the capacity of oligonucleotide-directed triple helices to block the progress of primer extension by DNA polymerase. Occupancy of the major groove of a double-helical DNA substrate obstructed Klenow fragment progress at sites that map near the proximal boundary between duplex and triplex. Among a family of related third-strand oligonucleotides that all stably occupied the target duplex in the absence of polymerase, those forming longer triplexes were more effective polymerase inhibitors than shorter complexes. Kinetic analysis revealed that the triple-helical complex provided an effective blockade for times of at least 20 min. These observations provide the basis for considering and further dissecting repair DNA polymerase function and mechanism by using various defined local three-stranded DNA structures as probes.

Phosphorothioate oligonucleotide-directed triple helix formation.

Phosphorothioate oligodeoxyribonucleotides were tested for their ability to recognize double-helical DNA in two distinct triple helix motifs. Purine-rich oligonucleotides containing a diastereomeric mixture of phosphorothioate or stereoregular (all RP) phosphorothioate linkages are shown to form triple-helical complexes with affinities similar to those of the corresponding natural phosphodiester oligonucleotides. In contrast, pyrimidine-rich phosphorothioate oligonucleotides containing a mixture of diastereomeric or stereoregular (all RP) linkages do not bind to double-helical DNA with measurable affinity. These observations have implications for triple helix structure and for biological applications.

Cloning of mid-G1 serum response genes and identification of a subset regulated by conditional myc expression.

The emergence of cells from a quiescent G0 arrested state into the cell cycle is a multistep process that begins with the immediate early response to mitogens and extends into a specialized G1 phase. Many immediate early serum response genes including c-fos, c-myc, and c-jun are transcriptional regulators. To understand their roles in regulating cell cycle entry and progression, the identities of their regulatory targets must be determined. In this work we have cloned cDNA copies of messenger RNAs that are either up- or down-regulated at a mid-G1 point in the serum response (midserum-response [mid-SR]). The mid-SR panel is expected to include both direct and indirect targets of immediate early regulators. This expectation was confirmed by the identification of several transcriptional targets of conditional c-myc activity. In terms of cellular function, the mid-SR class is also expected to include execution genes needed for progression through G1 and into S-phase. DNA sequence data showed that the mid-SR panel included several genes already known to be involved in cell cycle progression or growth transformation, suggesting that previously unknown cDNAs in the same group are good candidates for other G1 execution functions. In functional assays of G0-->S-phase progression, c-myc expression can bypass the requirement for serum mitogens and drive a large fraction of G0 arrested cells through G1 into S-phase. However, beyond this general similarity, little is known about the relation of a serum-driven progression to a myc-driven progression. Using the mid-SR collection as molecular reporters, we found that the myc driven G1 differs qualitatively from the serum driven case. Instead of simply activating a subset of serum response genes, as might be expected, myc regulated some genes inversely relative to serum stimulation. This suggests that a myc driven progression from G0 may have novel properties with implications for its action in oncogenesis.

Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

Skeletal muscle phenotypes initiated by ectopic MyoD in transgenic mouse heart.

Forced expression of the myogenic regulatory gene MyoD in many types of cultured cells initiates their conversion into skeletal muscle. It is not known, however, if MyoD expression serves to activate all or part of the skeletal muscle program in vivo during animal development, nor is it known how limiting the influences of cellular environment may be on the regulatory effects of MyoD. To begin to address these issues, we have produced transgenic mice which express MyoD in developing heart, where neither MyoD nor its three close relatives--myogenin, Myf-5, and MRF4/herculin/Myf-6--are normally expressed. The resulting gross phenotype in offspring from multiple, independent transgenic founders includes abnormal heart morphology and ultimately leads to death. At the molecular level, affected hearts exhibit activation of skeletal muscle-specific regulatory as well as structural genes. We conclude that MyoD is able to initiate the program that leads to skeletal muscle differentiation during mouse development, even in the presence of the ongoing cardiac differentiation program. Thus, targeted misexpression of this tissue-specific regulator during mammalian embryogenesis can activate, either directly or indirectly, a diverse set of genes normally restricted to a different cell lineage and a different cellular environment.

Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting.

We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference leads to significantly improved genomic sequencing, methylation analysis, and in vivo footprinting with LMPCR. These improvements include representation of all bands with more uniform intensity, clear visualization of previously difficult regions of sequence, and reduction in the occurrence of spurious bands. It also simplifies the use of DNase I cut DNA for LMPCR footprinting.

c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.

Tissue-specific expression from a compound TATA-dependent and TATA-independent promoter.

We have found that the mouse metallothionein-I (MT-I) gene promoter functions in an unusual, compound manner. It directs both TATA-dependent and TATA-independent modes of transcription in vivo. The TATA-dependent message is initiated at the previously characterized +1 transcription start site and is the predominant species in most tissues. In many cell types it is metal inducible. The TATA-independent initiation sites are distributed over the 160 bp upstream of the previously characterized +1 start site, and the RNA products are present in all tissues examined. Only in testis, however, do the TATA-independent transcripts predominate, accumulating to highest levels in pachytene-stage meiotic cells and early spermatids. Unlike the TATA-dependent +1 transcript, these RNAs are not induced by metal, even in cultured cells in which the +1 species is induced. Transfection studies of site-directed mutants show that destruction of the TATA element drastically alters the ratio of the two RNA classes in cells in which the +1 transcripts normally dominates. In TATA-minus mutants, the TATA-independent RNAs become the most prevalent, although they remain refractory to metal induction. Thus, the MT-I promoter utilizes two different types of core promoter function within a single cell population. The two different types of core promoter respond very differently to environmental stimuli, and the choice between them appears to be regulated in a tissue-specific fashion.

Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA.

Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k2obs, is 1.8 x 10(3) +/- 1.9 x 10(2) L.(mol of oligomer-1.s-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.

Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin.

Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family.

The C proteins, C1 and C2, are major constituents of the heterogeneous nuclear RNA (hnRNA) ribonucleoprotein (hnRNP) complex in vertebrates. C1 and C2 are antigenically related phosphoproteins that are in contact with hnRNA in intact cells and bind to RNA tightly in vitro. A cDNA clone for the C proteins was isolated by immunological screening of a human lambda gt11 expression vector cDNA library with monoclonal antibodies. The lacZ-cDNA fusion protein is recognized by two different anti-C protein monoclonal antibodies. HeLa cell mRNA that was hybrid-selected with the cDNA clone (1.1 kilobases long) was translated in vitro and yielded both the C1 and C2 proteins (41 and 43 kDa, respectively). RNA blot analysis showed strong hybridization to two polyadenylylated transcripts, of about 1.4 kb and 1.9 kb, in human cells. Genomic DNA blot analysis showed multiple hybridizing restriction fragments in human and mouse, and homologous DNA sequences are found across eukaryotes from human to yeast. These findings suggest that the sequences encoding the hnRNP C proteins are members of a conserved gene family and they open the way for detailed molecular and genetic studies of these proteins.

Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA.

Anti-sense thymidine kinase (TK) RNA was expressed as part of a chimeric dihydrofolate reductase (DHFR) anti-sense TK transcript. High level expression was obtained by selection of cells resistant to progressively higher levels of methotrexate, provided by overproduction of DHFR. The result was a concomitant increase in intracellular anti-sense TK RNA level owing to its presence on the same transcription unit as DHFR. In several cell lines expressing high levels of anti-sense TK RNA, thymidine kinase activity was reduced by 80%-90%. RNA:RNA duplexes were detected in the nuclear fraction. The results suggest a mechanism for diminution of TK activity; anti-sense RNA hybridizes with sense TK RNA in the nucleus, and duplex containing TK transcripts fail to enter the cytoplasm with normal efficiency.