Defective T-cell activation is associated with augmented transforming growth factor Beta sensitivity in mice with mutations in the Sno gene.

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo.

Proto-oncogene Sno expression, alternative isoforms and immediate early serum response.

The mouse Sno gene, a Ski proto-oncogene homolog, expresses two isoforms, SnoN and SnoN2 (also called sno -dE3), which differ from each other in a location downstream from the site of alternative splicing previously described in the human SNO gene. SnoN2 is missing a 138 nt coding segment present in mouse SnoN and human SNON . We have cloned and sequenced the human ortholog of mouse SnoN2 , the existence of which was predicted from conservation of the alternative splice donor site that produces the SnoN2 isoform. Mouse SnoN2 and SnoN are expressed throughout embryonic development, in neonatal muscle and in many adult tissues. SnoN2 is the major species in most tissues, but SnoN and SnoN2 are expressed at approximately equal levels in brain. In human tissues, SNON2 is the less abundantly expressed isoform. Expression of mouse SnoN and SnoN2 mRNAs is induced with immediate early kinetics upon serum stimulation of quiescent fibroblasts, even in the presence of the protein synthesis inhibitor cycloheximide, while Ski is not. Interestingly, although both isoforms of Sno are induced, SnoN2 induction is much higher than SnoN . These data are consistent with a role for Sno in the response to proliferation stimuli.

Expression of basic helix-loop-helix transcription factors in explant hematopoietic progenitors.

The basic helix-loop-helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl-1, E12/E47, and ld-1, in individual lineage-defined progenitors and hematopoietic growth factor-dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage-defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT-PCR. SCL and E12/E47 were expressed in the vast majority of tri-, bi-, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl-1 showed a more restricted pattern of expression, although expression was seen in some bi- and unilineage progenitors. No expression was detected in erythroid, erythroid-megakaryocyte-macrophage, macrophage-neutrophil, macrophage, or megakaryocytic progenitors. Id-1, an inhibitory bHLH transcription factor, was also widely expressed in all bi- and unilineage progenitors; only the trilineage erythroid-megakaryocyte-macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id-1, but not E12/E47, mRNA was increased in FDC-P1 and MO7E hematopoietic cell lines after exposure to IL-3 or GM-CSF. Id-1, E12, and lyl-1 showed marked variation at different points in cell cycle in isoleucine-synchronized FDC-P1 cells. These results suggest that SCL, lyl-1, E12/E47, and Id-1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle.

The ski/sno protooncogene family in hematopoietic development.

The ski/sno protooncogenes encode nuclear proteins that may act as transcription factors. We examined ski and sno mRNA expression in hemolymphopoietic lineages. The ski protooncogene is expressed in B- and T-lineage cells, mature macrophages, and mast cells. In normal murine marrow-derived progenitors analyzed by single-cell reverse transcription-polymerase chain reaction (RT-PCR), ski expression is limited to dual-lineage megakaryocyte/erythrocyte colony-starts. Expression of sno is more limited than ski in mature cells; it is expressed in T lymphopoietic cells, but not in B-lineage cells. The sno protooncogene is expressed more widely than ski in myeloid progenitors, as it is found consistently in tri-, dual-, and single-lineage progenitors. Both ski and sno are cell cycle-regulated in synchronized factor-dependent mouse myeloid cells. Expression of ski mRNA peaks in mid G1 in cells synchronized by isoleucine deprivation in the presence of growth factor, but falls off rapidly when growth factor is withdrawn. Expression of sno mRNA is maximal in early to mid G1 and then oscillates as the cells continue through cycle. These results suggest that the ski/sno protooncogenes play a role in hematopoiesis, growth factor responses, and cell cycle-regulation, with the two members of the family showing differing properties.

SnoI, a novel alternatively spliced isoform of the ski protooncogene homolog, sno.

We have cloned and sequenced a novel human isoform of sno, snoI for insertion. SnoI contains 1330 nucleotides inserted in place of 7 nucleotides of the snoN mRNA. Sno is a member of the ski protooncogene family, which has been implicated in muscle development. The two previously known sno alternatively spliced isoforms are snoN (684 amino acids), and snoA (415 amino acids); snoI encodes a truncated isoform of 399 amino acids (44,298 MW). Southern blot experiments show that snoI contains a third alternative exon from the sno gene; a single sno gene can express all three isoforms of sno by alternative splicing. All three isoforms contain the region that is most similar to the ski proto-oncogene. The relationship between snoI and snoN is analogous to that between delta fosB and fosB, where a truncated form of the fosB transcription factor is produced by alternative splicing. We find conservation of human snoI-specific sequences in several mammalian species, in monkey, dog, cow, rabbit and pig, but not in rodents, whereas the common portion of the sno gene is conserved in all vertebrate species tested. SnoN, snoA, and ski mRNAs accumulate in many human tissues including skeletal muscle; the snoI alternative mRNA accumulates more specifically in skeletal muscle. SnoI is also expressed in rhabdomyosarcoma tumor, a tumor that contains differentiated skeletal muscle. The tissue-specific alternative splicing of human snoI, an mRNA in the ski/sno gene family, and the presence of sno mRNAs in muscle are consistent with a proposed role for the sno oncogene in muscle gene regulation.

Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice.

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that "niches" need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

Ectopic expression of MyoD1 in mice causes prenatal lethalities.

A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether MyoD1 is a dominant muscle regulator in vivo, mouse fertilized eggs were microinjected with a beta-actin/MyoD1 gene. Ectopic expression of MyoD1 during mouse embryogenesis led to embryonic lethalities, the cause of which is not known. Transgenic embryos died before midgestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control littermates, differentiated normally into tissues representative of all three germ layers. In most transgenic embryos there was no indication of myogenic conversion. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. Forced expression of MyoD1 was associated with the activation of myogenin and MLC2 (but not myf5 or MRF4) genes in non-muscle cell types, demonstrating the dominant regulatory function of MyoD1 during development. These results demonstrate that ectopic MyoD1 expression and activation of myogenin and MLC2 have no significant effects in the determination of cell lineages or the developmental fate of differentiated mesodermal and ectodermal cell lineages.

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.

The consequences of a constitutive expression of MyoD1 in ES cells and mouse embryos.

A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones tested, carrying single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 genes but not the endogenous MyoD1, MRF4, myf5, skeletal muscle actin or myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers was observed only when the transfected cells were allowed to differentiate, via embryoid bodies, in low mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and myf5 genes and in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of MyoD1 was not required for myogenesis. Interestingly, no preferential myogenesis was observed when the transfected ES cells were allowed to differentiate in vivo to teratocarcinomas. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function. Second, MyoD1 function in ES cells, even under environmental conditions that favour differentiation, is not dominant (incomplete penetrance). Third, that the exogenous MyoD1 transactivates the endogenous myogenin and MLC2 genes in ES cells. No live transgenic mice could be produced following microinjection of the beta-actin/MyoD1 gene into the pronuclei of fertilized eggs. Transgenic embryos died before mid gestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control litermates, differentiated into tissues representative of all three germ layers. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. These results demonstrate again that MyoD1 is not a dominant regulatory factor.

Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway.

Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.

A novel hybrid alpha-tropomyosin in fibroblasts is produced by alternative splicing of transcripts from the skeletal muscle alpha-tropomyosin gene.

A single alpha-tropomyosin gene in the Japanese quail exhibits tightly controlled expression of multiple isoforms by an alternative splicing mechanism. We have isolated two full length cDNA clones, cC401 and cC402, from quail embryonic myofibers which differentiated in culture. cC402 is the major skeletal muscle alpha-tropomyosin, expressed in adult skeletal muscle and in embryonic muscle culture. cC401 exhibits a novel hybrid primary structure; smooth muscle structure at the carboxyl terminus is combined with skeletal muscle primary structure for the amino-terminal 90% of the protein. The cC401-type tropomyosin, unlike other smooth muscle or cytoskeletal tropomyosins, thus retains two muscle-type troponin-binding regions, for troponin I at amino acid residues 41-80 and for troponin T near amino acid residue 190, while switching to a different troponin binding site at residues 258-284. cC401-type mRNA is expressed at high levels in cultured skin fibroblasts and at low levels in cultured embryonic myoblasts and myofibers, but not in adult muscle, liver, or embryonic skin. A single quail genomic clone encodes both sets of alternative exons found in cC401 and cC402; the different transcripts from this gene share the same 5' ends. Two additional alpha-tropomyosin isoforms have been detected in quail tissues by S1 mapping experiments. Comparison of the cC401 protein with 11 other tropomyosin protein sequences shows that the quail alpha-tropomyosin gene has three different pairs of mutually exclusive exons which are alternatively spliced in nonrandom combinations in specific tissues. The implications of these findings are discussed in relation to mechanisms that regulate alternative splicing.

Closely related alpha-tropomyosin mRNAs in quail fibroblasts and skeletal muscle cells.

We describe the analysis of two quail cDNA clones representing distinct but closely related alpha-tropomyosin mRNAs. cDNA clone cC101 corresponds to a 1.2-kilobase RNA which accumulates to high levels during myoblast differentiation and which encodes the major isoform of skeletal muscle alpha-tropomyosin. cDNA clone cC102 corresponds to a 2-kilobase RNA which is abundant in cultured embryonic skin fibroblasts and which encodes one of two alpha-tropomyosin-related fibroblast tropomyosins of 35,000 and 34,000 daltons apparent molecular mass (class 1 tropomyosins). The cC102 protein is unique among reported nonstriated-muscle tropomyosins in being identical in amino acid sequence to the major isoform of skeletal muscle alpha-tropomyosin over an uninterrupted stretch of at least 183 amino acids (residues 75-257). The two protein sequences differ in the COOH-terminal region beginning with residue 258. Because the cC101 and cC102 RNAs share an extensive region (at least 373 nucleotides) of nucleotide sequence identity upstream of the codon for residue 258, they are likely derived from a single gene by alternative RNA splicing, as was recently proposed in the case of related beta-tropomyosin mRNAs in human fibroblasts and skeletal muscle (MacLeod, A. R., Houlker, C., Reinach, R. C., Smillie, L. B., Talbot, K., Modi, G., and Walsh, F. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7835-7837). No alpha-tropomyosin-related RNAs are abundant in undifferentiated myoblasts. This suggests the possibility of a fibroblast-specific function, as opposed to a general nonmuscle-cell function for class 1 tropomyosins and also has implications for the regulation of alpha-tropomyosin gene expression during embryonic development.

Isolation of mutants of an animal virus in bacteria.

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.