Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7520-7524, August 1995 Cell Biology Transdifferentiation of chicken embryonic cells into muscle cells by the 3' untranslated region of muscle tropomyosin THOMAS J. L'ECUYER*, PAUL C. TOMPACHt, ERIC MORRISt, AND ALICE B. FULTON*§ Departments of tBiochemistry, tOral and Maxillofacial Surgery, and *Pediatrics, University of Iowa, Iowa City, IA 52242 Communicated by Sheldon Penman, Massachusetts Institute of Technology, Cambridge, MA, May 3, 1995 ABSTRACT Transfection with a plasmid encoding the 3' Drosophila pattern formation is influenced by the 3' UTRs untranslated region (3' UTR) of skeletal muscle tropomyosin of nanos (23) and bicoid (24). The posterior determinant induces chicken embryonic fibroblasts to express skeletal nanos, important in abdominal segmentation, exerts its effect tropomyosin. Such cells become spindle shaped, fuse, and by its 3' UTR inhibiting maternal hb gene expression. Dro- express titin, a marker of striated muscle differentiation. sophila embryos acquire anterior-position determination by a Skeletal muscle tropomyosin and titin organize in sarcomeric gradient of bicoid protein; this gradient requires the 3' UTR arrays. When the tropomyosin 3' UTR is expressed in osteo- of bicoid mRNA. blasts, less skeletal muscle tropomyosin is expressed, and titin A 3' UTR can affect mineral metabolism. Eukaryotic sel- expression is delayed. Some transfected osteoblasts become enoproteins have sites within the 3' UTR that are critical for spindle shaped but do not fuse nor organize these proteins into selenocysteine incorporation in the coding region (25). An sarcomeres. Transfected cells expressing muscle tropomyosin iron response element within the ferritin and transferrin organize muscle and nonmuscle isoforms into the same struc- receptor transcripts allows the expression of these genes to be tures. Thus, the skeletal muscle tropomyosin 3' UTR induces controlled by iron availability (26, 27). The iron response transdifferentiation into a striated muscle phenotype in a element for transferrin receptor, to which a protein binds, is cell-type-specific context. found within the 3' UTR of its transcript. A different function for 3' UTRs is revealed by localization The 3' untranslated region (3' UTR) of transcribed RNA has of actin transcripts in myoblasts, which is determined by the 3' recently been demonstrated to have important biological func- UTR of its mRNA; this region may function as a "zip code" tions. The 3' UTRs of two important protooncogenes, c-myc (28). The 3' UTR also determines localization in vivo for and c-fos, have AU-rich elements that act to destabilize the transcripts of the chloroplast genes rbcl and psaB in Chlamy- mRNA domonas (29). full-length by facilitating the removal of the poly(A) The most surprising consequence of a 3' UTR was observed site; in this fashion the 3' UTRs of protooncogenes control the for the muscle structural proteins troponin I, tropomyosin, and amount of their own mRNA that is available for translation cardiac actin. The 3' UTRs of these transcripts have been (1-6). Similarly, the cytokine granulocyte/macrophage colo- shown by genetic complementation to permit differentiation in ny-stimulating factor (GM-CSF) has a region within its 3' UTR a mutant myoblastic cell line that is defective in differentiation that causes degradation of the message (7, 8). Since the (30). Wild-type muscle cells were augmented in their differ- half-life of this mRNA controls its gene expression level, the entiation by introduction of these 3' UTR sequences, and 3' UTR has a profound influence on GM-CSF gene expres- proliferation of 10T'/2 fibroblasts was suppressed, suggesting sion. Interleukin 1 increases GM-CSF expression by stabilizing that the 3' UTRs of certain differentiation-specific RNAs are its mRNA, perhaps by binding to the 3' UTR of GM-CSF trans-acting regulators in a feedback loop that can inhibit mRNA. The 3' UTR of human papillomavirus acts to desta- proliferation and promote differentiation. The 3' UTR of bilize late mRNAs as well, negatively influencing its own gene a-tropomyosin also suppressed tumor growth in mutant myo- expression (9). Another example of 3' UTRs influencing gene genic cells that form tumors in mice, further suggesting that expression by destabilizing their mRNAs occurs in a highly untranslated RNAs can function as regulators of cell prolif- unstable set of transcripts in soybeans, the auxin-up RNAs eration (31). The work to be described below extends these last (10). In Xenopus oocytes, the 3' UTR of a transcript called observations by showing that 3' UTRs can transdifferentiate XRh box 2B causes endonuclease activity ofspecific nucleotide primary chicken fibroblasts into muscle cells. sequences, providing another example of how this region can negatively influence gene expression, but of sequences other than its own (11). Numerous other transcripts have their MATERIALS AND METHODS stability decreased through elements in the 3' UTR (12, 13). Cell Culture. Fibroblasts were cultured from day 12 chicken Expression is also controlled by decreases in translatability (14, embryos by autodigestion of skin as described (32). Cells were 15). The role of the 3' UTR on translation has recently been grown on 100-mm dishes in Dulbecco's modified Eagle's reviewed (16, 17). medium (DMEM), supplemented with 10% horse serum, In other cases, a 3' UTR has a positive influence on gene 2.5% chicken embryonic extract, nonessential amino acids, expression. The 3' UTR of tobacco mosaic virus mRNA forms minimum essential medium vitamins, Hepes as a buffer, and a tertiary structure that includes a pseudoknot. Adding this 3' gentamicin until they reached confluency, at which time the UTR to foreign transcripts dramatically increases their expres- culture was passed by trypsinization onto collagen-coated glass sion in prokaryotes and eukaryotes (18, 19), by increases in coverslips at a density of 1.25 x 105 cells per dish. After cells translational efficiency and mRNA stability. Other mRNAs reached 80-90% confluency on coverslips, fibroblasts were also show increases in stability (20) or translational efficiency used for transfection experiments. Osteoblast cultures were with given 3' UTRs (21, 22). prepared by obtaining calvarial explants from day 12 chicken embryos. The explants were placed in 35-mm tissue culture The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: 3' UTR, 3' untranslated region. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 7520 Downloaded by guest on October 1, 2021 Cell Biology: L'Ecuyer et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7521 plates with culture medium consisting of high glucose DMEM Nordic Immunologicals (Tilburg, The Netherlands). An anti- supplemented with 10% fetal bovine serum and containing 50 body to f3-galactosidase was obtained from Promega and used units of penicillin per ml, 50 jig of streptomycin per ml, 2 ,ug at 1:500 dilution. of amphotericin per ml, 50 ,ug of ascorbic acid per ml, 250 ,ug of glutamine per ml, and 20 mM Hepes. Outgrowths of cells from the bone fragments appeared within 4-5 days and formed RESULTS confluent monolayers at 7-10 days. After the bone explants Tropomyosin isoforms assemble by different pathways in were removed, the confluent cells were trypsinized and plated skeletal muscle and in nonmuscle cells (40). When we sought on collagen-coated coverslips in 35-mm culture plates at 1 x to examine the assembly of muscle-specific tropomyosin in 105 cells per ml. All cells are maintained at 37°C in a chicken embryonic fibroblasts by transfecting the cells with a humidified incubator with 5% CO2. plasmid encoding chicken skeletal muscle a-tropomyosin, an Production of Construct for Transfection. A plasmid con- unexpected result was obtained. Passaged chicken embryonic taining the avian skeletal a-tropomyosin cDNA was provided fibroblasts that expressed skeletal tropomyosin from a plasmid by S. Hitchcock-DeGregori (33) and was cloned into the during a transient transfection became spindle shaped and eukaryotic expression vector pCMV5 (34). The 3' UTR of this elongated (Fig. 1A). Many of the spindle-shaped cells that cDNA was amplified by designing PCR primers to the 5' and express skeletal muscle tropomyosin also express titin, the 3 3' ends of this sequence [excluding the poly(A)denylation mD marker of striated muscle differentiation (Fig. 1B). Tro- sequence] and performing PCR using full-length skeletal pomyosin and titin were found in fibrillar longitudinal strands, a-tropomyosin as a template. Nucleotides encoding unique and in some cells the proteins were organized in sarcomeric restriction sites were added to either end of the primers to arrays, as early as 24 hr after transfection. In areas where allow directional cloning into a vector (pCMV5) cut with the several cells expressing tropomyosin were close, they appeared same restriction enzymes. A 300-bp fragment was released to have fused, as multinucleated cells with as many as five from DNA purified from bacteria transformed with this nuclei were observed. Cultures allowed to express muscle construct after digestion with the unique restriction enzymes tropomyosin for 72 hr showed a higher frequency of multinu- flanking the cloning site, as expected. All cloning procedures cleated cells with titin in were adapted from described protocols (35). Control trans- periodic arrays. Dishes containing fections were performed with the vector pCMV5 alone and cells that had been mock-transfected showed no cells express- with pRSV-,Bgal, a construct encoding the lacZ gene (gift of R. ing muscle tropomyosin or titin. By all criteria used, the Singer; ref. 28), allowing the determination of whether cells fibroblasts had transdifferentiated into striated muscle cells. exposed to transfection cocktail take up and express foreign Numerous examples point to connections between cell DNA. shape and gene expression (41), so it was possible that this DNA Transfection.