Organization and Myogenic Restricted Expression of the Murine Serum Response Factor Gene

Serum response factor (SRF), a member of an ancient family of DNA-binding proteins, is generally assumed to be a ubiquitous transcription factor involved in regulating growth factor-responsive genes. However, avian SRF was recently shown (Croissant, J. D., Kim, J.-H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250–264) to be preferentially expressed in myogenic lineages and is required for regulating post-replicative muscle gene expression. Given the central importance of SRF for the muscle tissue-restricted expression of the striated α-actin gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the murine SRF genomic locus, which has seven exons interrupted by six introns, with the entire locus spanning 11 kilobases. Murine SRF transcripts were processed to two 3′-untranslated region polyadenylation signals, yielding 4.5- and 2.5-kilobase mRNA species. Murine SRF mRNA levels were the highest in adult skeletal and cardiac muscle, but barely detected in liver, lung, and spleen tissues. During early mouse development,in situ hybridization analysis revealed enrichment of SRF transcripts in the myotomal portion of somites, the myocardium of the heart, and the smooth muscle media of vessels of mouse embryos. Likewise, murine SRF promoter activity was tissue-restricted, being 80-fold greater in primary skeletal myoblasts than in liver-derived HepG2 cells. In addition, SRF promoter activity increased 6-fold when myoblasts withdrew from the cell cycle and fused into differentiated myotubes. A 310-base pair promoter fragment depended upon multiple intact serum response elements in combination with Sp1 sites for maximal myogenic restricted activity. Furthermore, cotransfected SRF expression vector stimulated SRF promoter transcription, whereas dominant-negative SRF mutants blocked SRF promoter activity, demonstrating a positive role for an SRF-dependent autoregulatory loop. Serum response factor (SRF), a member of an ancient family of DNA-binding proteins, is generally assumed to be a ubiquitous transcription factor involved in regulating growth factor-responsive genes. However, avian SRF was recently shown (Croissant, J. D., Kim, J.-H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250–264) to be preferentially expressed in myogenic lineages and is required for regulating post-replicative muscle gene expression. Given the central importance of SRF for the muscle tissue-restricted expression of the striated α-actin gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the murine SRF genomic locus, which has seven exons interrupted by six introns, with the entire locus spanning 11 kilobases. Murine SRF transcripts were processed to two 3′-untranslated region polyadenylation signals, yielding 4.5- and 2.5-kilobase mRNA species. Murine SRF mRNA levels were the highest in adult skeletal and cardiac muscle, but barely detected in liver, lung, and spleen tissues. During early mouse development,in situ hybridization analysis revealed enrichment of SRF transcripts in the myotomal portion of somites, the myocardium of the heart, and the smooth muscle media of vessels of mouse embryos. Likewise, murine SRF promoter activity was tissue-restricted, being 80-fold greater in primary skeletal myoblasts than in liver-derived HepG2 cells. In addition, SRF promoter activity increased 6-fold when myoblasts withdrew from the cell cycle and fused into differentiated myotubes. A 310-base pair promoter fragment depended upon multiple intact serum response elements in combination with Sp1 sites for maximal myogenic restricted activity. Furthermore, cotransfected SRF expression vector stimulated SRF promoter transcription, whereas dominant-negative SRF mutants blocked SRF promoter activity, demonstrating a positive role for an SRF-dependent autoregulatory loop. Serum response factor (SRF), 1The abbreviations used are: SRF, serum response factor; mSRF, murine serum response factor; SRE, serum response element; bp, base pair(s); kbp, kilobase pair(s); GST, glutathione S-transferase; PCR, polymerase chain reaction. 1The abbreviations used are: SRF, serum response factor; mSRF, murine serum response factor; SRE, serum response element; bp, base pair(s); kbp, kilobase pair(s); GST, glutathione S-transferase; PCR, polymerase chain reaction. a member of an ancient family of DNA-binding proteins, contains a highly conserved DNA-binding/dimerization domain of 90 amino acids, termed the MADS box (1Schwarz-Sommer Z. Huijser P. Nacken W. Saedler H. Sommer H. Science. 1990; 250: 931-936Crossref PubMed Scopus (652) Google Scholar). The structure of the MADS box domain, recently elucidated by Pellegrini et al. (2Pellegrini L. Tans S. Richmond T.J. Nature. 1995; 376: 490-498Crossref PubMed Scopus (297) Google Scholar), was assembled before the divergence of plants and animals. The identical MADS box structures were present in yeast transcription factors MCM1 and ARG80, a large number of homeotic like plant proteins, and invertebrate and vertebrate SRFs (reviewed in Ref. 3Shore P. Sharrocks A.D. Eur. J. Biochem. 1995; 229: 1-13Crossref PubMed Scopus (719) Google Scholar). All of these transcription factors, through their common MADS boxes, virtually bind to the same DNA sequences (1Schwarz-Sommer Z. Huijser P. Nacken W. Saedler H. Sommer H. Science. 1990; 250: 931-936Crossref PubMed Scopus (652) Google Scholar) and interact with similar kinds of co-accessory regulatory factors (reviewed in Refs. 4Treisman R. Curr. Opin. Genet. 4: 96-101Crossref PubMed Scopus (618) Google Scholar and 5Olson E.N. Perry M. Schultz R.A. Dev. Biol. 1995; 172: 2-14Crossref PubMed Scopus (314) Google Scholar). Molecular dissection of human SRF revealed phosphorylation sites in the N-terminal domain that influence DNA binding, whereas sequences downstream of the MADS box contain the C-terminal transcription activation domain (6Lee T.-C. Shi Y. Schwartz R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9814-9818Crossref PubMed Scopus (197) Google Scholar, 7Johansen F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar). These extra MADS box sequences are well conserved in vertebrate SRF species, but have diverged from lower animal and plant species. Earlier studies (8Treisman R. Cell. 1985; 42: 889-902Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 9Greenberg M.E. Siegfried Z. Ziff E.B. Mol. Cell. Biol. 1987; 7: 1217-1225Crossref PubMed Scopus (155) Google Scholar, 10Christy B. Nathans D. Mol. Cell. Biol. 1989; 9: 4889-4895Crossref PubMed Scopus (147) Google Scholar) have demonstrated that SRF-binding sites, termed serum response elements (SRE; GG(A/T)6CC), play a primary role in regulating early response genes such and (reviewed in Ref. R. Biol. 1990; Google Scholar). factor through the to be through the of with an and through with a the of the A. J. PubMed Scopus Google J. 1992; PubMed Scopus Google Scholar). The domain M. A. Science. 1989; PubMed Scopus Google Scholar) and S. R. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar) that are of R. S. J. S. R. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar) and interact with the C-terminal portion of the MADS box P. Sharrocks A.D. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). Furthermore, MADS box a S. Science. 1992; PubMed Scopus Google Scholar), has shown to the DNA activity of SRF the In addition, and the of SRF to the promoter in response to S. Science. 1992; PubMed Scopus Google Scholar). SRF an role in regulating post-replicative muscle gene expression. The multiple in the of vertebrate striated α-actin genes are required for myogenic expression Prywes R. R. L. Mol. Cell. Biol. 1989; 9: PubMed Scopus Google Scholar, T.J. J. 1989; PubMed Scopus Google Scholar, M. R. 1989; Google Scholar, Schwartz R.J. Mol. Cell. Biol. 1990; PubMed Google Scholar). of the of the avian striated α-actin revealed that these in combination with and were for transcription Schwartz R.J. Mol. Cell. Biol. 1990; PubMed Google Scholar, Schwartz R.J. J. Biol. 1994; Full Text PDF PubMed Google Scholar). T.-C. P. Schwartz R.J. Mol. Cell. Biol. PubMed Google Scholar) and F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google A. D. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar) have shown that are in to the sequences the skeletal α-actin a promoter to be muscle Mol. Cell. Biol. 1989; 9: PubMed Scopus Google Scholar). SRF the avian skeletal α-actin promoter in by a factor for the F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar, A. D. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar). SRF mRNA and primary myoblasts from the cell cycle and In addition, SRF to the in differentiated L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar). the in SRF mRNA to the of α-actin gene activity Schwartz R.J. J. Biol. PubMed Scopus Google Scholar). et al. M. A. J. Biol. 1992; PubMed Scopus Google Scholar) demonstrated that of SRF the of myogenic an early In addition, et al. L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar) demonstrated that a dominant-negative SRF in DNA but of with SRF the activity of the skeletal α-actin gene promoter in myogenic and blocked SRF has a role in α-actin gene transcription skeletal muscle and J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that a promoter region of the murine SRF gene was to serum in and that the and sites present this promoter region were is generally assumed that SRF is a ubiquitous transcription has to the for the muscle expression of Given the central importance of SRF for the muscle tissue-restricted expression of the gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the SRF genomic Murine SRF gene activity was of the expression of MADS Y. B. Dev. 1992; PubMed Scopus Google Scholar), being restricted to cell from such and smooth to a to cell of SRF was virtually in such the liver, lung, and the for the expression of SRF, we the elements in the SRF promoter the of the SRF promoter region of the the required for muscle-restricted expression. Furthermore, dominant-negative SRF mutants blocked SRF promoter activity in muscle cells. SRF gene activity to be an autoregulatory in which two in the promoter were required for the SRF expression in skeletal muscle cells. A fragment of mouse SRF to of human SRF from was used to a mouse were with the mouse SRF fragment the and were and that of these were The fragment from the of of these to was used to the same and seven were The 3′-untranslated region sequences were by with the fragment of the mouse SRF genomic which to of human SRF The were and the S. The mouse genomic in the was a from of were by with the fragment to of the human SRF and analysis of these with from the murine SRF that the SRF from this were and The were by the and genomic DNA was from adult mouse to and P. Biochem. 1987; PubMed Scopus Google Scholar). of was and The and were to the The region and the region were in and The used for analysis was to of The was with and and of the was with of skeletal muscle in and The was and with of in and for The was with of with and A the same was used In situ hybridization was of mouse by et al. L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar). A murine SRF to the 3′-untranslated region into was with the to were and were processed for with and by and The was by the fragment and to the of the SRF gene and and sites of the and was with and with of the were by DNA polymerase The DNA was with which the The SRF promoter region and of the vector were and into fragment of and were from a by into The from these and of the SRF promoter were the of to and were into the by chain were in a of polymerase of the and polymerase The and downstream were with sites, their Sp1 The for were for and an of for for and for The was for The of the the were with and the of All were by primary skeletal myoblasts were from 11 muscle T.-C. Y. Schwartz R.J. 1994; 9: Google Scholar). were in serum and and HepG2 were in serum before and were with of the DNA with of by to the were with SRF cotransfected with of SRF expression vector SRF promoter were with of a SRF dominant-negative were and activity was to in a activity was for the the promoter activity was to the were to al. R.J. R. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). The of was by the used of the from myotubes. The was for with of in and and and were in the for and of the was and for a were a and in The was and The SRF promoter fragment from to to the was and used for of was for with of in of The was for an of The and were to and and the was with of for 90 The was by the of of and and for and the were in of the was and A mouse genomic was with the human SRF M. R. R. Cell. Full Text PDF PubMed Scopus Google Scholar) to the conserved MADS box of mouse genomic were and of these the SRF The SRF gene and of seven exons the SRF of amino and The the to the shown in P. 1990; PubMed Scopus Google Scholar). The of the exons from to However, which of the two polyadenylation sequences are the was in The was in the downstream of the The of region and of the The conserved MADS box region was by the and by the The transcription activation domain was exons The was in the and of the and serum response for amino are identical amino the same amino The MADS box domain is by The for phosphorylation by human of the murine SRF of exons in and of in are shown in the and The the is in for exons and in for The interrupted by the is shown in the in a The of exons in and of in are shown in the and The the is in for exons and in for The interrupted by the is shown in the of the mouse SRF amino with the and sequences revealed a of The MADS box region was identical in vertebrate SRF species. SRF is to human SRF than to The region of is and similar to the and amino sequences of human SRF, The of the region was conserved for these two species. However, in with ancient such the sequences were to the MADS of the N-terminal domain revealed the of a conserved in the region in human and mouse which was in the A of divergence and SRF was in the N-terminal domain with the domain, which is involved in The was by A from the of the murine which to to was to mouse muscle and with of analysis that the of the SRF transcripts were from the of the which we P. Schwartz R.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The for transcripts was downstream of that for human SRF M. R. R. Cell. Full Text PDF PubMed Scopus Google Scholar). sites were detected downstream of the of the genomic sequences of the revealed a box to The box the for and a tissue-restricted transcription are two and In addition, are two and sites for gene and are the sites are sites for factors, and are present the SRF promoter how SRF role in regulating striated α-actin and how might SRF contribute to tissue-restricted the expression of SRF, from was by shown in a region downstream of the MADS box detected SRF transcripts of and which were expressed in such skeletal and cardiac muscle to a in lung, and which are from the barely expressed SRF mRNA of the genomic revealed the of two polyadenylation from the by of these two polyadenylation for mRNA have to the for the two SRF species. this we used a that the polyadenylation which detected the species, shown in B. the two from of the two polyadenylation for with the SRF SRF mRNA was detected in skeletal and cardiac muscle and but in that the expression of SRF is tissue-restricted T.-C. Y. Schwartz R.J. 1994; 9: Google Scholar). During vertebrate expression of striated α-actin transcripts an early for of and smooth muscle cell SRF mRNA expression were restricted to early and smooth muscle cell In mouse SRF transcripts were in the of the and the but were in the revealed levels of SRF expression in the and the of the shown in B. SRF was detected levels in the myotomal portion of and in the smooth muscle the levels of SRF was detected in the and in the SRF was barely detected in the and in levels of hybridization in These in situ hybridization demonstrated that SRF gene expression was and restricted to the cardiac and skeletal muscle cell with the early expression of the α-actin genes in the the for the muscle expression and to the we primary and the human cell with SRF promoter The was 80-fold in than in HepG2 A to the of a of in a in the promoter activity in myotubes. The was in than in HepG2 cells. A of to the promoter activity in and by in HepG2 cells. A to the promoter activity by in but in HepG2 cells. were revealed by to which in a in the promoter activity in and a in HepG2 cells. are present this A of to which of the two and and sites, the promoter activity by in myotubes. which contains a and the box was than the in myotubes. The was in HepG2 which levels of A of to which the promoter activity to levels in that the were required for SRF promoter activity. of promoter analysis that the present the SRF promoter were required for activity. with expressed SRF were used to the two present in the SRF promoter bind and were well the levels of In addition, the box region was but of was the two but and for and of these factors are expressed in skeletal muscle 7Johansen F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar and R. P. A. W. M. Mol. Cell. Biol. 1995; PubMed Scopus Google in Ref. R. Biol. 1990; Google Scholar). demonstrated of expressed SRF, we the of from with from the SRF to and were with the from myotubes. A of and and a were with the from the cell to a similar of SRF when a was used for J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and were by a of cardiac and and from the SRF but by and and Furthermore, and were by SRF but by that these two contain SRF was a this was in from myoblasts and in this was by and the skeletal but by and cardiac which bind was by and by SRF was similar to with to SRF binding, that bind have the of SRF to and by in which was the and for SRF to the the role of and in the muscle expression of SRF, analysis of these was in the of the which the of SRF, was for these of the activity of the that the have for In to the the increased the activity of the promoter by However, of and in in the promoter activity, that of the two is required for the promoter activity in myotubes. In the SRF were two Sp1 sites and these Sp1 sites were sites for the transcription factor Sp1 sites from contain sites and bind Sp1 and the Sp1 sites from the SRF promoter bind Sp1 and present in were with the Sp1 These were by a of Sp1 and and Sp1 sites, but Sp1 The of these was by an The were by the of Sp1 but by that the contain Sp1 and the Sp1 contains an of Sp1 was and J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) by analysis that the two Sp1 sites present in the murine SRF promoter were for serum of the promoter in but Sp1 sites the promoter activity. The role of these Sp1 sites in the muscle expression of the SRF promoter was by Sp1 the of Sp1 from of the Sp1 increased the promoter activity by whereas a the Sp1 the promoter activity by avian SRF and and SRF DNA activity were shown to when primary myoblasts were to from the cell cycle and to F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar, L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar). wanted to determine the of SRF gene activity was by the activity of SRF in primary myogenic The SRF promoter fragment from to to a gene activity in but was in sequences required for the of SRF gene activity are the promoter The of SRF the of multiple in the SRF and the of SRF to these the that SRF we of the SRF promoter cotransfected with SRF expression in of an SRF in to a in SRF promoter activity dominant-negative SRF mutants SRF promoter in myotubes. The F.-E. Prywes R. 1995; PubMed Scopus Google Scholar) with SRF and with SRF by DNA In addition, dominant-negative SRF in which the C-terminal transcription activation domain was a by through DNA binding, but is of of with in and in SRF promoter activity, that the myogenic of SRF promoter activity was by SRF by and was to the SRF promoter promoter activity was by these dominant-negative SRF These that SRF promoter and that this is through and The that and the of ancient genes in the was recently Schwartz R.J. in and Scholar). The of in the of genes be by the that in and in the of genes that and in the but were the of the Nature. PubMed Scopus Google Scholar) that for of a gene might be by the of with the of The of the structure of the SRF MADS box demonstrated a DNA-binding a and a involved in (2Pellegrini L. Tans S. Richmond T.J. Nature. 1995; 376: 490-498Crossref PubMed Scopus (297) Google Scholar). and that the murine SRF gene of seven exons interrupted by six are well conserved the SRF T.J. J. PubMed Scopus Google Scholar) and murine SRF genes. The was to the N-terminal which base the of an from the in In with the genomic of E.N. Mol. Cell. Biol. PubMed Scopus Google Scholar), which was conserved and the plant gene H. M. Y. H. Mol. Biol. 1995; PubMed Scopus Google Scholar), the the N-terminal whereas the was to the C-terminal of MADS in animal and plant SRFs and the in genes. in MADS genes the of primary of the MADS box region and gene might have in the of the MADS to the of plants and that these MADS box have to such of in homeotic in and of structures in the divergence of SRF of to be to in the expression of the of animal the SRF of SRF J. R. M. Google Scholar), was the in which was to the MADS box The of SRF expression was from that of and murine which are in structure and expression. SRF was to the M. J. U. J. U. M. 1994; Google Scholar), whereas vertebrate like of their (reviewed in Ref. 5Olson E.N. Perry M. Schultz R.A. Dev. Biol. 1995; 172: 2-14Crossref PubMed Scopus (314) Google Scholar), were to avian and murine skeletal and cardiac muscle and and L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar). SRF play a central role in regulating genes that are expressed cell have shown that has a striated muscle expression and the a of SRF promoter is generally assumed that SRF a role a factor with factors, we have shown that SRF activity increased the of cell to in the of SRF in primary myoblasts F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar). of early avian L. J. Prywes R. Schwartz R.J. Dev. Biol. PubMed Scopus Google Scholar) and murine tissue-restricted expression of SRF which increased the of SRF in the myotomal portion of somites, cardiac and smooth muscle cells. The expression of SRF in these levels through During primary in SRF promoter activity, and of myoblasts and the of genes T.-C. Y. Schwartz R.J. 1994; 9: Google Scholar). that of and were required to SRF promoter activity, that and were of SRF from a vector increased SRF promoter activity in In the dominant-negative mutants of SRF, and the muscle expression of SRF and genes. D. Kim, Eichele, L. Goering, J. Lough, R. Prywes, and R. J. Schwartz, for SRF has a primary role in muscle expression of SRF was from of promoter activity, in which the SRF promoter activity was of greater in primary than in HepG2 cells. SRF promoter activity be to the of elements in the SRF promoter in the promoter the is that a the region the SRF activity in cells. for the of SRF promoter activity in be the of SRF factors from this cell SRF Furthermore, a SRF promoter a SRE, which was in was in HepG2 that the SRF promoter activity was The were required for the of expression in The activity of the SRF promoter in was shown to be the ubiquitous whereas the and Sp1 sites were J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In and Sp1 sites were required for promoter activity J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and muscle tissue-restricted activity. SRF promoter analysis demonstrated that SRF was but for the SRF Furthermore, of the SRF promoter by SRF for the muscle expression of the SRF The expression of SRF might be by the of with of sequences to and the promoter activity, that the of these sequences with and be required for the activity of the the two sites present in the SRF the Sp1 to be for SRF expression. et al. L. Dev. 1990; 4: PubMed Scopus Google Scholar) have shown that a SRF, the ubiquitous transcription factor and the cell myogenic factor is required for human cardiac α-actin gene expression. by which SRF might gene expression be by with the activity of of muscle a expressed Y. Cell. Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that the has shown to bind F.-E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar, T.-C. Schwartz R.J. J. 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