Identification and Characterization of the Cell Type-specific and Developmentally Regulated α7 Integrin Gene Promoter

Expression of α7 is mainly confined to skeletal and cardiac muscle in which it appears to be the major laminin-binding integrin. When myoblasts differentiate to myotubes, α7 mRNA and protein expression is up-regulated. To explore the mechanisms involved in the tissue-specific and developmentally regulated expression of α7, we isolated and characterized a genomic clone containing ~2.8 kilobase pairs (kb) of the 5′-flanking region of the murine α7 gene. The 5′-flanking region lacks both TATA and CCAAT boxes but contains five putative Sp1 binding sites located in a CpG island. Two transcription start sites, located near an initiator-like sequence, are 176 and 170 base pairs upstream of the translation start site. There are numerous binding sites for developmental and cell type-specific transcription factors, including AP-1, AP-2, GATA, and several AT-rich sites. There are also eight consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The ~2.8-kb 5′-flanking region was an active promoter in C2C12 skeletal myoblasts and exhibited increased expression upon conversion to myotubes but was inactive in HtLM2 cells, a mouse breast carcinoma epithelial cell line that does not express α7. Deletion analysis identified both positive and negative regulatory elements within the ~2.8-kb fragment. In 10T1/2 fibroblasts the ~2.8-kb α7 promoter was trans-activated by the myogenic basic helix-loop-helix proteins myogenin and MyoD but not by MRF4 and myf5. These results suggest that muscle-specific transcription factors play a role in regulating the cell-type expression of the α7 gene during development. Expression of α7 is mainly confined to skeletal and cardiac muscle in which it appears to be the major laminin-binding integrin. When myoblasts differentiate to myotubes, α7 mRNA and protein expression is up-regulated. To explore the mechanisms involved in the tissue-specific and developmentally regulated expression of α7, we isolated and characterized a genomic clone containing ~2.8 kilobase pairs (kb) of the 5′-flanking region of the murine α7 gene. The 5′-flanking region lacks both TATA and CCAAT boxes but contains five putative Sp1 binding sites located in a CpG island. Two transcription start sites, located near an initiator-like sequence, are 176 and 170 base pairs upstream of the translation start site. There are numerous binding sites for developmental and cell type-specific transcription factors, including AP-1, AP-2, GATA, and several AT-rich sites. There are also eight consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The ~2.8-kb 5′-flanking region was an active promoter in C2C12 skeletal myoblasts and exhibited increased expression upon conversion to myotubes but was inactive in HtLM2 cells, a mouse breast carcinoma epithelial cell line that does not express α7. Deletion analysis identified both positive and negative regulatory elements within the ~2.8-kb fragment. In 10T1/2 fibroblasts the ~2.8-kb α7 promoter was trans-activated by the myogenic basic helix-loop-helix proteins myogenin and MyoD but not by MRF4 and myf5. These results suggest that muscle-specific transcription factors play a role in regulating the cell-type expression of the α7 gene during development. INTRODUCTIONThe integrin superfamily is made up of a number of transmembrane heterodimeric receptors that mediate cell-extracellular matrix and cell-cell interactions (1Hynes R.O. Cell. 1992; 69: 11-25Google Scholar). Each integrin is composed of noncovalently paired α and β subunits. Regulation of the genes expressing these extracellular matrix receptors is important in a number of processes including development, migration, and invasion (2Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Google Scholar, 3Rosales C. O'Brien V. Kornberg L. Juliano R. Biochim. Biophys. Acta. 1995; 1242: 77-98Google Scholar, 4Bosman F.T. Histochem. J. 1993; 25: 469-477Google Scholar).Previously, we have reported that human and mouse melanoma cells express an integrin complex designated α7β1 (5Kramer R.H. McDonald K.A. Vu M.P. J. Biol. Chem. 1989; 264: 15642-15649Google Scholar, 6Kramer R.H. Vu M.P. Cheng Y.P. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Google Scholar). This integrin receptor binds to the E8 fragment of laminin-1 and mediates cell adhesion to this ligand (6Kramer R.H. Vu M.P. Cheng Y.P. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Google Scholar). In melanoma, the expression of the α7 integrin receptor is elevated in the nonmetastatic phenotype but is lacking in metastatic cells, suggesting that absence of this laminin adhesion receptor can play a role in melanoma tumor cell dissemination. 1D. M. Ramos, N. Waleh, M. P. Vu, B. L. Ziober, C. C. Yao, and R. H. Kramer, submitted for publication. Expression of α7, as detected by reverse transcriptase-PCR, 2The abbreviations used are: PCRpolymerase chain reactionbHLHbasic helix loop helixCATchloramphenicol acetyltransferasebpbase pair(s)kbkilobase pair(s)Pipes1,4-piperazinediethanesulfonic acidInrinitiator sequence. has also been reported to be present in a number of tissues including for example stomach and uterus (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar). However, it has not yet been determined if any of the α7 protein isoforms are expressed in these tissues. In myoblasts and cardiomyocytes, α7 appears to be the predominant laminin-binding integrin expressed (8Kaufman S.J. Foster R.F. Haye K.R. Faiman L.E. J. Cell Biol. 1985; 100: 1977-1987Google Scholar, 9Mark H. Durr J. Sonnenberg A. Mark K. Deutzmann R. Goodman S.L. J. Biol. Chem. 1991; 266: 23593-23601Google Scholar). In vitro, basal levels of α7 expression are seen in replicating rat and mouse myoblasts (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar, 10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Google Scholar, 11Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell Biol. 1992; 117: 643-650Google Scholar). Expression of α7 is increased upon differentiation of myoblasts to myotubes. This up-regulation is paralleled by increased expression of myogenin (11Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell Biol. 1992; 117: 643-650Google Scholar), a member of the muscle-specific basic helix-loop-helix (bHLH) transcription factor family, suggesting that these factors play a regulatory role in α7 expression during muscle development.At embryonic day 7.5, α7 can be detected in the ectoplacental cone (differentiating murine trophoblast), where it is believed to play a role in trophoblast adhesion and/or differentiation. The use of laminin fragments suggested that α7β1 may be one of the major trophoblast laminin-binding integrins involved in embryo implantation (12Sutherland A.E. Calarco P.G. Damsky C.H. Development. 1993; 119: 1175-1186Google Scholar). Recently, the cDNAs for rat and mouse α7 have been isolated and shown to be alternatively spliced in both the extracellular (giving rise to two isoforms, X1 and X2) and cytoplasmic (producing three isoforms, A, B, and C) domains. Subsequent work has shown that these alternatively spliced forms are also developmentally regulated in muscle (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar, 10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Google Scholar, 13Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. J. Cell. Sci. 1993; 106: 1139-1152Google Scholar).Taken together, these results indicate that expression of α7 plays a role in embryo implantation and muscle development. Furthermore, the developmental and restricted tissue expression (in skeletal muscle and cardiac muscle) of the α7 subunit suggests that the elements governing its expression are complex. As an approach to understanding the mechanisms regulating cell type- and differentiation-specific expression of the α7 subunit, we isolated and characterized the promoter region for the murine α7 integrin gene. INTRODUCTIONThe integrin superfamily is made up of a number of transmembrane heterodimeric receptors that mediate cell-extracellular matrix and cell-cell interactions (1Hynes R.O. Cell. 1992; 69: 11-25Google Scholar). Each integrin is composed of noncovalently paired α and β subunits. Regulation of the genes expressing these extracellular matrix receptors is important in a number of processes including development, migration, and invasion (2Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Google Scholar, 3Rosales C. O'Brien V. Kornberg L. Juliano R. Biochim. Biophys. Acta. 1995; 1242: 77-98Google Scholar, 4Bosman F.T. Histochem. J. 1993; 25: 469-477Google Scholar).Previously, we have reported that human and mouse melanoma cells express an integrin complex designated α7β1 (5Kramer R.H. McDonald K.A. Vu M.P. J. Biol. Chem. 1989; 264: 15642-15649Google Scholar, 6Kramer R.H. Vu M.P. Cheng Y.P. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Google Scholar). This integrin receptor binds to the E8 fragment of laminin-1 and mediates cell adhesion to this ligand (6Kramer R.H. Vu M.P. Cheng Y.P. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Google Scholar). In melanoma, the expression of the α7 integrin receptor is elevated in the nonmetastatic phenotype but is lacking in metastatic cells, suggesting that absence of this laminin adhesion receptor can play a role in melanoma tumor cell dissemination. 1D. M. Ramos, N. Waleh, M. P. Vu, B. L. Ziober, C. C. Yao, and R. H. Kramer, submitted for publication. Expression of α7, as detected by reverse transcriptase-PCR, 2The abbreviations used are: PCRpolymerase chain reactionbHLHbasic helix loop helixCATchloramphenicol acetyltransferasebpbase pair(s)kbkilobase pair(s)Pipes1,4-piperazinediethanesulfonic acidInrinitiator sequence. has also been reported to be present in a number of tissues including for example stomach and uterus (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar). However, it has not yet been determined if any of the α7 protein isoforms are expressed in these tissues. In myoblasts and cardiomyocytes, α7 appears to be the predominant laminin-binding integrin expressed (8Kaufman S.J. Foster R.F. Haye K.R. Faiman L.E. J. Cell Biol. 1985; 100: 1977-1987Google Scholar, 9Mark H. Durr J. Sonnenberg A. Mark K. Deutzmann R. Goodman S.L. J. Biol. Chem. 1991; 266: 23593-23601Google Scholar). In vitro, basal levels of α7 expression are seen in replicating rat and mouse myoblasts (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar, 10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Google Scholar, 11Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell Biol. 1992; 117: 643-650Google Scholar). Expression of α7 is increased upon differentiation of myoblasts to myotubes. This up-regulation is paralleled by increased expression of myogenin (11Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell Biol. 1992; 117: 643-650Google Scholar), a member of the muscle-specific basic helix-loop-helix (bHLH) transcription factor family, suggesting that these factors play a regulatory role in α7 expression during muscle development.At embryonic day 7.5, α7 can be detected in the ectoplacental cone (differentiating murine trophoblast), where it is believed to play a role in trophoblast adhesion and/or differentiation. The use of laminin fragments suggested that α7β1 may be one of the major trophoblast laminin-binding integrins involved in embryo implantation (12Sutherland A.E. Calarco P.G. Damsky C.H. Development. 1993; 119: 1175-1186Google Scholar). Recently, the cDNAs for rat and mouse α7 have been isolated and shown to be alternatively spliced in both the extracellular (giving rise to two isoforms, X1 and X2) and cytoplasmic (producing three isoforms, A, B, and C) domains. Subsequent work has shown that these alternatively spliced forms are also developmentally regulated in muscle (7Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Google Scholar, 10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Google Scholar, 13Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. J. Cell. Sci. 1993; 106: 1139-1152Google Scholar).Taken together, these results indicate that expression of α7 plays a role in embryo implantation and muscle development. Furthermore, the developmental and restricted tissue expression (in skeletal muscle and cardiac muscle) of the α7 subunit suggests that the elements governing its expression are complex. As an approach to understanding the mechanisms regulating cell type- and differentiation-specific expression of the α7 subunit, we isolated and characterized the promoter region for the murine α7 integrin gene.