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

Abstract

Expression of ␣7 is mainly confined to skeletal and cardiac muscle in which it appears to be the major laminin-binding integrin. When myoblasts differentiate to myotubes, ␣7 mRNA and protein expression is up-regulated. To explore the mechanisms involved in the tissuespecific and developmentally regulated expression of ␣7, we isolated and characterized a genomic clone containing ϳ2.8 kilobase pairs (kb) of the 5-flanking region of the murine ␣7 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 helixloop-helix family of muscle-specific transcription factors. The ϳ2.8-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 ␣7. Deletion analysis identified both positive and negative regulatory elements within the ϳ2.8-kb fragment. In 10T1/2 fibroblasts the ϳ2.8-kb ␣7 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 ␣7 gene during development.The integrin superfamily is made up of a number of transmembrane heterodimeric receptors that mediate cell-extracellular matrix and cell-cell interactions (1). Each integrin is composed of noncovalently paired ␣ and ␤ subunits. Regulation of the genes expressing these extracellular matrix receptors is important in a number of processes including development, migration, and invasion (2-4).Previously, we have reported that human and mouse melanoma cells express an integrin complex designated ␣7␤1 (5, 6). This integrin receptor binds to the E8 fragment of laminin-1 and mediates cell adhesion to this ligand (6). In melanoma, the expression of the ␣7 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.

Integrins in the mouse myotome: Developmental changes and differences between the epaxial and hypaxial lineage

“…MyoD has been shown to activate this integrin in cell culture models (Ziober and Kramer, 1996;Bergstrom et al, 2002). Our in vivo results show that ␣7 is up-regulated epaxially in elongated, mononucleated myocytes after these start expressing MyoD.…”

“…MyoD and myogenin are reported to activate expression of the ␣7 integrin subunit in C2C12 skeletal myoblasts (Ziober and Kramer, 1996). ␣7 is not present in the mouse myotome when myogenin starts being expressed (see above), but its up-regulation coincides in time with the appearance of MyoD (Velling et al, 1996).…”

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

“…Both of these families of proteins have a helix-loop-helix construction. E-box consensus binding motifs are present in the genes of many muscle proteins, including creatine kinase, myosin light chain, α7 integrin gene and the α and δ subunits of the acetylcholine receptor [11,24,31,32]. The three E-boxes surrounding the transcription-initiation site in the Phk γ subunit gene show a high degree of consensus (85-92 %) with E-box sequences in other muscle-specific genes, and appear to provide a strongly positive differentiation-dependent regulated expression.…”

“…Other members of this family include myogenin [29], myf5 [30] and MRF4 [20]. Acting in concert or individually, these factors have been implicated in the regulation of expression of an array of muscle-specific genes, including muscle creatine kinase [11], myosin light chain [31], α7-integrin gene [32] and the acetylcholine receptor α and δ subunits [24]. The regulatory potential of the three E-boxes closely proximal to the PhK-γ promoter at positions k114 to k109 (denoted here as Ep), j273 to j278 (denoted E " ) and j303 to j309 (denoted as E # ) was tested by the construction of pLuc plasmids containing varying complements of these sites.…”

TATA-less Mouse Vitronectin Gene Promoter: Characterization of the Transcriptional Regulatory Elements and a Nuclear Protein Binding Site on the Promoter.

“…The consensus sequence of the initiator is Py-Py-A-N-T/A-Py-Py (Py means pyrimidine, and N means A, G, C or T) (7, 28). Promoters of some integrin genes, such as al (33), a5 (34), and al (35) have a consensus sequence of the initiator.…”