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Mol Cell Biol. 1989 August; 9(8): 3269-3283

Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter.

T A Gustafson and L Kedes

Department of Medicine, Stanford University School of Medicine, Palo Alto, California.

ABSTRACT

5' Sequences of the human cardiac alpha-actin gene are involved in the tissue-specific and developmental regulation of the gene. Deletion analyses combined with transient expression experiments in muscle cells have demonstrated three primary regions of functional importance (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986; T. Miwa and L. Kedes, Mol. Cell. Biol. 7:2803-2813, 1987), and we have previously demonstrated binding of a protein indistinguishable from serum response factor (SRF) to the most proximal region (T.A. Gustafson, T. Miwa, L.M. Boxer, and L. Kedes, Mol. Cell. Biol. 8:4110-4119, 1988). In this report, we examine protein interaction with the remainder of the promoter. Gel shift and footprinting assays revealed that at least seven distinct nuclear proteins interacted with known and putative regulatory regions of the promoter. The transcription factor Sp1 bound to eight sites, as demonstrated by footprinting assays and gel shift analysis with purified Sp1. Purified CCAAT box-binding transcription factor CTF/NF-I and Sp1 were shown to interact with the far-upstream regulatory element at -410, and footprint analysis showed extensive overlap of these two sites. Two unidentified proteins with similar but distinct footprints interacted with the second region of functional importance at -140, which contains the second CArG motif [CC(A + T rich)6GG], and these proteins were shown to be distinct from SRF. SRF was found to bind to the remaining three CArG boxes, two of which were closely interdigitated with Sp1 sites. In addition, CArG box 4 was found to interact with SRF and another distinct protein whose footprint was contained within the SRF-binding site. Sequences surrounding the TATA box were also shown to bind proteins. Sp1 was shown to bind to a site immediately downstream from the TATA box and to a site within the first exon. Thus, each of the three functional upstream regions, as defined by transfection assays, was shown to interact with five factors: Sp1 and CTF/NF-I at the upstream site, two unidentified proteins at the central site, and SRF at the most proximal site. These results suggest that expression of the cardiac actin gene in muscle cells is controlled by complex interactions among multiple upstream and intragenic elements.

Mol Cell Biol. 1989 August; 9(8): 3269-3283




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