Multiple Sp1 Binding Sites in the Cardiac/Slow Twitch Muscle Sarcoplamsic Reticulum Ca2⁺-ATPase Gene Promoter Are Required for Expression in Sol8 Muscle Cells

Abstract

2؉transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1-like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.

Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells

“…SP-1 binds to GC box promoter element and selectively activates mRNA synthesis by RNA polymerase II Tjian, 1983, 1985). A number of closely regulated genes have been reported to possess promoters with a high frequency of G and C residues suggesting that SP-1 plays a key role in transcription of these genes (Baker et al, 1996;Mo at et al, 1996;Cohen et al, 1997;Dharmavarm et al, 1997). However, SP-1 binding a nity and transcription properties can be altered by interaction with other co-factors.…”

Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac α-Actin Promoter

“…It is likely that the functional synergism and physical interaction properties of the proteins described here operate also for the regulation of additional muscle genes controlled by the same set of proteins. We note that several muscle-specific genes are positively regulated by a combination of Sp1 and myogenic bHLH proteins, including the regulatory regions of HCA (33), troponin I (19), ␣ and ␦ subunits of the acetylcholine receptor (4, 37), muscle sarcoplasmic reticulum Ca 2ϩ -ATPase gene (2), and muscle phosphofructokinase P2 (18). Our findings support the hypothesis that the regulation operated by Sp1 and myogenic bHLH on the HCA promoter is a general mechanism that applies to many genes expressed in skeletal muscle cells.…”

Nuclear Factor of Activated T Cells Regulates Transcription of the Surfactant Protein D Gene (Sftpd) via Direct Interaction with Thyroid Transcription Factor-1 in Lung Epithelial Cells

“…Electrophoretic Mobility Shift Assays (EMSAs)-Nuclear extract from MLE-15 cells were prepared as previously described (52) except that nuclear extracts were stored before dialyses. 32 P-Labeled doublestranded DNA probes were made by a Klenow end-filling reaction using [␣-32 P]dCTP.…”

“…The DNase I footprinting assay contained 10 fmol (ϳ5,000 cpm) of the end-labeled fragment in 200 l of 1ϫ binding buffer (52) containing 150-and 300-g nuclear extracts, and a protein binding reaction was carried out for 30 min at 4°C. DNase I reaction conditions and purification of digested DNA were as described previously (52). Samples were heatdenatured and loaded on to an 8% sequencing gel.…”