A novel E box/AT-rich element is required for muscle-specific expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene.

The cardiac/slow twitch sarcoplasmic reticulum (SR) Ca2+-ATPase gene (SERCA2 ) encodes a calcium transport pump whose expression is regulated in a tissue- and development-specific manner. Previously we have identified two distinct positive regulatory regions (bp -284 to -72 and -1815 to -1105) as important for SERCA2 promoter activity. Here we demonstrate that the SERCA2 distal promoter region functions like an enhancer by activating a heterologous promoter (TK) in a muscle cell-specific manner. Through deletion analysis a core enhancer region was delimited to the -1467 to -1105 bp fragment. We identified the E box/AT-rich element located at -1115 bp as critical for maximal enhancer activity. Gel mobility shift studies revealed that this E box/AT-rich element specifically binds a protein which is induced during Sol8 myogenesis. This region includes two other cis -acting elements, CArG and MCAT, which also bind specific nuclear protein complexes from Sol8 myotubes. Mutagenesis of each of these sites resulted in decreased SERCA/TK-CAT promoter activity. Based on these data, we propose that the E box/AT-rich element may contribute along with CArG and MCAT elements to the overall activation and regulation of the SERCA2 gene promoter.     

A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants

The tissue-specific expression of transgenes is essential in plant breeding programmes to avoid the fitness costs caused by constitutive expression of a target gene. However, knowledge on the molecular mechanisms of tissue-specific gene expression and practicable tissue-specific promoters is limited. In this study, we identified the cis -acting elements of a tissue-specific promoter from rice, P_D54O , and tested the application of original and modified P_D54O and its cis -elements in the regulation of gene expression. P_D54O is a green tissue-specific promoter. Five novel tissue-specific cis -elements (LPSE1, LPSE2, LPSRE1, LPSRE2, PSE1) were characterized from P_D54O . LPSE1 activated gene expression in leaf and young panicle. LPSRE2 suppressed gene expression in leaf, root, young panicle and stem, and PSE1 suppressed gene expression in young panicle and stem. LPSRE1 and LPSE2 had dual roles in the regulation of tissue-specific gene expression; both functioned as activators in leaf, but LPSRE1 acted as a repressor in stem and LPSE2 as a repressor in young panicle and root. Transgenic rice plants carrying cry1Ac encoding Bacillus thuringiensis endotoxin, regulated by P_D54O , were resistant to leaf-folders, with no Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated P_D54O showed various tissue-specific expression patterns. Different fragments of P_D54O fused with the constitutive cauliflower mosaic virus 35S promoter suppressed 35S -regulated gene expression in various tissues. P_D54O , truncated P_D54O and the tissue-specific cis -elements provide useful tools for the regulation of tissue-specific gene expression in rice breeding programmes.