Transcriptional regulation of the rat phospholipse C-β3

Abstract

To understand the transcriptional regulation of the rat phospholipase C-β3 (PLC-β3) gene, the 5``-upstream region has been characterized. Sequence analysis of the 5``-upstream region showed that it contains a GC-rich region (-166 to +1: 79%) and multiple binding sites for the transcription factors Sp1, AP-1 and AP-2 but does not contain a canonical TATA box. It also showed that a 0.4 kb fragment including the proximal promoter, the first exon, and part of the first intron, contain several CpG dinucleotides, consistent with CpG island. These structural features, with the high G+C rich content, absence of TATA box and presence of the CpG island, show similarities to that of the promoters of various housekeeping genes. Primer extension analysis of total RNA isolated from rat glial cell C6Bu1 revealed that single transcription start site is located at a initiator (Inr) element similar to that found in the HIV promoter. Gel mobility shift and competitive mobility shift assays indicated that this in itiator element forms a DNA-protein complex with the HIV Inr-binding protein, LBP-1/CP2 or a homologue. Ectopic expression of LBP-1 can stimulate the promoter activity of the PLC-β3 gene when analyzed in transient co-transfection assay. These results indicated that LBP-1/CP2 can stimulate transcription through initiator element in the PLC-β3 promoter, although the exact mechanism remains unclear. In order to localize functional elements of the 5``-upstream region of the rat PLC-β3 gene, 5``-deletion fragments were cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. Transient transfection analyses of the 5``-deletion mutants identified a crucial promoter element located at -128 to -14. Gel mobility shift assay and DNase I footprinting analysis showed that the crucial promoter element contains four regulatory element, one A-box and three GC boxes. A-box (+112/+99) is identified as a novel regulatory element through sequence comparison with known regulator...