A Silencer Element for the Lipoprotein Lipase Gene Promoter and Cognate Double- and Single-Stranded DNA-Binding Proteins

Abstract

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.

Association of lipoprotein lipase gene polymorphisms with obesity and type 2 diabetes in an Asian Indian population

“…It is known that the nucleotides flanking this conserved element may influence binding affinity of Sp1 and Sp3. 22 Furthermore, this variant lies within the region of LSE-2, identified by Tanuma et al 23 as a potential LSE-2, and hence it is possible that this T-to-G change at À93 results specifically in the disruption of LSE-2, leading to increased expression of LPL.…”

Repression of MHC class II gene transcription in trophoblast cells by novel single-stranded DNA binding proteins

“…The majority of transcription factors recognize and bind to double-stranded DNA; however, there are a number of reports of single-stranded DNA binding proteins that have been implicated in both positive and negative regulation of gene expression (Pan et al, 1990;O'Neill et al, 1991;Kamada and Miwa, 1992;Altiok and Groner, 1993;Tanuma et al, 1995;Sun et al, 1995). Among these are cell type-specific single-stranded DNA binding proteins present in mammary epithelial cells that regulate β-casein gene transcription (Altiok and Groner, 1993), and factors in myoblasts and fibroblasts that negatively regulate α-actin gene transcription (Sun et al, 1995).…”

Heterogeneous nuclear ribonucleoprotein A/B and G inhibits the transcription of gonadotropin-releasing-hormone 1

“…Transcription factors generally bind to doublestranded DNA recognition sites distal to the TATA-box and influence promoter activity (Johnson, 1995;Tjian and Maniatis, 1994). Although traditional transcription factors are double-stranded DNA (dsDNA) binding proteins, in recent years, many sequencespecific, single-stranded DNA (ssDNA)/RNA (ssRNA)-binding proteins have been discovered that either activate Haas et al, 1995b;Tomonaga and Levens, 1996) or repress (Altiok and Groner, 1993;Gupta et al, 2003;Haas et al, 1995a;Kelm et al, 1999;Lindzey et al, 1994;Pan et al, 1990;Tanuma et al, 1995;Wilkison et al, 1990) transcriptional activity. Many such single-strand DNA-binding proteins are important for gene transcriptional regulation.…”