Common double- and single-stranded DNA binding factor for a sterol regulatory element.

Abstract

A cis-acting element necessary for sterol regulation, SRE-1, has previously been identified in the promoters of the low density lipoprotein receptor, hydroxymethylglutaryl (HMG)-CoA reductase, and HMG-CoA synthase genes. In this report we describe a nuclear factor, SRE-BF, isolated from Chinese hamster ovary nuclear extracts, that binds to the SRE-1 octanucleotide sequence. In addition to sequencespecific binding to SRE-1, as indicated by competition analysis with double-stranded DNA fragments, single-stranded oligomer DNA sequences also compete for binding in a sequencespecific fashion. Photochemical cross-linking experiments suggest that a common protein factor, with apparent molecular mass of 4549 kDa, recognizes both single-stranded and double-stranded SRE-1. A conserved DNA sequence has been identified in the 5' flanking regions of several sterol-regulated genes: the genes encoding the LDL receptor (4, 5), hydroxymethylglutaryl (HMG)-CoA reductase (6), and HMG-CoA synthase (2). This octanucleotide region, GTGCGGTG sterol regulatory element 1 (SRE-1), has been shown to be an essential regulatory sequence in all of these promoters. Deletion or point mutations within this region significantly reduce levels of transcription under conditions of sterol depletion for the HMG-CoA reductase (6), HMG-CoA synthase (2, 7) and LDL receptor genes (3).This region has been studied by a number of investigators. Gil et al. (8) reported cloning NF-1-like proteins that bound to TGG-containing sequences, which are also found in the SRE-1 region. Recently, a 19-kDa protein has been cloned (9) that binds to one of the two strands encoding the SRE-1 region of the HMG-CoA reductase promoter. This protein has no demonstrable affinity for the double-stranded sequence and is upregulated in the presence of sterols.In this study we sought to characterize the nuclear proteins that bind to SRE-1 in order to understand the role of SRE-1 in sterol-mediated transcriptional regulation. The relationship between factor binding and the sterol regulatory activity of SRE-1 mutants was investigated. A 45-to 49-kDa SRE-1-specific DNA binding activity was identified that binds to the native, double-stranded DNA element and, surprisingly, binds preferentially to one of the two single strands of SRE-1. MATERIALS AND METHODSPlasmids. Plasmids were constructed by standard techniques (10) and their structures were verified by DNA sequence analysis. pHMG-SRE resulted from ligation of a synthetic oligonucleotide containing the 20-base sequence (-141 through -160, see Table 1) from the hamster HMGCoA reductase promoter (9) into the BamHI/Xba I sites in pUC19. pLDL-SRE contains a 35-base sequence (-38 through -72) from the promoter region (repeat 2) of the human LDL receptor ligated into the BamHI/Xba I sites of pUC19.Oligonucleotides. Synthetic oligomers (Table 1). were prepared commercially by Oligos Etc. (Guilford, CT) on a modified Biotix (Danbury, CT) synthesizer using 13-cyanoethyl phosphoramidite chemistry. Oligomer SRE-H contains a 20-base se...

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

“…The estrogen receptor binds to both the double-and single-stranded forms of the estrogenresponsive element, showing higher affinity for the singlestranded form (21). This affinity preference is similar to that of the sterol regulatory element and its binding factor (41). The LSE-BF bound to both single-and double-stranded LSE-1 probes in the same sequence-specific fashion and with the same affinity.…”

“…Most eukaryotic sequence-specific DNA binding proteins recognize native double-stranded sequences, but there are several reports of single-stranded protein-DNA complexes that are thought to be involved in the regulation of transcription (5,9,20,21,27,31,33,(39)(40)(41)44). The estrogen receptor binds to both the double-and single-stranded forms of the estrogenresponsive element, showing higher affinity for the singlestranded form (21).…”

The Different Activities of the Two Activation Domains of the Brn-3a Transcription Factor Are Dependent on the Context of the Binding Site

“…Similar preferential binding to one single strand compared with the corresponding double-stranded sequence or the complementary single strand has been reported for proteins binding to specific regulatory sites in a number of other cases. These include proteins binding to the sterol regulatory element in the hydroxymethylglutaryl-CoA reductase and synthase genes (Stark et al, 1991), an inhibitory element in the growth hormone gene promoter (Pan et al, 1990), a regulatory region of the adipsin gene promoter (Wilkison et al, 1990) and an inhibitory region in the androgen receptor gene promoter (Grossman and Tindall, 1995). Similar preferential binding to one single strand has also been reported for the transcription factor Myef-2 (Haas et al, 1995) and for the estrogen receptor either alone or in association with another factor (Mukherjee and Chambon, 1990).…”

Identification of Translin and Trax as Components of the GS1 Strand‐Specific DNA Binding Complex Enriched in Brain

“…Although it has been widely assumed that the DNA binding domains of transcription regulatory factors bind selectively to double-stranded versions of their response elements, several recent studies have provided evidence for atypical transcription factors that bind preferentially to individual strands of these cis elements (Lannigan and Notides, 1989;Rajavashisth et al, 1989;Pan et al, 1990;Wilkinson et al, 1990;Stark et al, 1992;Coles et al, 1994;Flink and Morkin, 1995;Hass et al, 1995;Shimura et al, 1995;Smidt et al, 1995). To assess whether unconventional transcription factors of this type may target individual strands of the Egr response element, we have, in previous studies, performed gel-shift studies on brain extracts, using individual strands of the Egr response element as probes.…”