Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization.
This paper describes the purification and characterization of a sterol regulatory element binding protein (SREBP) that recognizes the SRE-1 sequence in the 5' flanking region of the gene for the low density lipoprotein (LDL) receptor. The protein was purified more than 38,000-fold from nuclear extracts of human HeLa cells by ion exchange, gel filtration, and DNA-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation revealed a cluster of bands at 59-68 kDa, each of which bound to the SRE-1 element as revealed by cross-linking experiments. Binding of SREBP correlated perfectly with transcriptional activity in a series of 16 sterol regulatory elements with point mutations. In the LDL receptor promoter, the 10-base pair SRE-1 is embedded in a 16-base pair sequence designated Repeat 2, which is adjacent to Repeat 3, a binding site for nuclear factor Sp1. Oligonucleotides containing Repeat 2 + 3 bound SREBP and Sp1 as revealed by mobility shift assays. SREBP produced a DNase I footprint over the SRE-1 sequence, which was immediately adjacent to the footprint produced by Sp1. The current data are consistent with the concept that SREBP acts in concert with Sp1 to achieve high level, sterol-suppressible transcription of the gene for the LDL receptor.
Wang
X
X
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Briggs
M R
MR
Hua
X
X
Yokoyama
C
C
Goldstein
J L
JL
Brown
M S
MS
D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011973:Receptors, LDL / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011973:Receptors, LDL; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors