Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter: II. Purification and characterization

Xiaodong Wang, Michael R. Briggs, Xianxin Hua, Chieko Yokoyama, Joseph L. Goldstein, Michael S. Brown

Research output: Contribution to journalArticlepeer-review

265 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14497-14504
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number19
StatePublished - Jul 5 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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