Sterol-dependent repression of low density lipoprotein receptor promoter mediated by 16-base pair sequence adjacent to binding site for transcription factor Sp1.
A 42-base pair sequence from the 5' flanking region of the low density lipoprotein receptor gene was shown previously to confer sensitivity to sterol-mediated repression when inserted into the herpes simplex virus thymidine kinase promoter. This sequence contains two contiguous 16-base pair repeats, designated repeats 2 and 3, which differ from each other at four positions. In the current study we have analyzed separately the functions of repeats 2 and 3 by altering their sequences, inserting them into the -60 position of the thymidine kinase promoter, and introducing the hybrid promoters into hamster cells by transfection. These studies show that repeat 3 is a constitutive positive transcriptional element that acts in the absence or presence of sterols. Repeat 2 confers strong repression upon repeat 3 when sterols are present. In vitro DNase footprinting and gel retardation assays show that repeat 3, but not repeat 2, binds purified Sp1, a positive transcription factor. Mutants of repeat 3 that abolish transcriptional activity in vivo abolish Sp1 binding in vitro. We suggest that the low density lipoprotein receptor is regulated by a push-pull mechanism in which sterol-regulated binding of a protein to repeat 2 silences the activity of the adjacent Sp1-binding site in repeat 3.
Dawson
P A
PA
Department of Molecular Genetics, University of Texas Health Science Center at Dallas 75235.
Hofmann
S L
SL
van der Westhuyzen
D R
DR
Südhof
T C
TC
Brown
M S
MS
Goldstein
J L
JL
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor; D013261:Sterols / Q000494:pharmacology*; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D011973:Receptors, LDL; D016329:Sp1 Transcription Factor; D013261:Sterols; D014157:Transcription Factors; D013937:Thymidine Kinase