New insights into cellular cholesterol acquisition: promoter analysis of human HMGCR and SQLE, two key control enzymes in cholesterol synthesis.
The two control points of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) are known targets of the transcription factor sterol-regulatory element binding protein-2 (SREBP-2). Yet the location of the sterol-regulatory elements (SREs) and cofactor binding sites, nuclear factor-Y (NF-Y) and specificity protein 1 (Sp1), have not been satisfactorily mapped in the human SQLE promoter, or at all in the human HMGCR promoter.



We used luciferase reporter assays to screen the sterol-responsiveness of a library of predicted SRE, Sp1 and NF-Y site mutants and hence identify bone fide binding sites. We confirmed SREs via an electrophoretic mobility shift assay (EMSA) and ChIP-PCR.



We identified two SREs in close proximity in both the human HMGCR and SQLE promoters, as well as one NF-Y site in HMGCR and two in SQLE. In addition, we found that HMGCR expression is highly activated only when SREBP-2 levels are very high, in contrast to the low density lipoprotein receptor (LDLR), a result reflected in mouse models used in other studies.



Both HMGCR and SQLE promoters have two SREs that may act as a homing region to attract a single SREBP-2 homodimer, with HMGCR being activated only when there is absolute need for cholesterol synthesis. This ensures preferential uptake of exogenous cholesterol via LDLR, thereby conserving energy.



We provide the first comprehensive investigation of SREs and NF-Ys in the human HMGCR and SQLE promoters, increasing our fundamental understanding of the transcriptional regulation of cholesterol synthesis.
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
Vicky
V
Howe
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
Laura J
LJ
Sharpe
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
Anika V
AV
Prabhu
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia. Electronic address: aj.brown@unsw.edu.au.
Andrew J
AJ
Brown
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000378:metabolism
D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; C409164:NDUFB7 protein, human; D009687:Nuclear Proteins; D051782:Sterol Regulatory Element Binding Protein 2; C402244:nuclear factor Y; D002784:Cholesterol; C553220:HMGCR protein, human; D006903:Hydroxymethylglutaryl CoA Reductases; D008156:Luciferases; D009247:NADH, NADPH Oxidoreductases
HMGCR; NF-Y; SQLE; SRE; SREBP-2; Transcriptional regulation