Promoter analysis of the DHCR24 (3β-hydroxysterol Δ(24)-reductase) gene: characterization of SREBP (sterol-regulatory-element-binding protein)-mediated activation.
DHCR24 (3β-hydroxysterol Δ(24)-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at -98/-90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5'-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.
Servicio de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain.
Lidia A
LA
Daimiel
María E
ME
Fernández-Suárez
Sara
S
Rodríguez-Acebes
Lorena
L
Crespo
Miguel A
MA
Lasunción
Diego
D
Gómez-Coronado
Javier
J
Martínez-Botas
D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D013196:Dihydrotestosterone / Q000494:pharmacology; D004357:Drug Synergism; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D051780:Sterol Regulatory Element Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
C492214:KLF5 protein, human; D051741:Kruppel-Like Transcription Factors; D008077:Lipoproteins, LDL; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013196:Dihydrotestosterone; D008148:Lovastatin; D008156:Luciferases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C499055:DHCR24 protein, human