Functional single-nucleotide polymorphism in acetyl-CoA carboxylase ACACB gene promoter.
The acetyl-CoA carboxylase isoform ACC2 expressed in the liver generates malonyl-CoA, which primarily regulates fatty acid oxidation through inhibition of the mitochondrial carrier carnitine palmitoyl-CoA transferase-I. Activity is initiated by sterol regulatory element-binding protein-1 (SREBP-1) binding to steroid response elements SRE in ACACB gene promoter P-II. We proposed that sequence variation in the promoter might affect expression. We investigated the effect of a single-nucleotide polymorphism -368 C/T (rs16939972) in ACACB P-II on activity in transfected HepG2 hepatoma cells. The T-allele construct showed significantly lower activity than the C-allele (p = 0.016) but only in the presence of SREBP-1a overexpression. Electrophoretic mobility shift assays showed that HepG2 nuclear proteins bound specifically to both allele probes, but with higher affinity to the T-allele. We tested competition for nuclear protein binding between the T-allele probe and unlabeled probes containing consensus sequences for six candidate transcription factors plus SREBP-1a. The SREBP-1a competitor probe had no effect on the shifted complex. GATA, c-Myb, and GR competitor probes abolished the complex; however, these proteins were undetectable in mass spectrometry of gel extracts from shifted bands. In conclusion, the -368 C/T single-nucleotide polymorphism in ACACB P-II binds HepG2 nuclear proteins that affect promoter activity in an allele-specific fashion.
Lee
Alex K
AK
Nutritional Sciences Division, King's College London, London, United Kingdom.
Kyriakou
Theodosios
T
Weston
Andrew J
AJ
O'Dell
Sandra D
SD
D000103:Acetyl-CoA Carboxylase / Q000235:genetics*; D000483:Alleles; D001483:Base Sequence; D056945:Hep G2 Cells; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D051780:Sterol Regulatory Element Binding Protein 1 / Q000378:metabolism
D051780:Sterol Regulatory Element Binding Protein 1; C520105:ACACB protein, human; D000103:Acetyl-CoA Carboxylase