Figure 3. rs12740374 alters a C/EBP transcription factor binding site.

a, The human DNA sequence surrounding rs12740374, major and minor alleles, and orthologous DNA sequence in mouse. The major allele of rs12740374 disrupts one of two core elements (position 2-3, 8-9) in the predicted consensus binding site on which a C/EBP dimer binds²¹. b, Electrophoretic mobility shift assays (EMSA) with labeled probes matching the C/EBP consensus binding site¹⁸, the rs12740374 minor allele (T) sequence, and the rs12740374 major allele (G) sequence. Competition assays were performed with 100-fold excess of cold probe. Either of two C/EBPα antibodies was used to compete for binding and/or shift the protein-DNA complex. c, Relative firefly luciferase expression from constructs with haplotypes of 2.1 kb region transfected into Hep3B cells. Single nucleotide alterations were introduced into constructs as indicated, altering rs12740374 and the three other core recognition nucleotides in the predicted C/EBP binding site. d, e, Relative firefly luciferase expression from constructs with haplotypes of 6.1 kb region transfected into (d) Hep3B human hepatoma cells with or without concomitant transduction with A-C/EBP (dominant negative C/EBP) cDNA via lentivirus and (e) NIH 3T3 fibroblasts with or without concomitant transduction with C/EBP-α cDNA via lentivirus. f, Relative SORT1 expression, determined as a ratio with B2M expression by qRT-PCR, in Hep3B cells [homozygous major (GG) at rs12740374] or SK-HEP-1 human hepatoma cells [heterozygous (GT) at rs12740374] with or without concomitant transduction with A-C/EBP cDNA via lentivirus. Error bars show s.e.m., N = 3 for each experiment.