Characterization of the bidirectional promoter region between the human genes encoding VLCAD and PSD-95.
Bidirectional promoters are widely known among lower organisms but rare in mammals. A shared promoter between the two human genes encoding very long chain acyl-CoA dehydrogenase (VLCAD) and postsynaptic density protein 95 (PSD-95) is an ideal model to investigate bidirectional transcription in mammals. VLCAD associates with the inner mitochondrial membrane and catalyzes the initial step in mitochondrial long-chain fatty acid beta-oxidation. PSD-95, a component protein of the PSD, plays an essential role in clustering the transmembrane proteins in synaptic membranes. Interestingly, the human genes encoding VLCAD (ACADVL) and PSD-95 (DLG4) are adjacently located in the head-to-head orientation on chromosome 17p. The transcribed regions of the two genes overlap, while the two transcription start sites stand approximately 220 bp apart. To analyze the common transcriptional control region shared by the two genes, we generated serial promoter partial deletion constructs using firefly luciferase as the reporter gene. Our results showed that the essential promoter activity of PSD-95 is carried within an approximately 400-bp region, which covers the entire approximately 270-bp minimal promoter of VLCAD. The results from di-(2-ethylhexyl) phthalate (DEHP)-treated HepG2 cells revealed that the minimal VLCAD promoter is able to up-regulate VLCAD expression in response to DEHP treatment. Site-directed mutagenesis experiments showed that a mutated activator protein 2-binding site markedly reduced the transcriptional activity of both promoters and abolished the minimal VLCAD promoter's response to DEHP treatment.
Zhang
Li-Feng
LF
Kimberly H Courtwright & Joseph W Summers Institute of Metabolic Disease, Baylor University Medical Center, Dallas, TX 75226, USA.
Ding
Jia-Huan
JH
Yang
Bing-Zhi
BZ
He
Guo-Cheng
GC
Roe
Charles
C
D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics*; D017931:DNA Primers; D004051:Diethylhexyl Phthalate; D000076122:Disks Large Homolog 4 Protein; D040461:Gene Components; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA; D015854:Up-Regulation
C501444:DLG4 protein, human; D017931:DNA Primers; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C033652:postsynaptic density proteins; D004051:Diethylhexyl Phthalate; D008156:Luciferases; D044942:Acyl-CoA Dehydrogenase, Long-Chain