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Am J Physiol Gastrointest Liver Physiol 284: G248-G254, 2003. First published October 16, 2002; doi:10.1152/ajpgi.00391.2002
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Vol. 284, Issue 2, G248-G254, February 2003

Regulation of apo A-IV transcription by lipid in newborn swine is associated with a promoter DNA-binding protein

Song Lu, Ying Yao, Heng Wang, Songmei Meng, Xiangying Cheng, and Dennis D. Black

Children's Foundation Research Center of Memphis at Le Bonheur Children's Medical Center, University of Tennessee Health Science Center, Memphis, Tennessee 38103

Dietary lipid acutely upregulates apolipoprotein (apo) A-IV expression by sevenfold at the pretranslational level in neonatal swine jejunum. To determine the mechanism of this regulation, two-day-old female swine received intraduodenal infusions of low- and high-triacylglycerol (TG) isocaloric diets for 24 h. Nuclear runoff assay confirmed apo A-IV gene transcriptional regulation by the high-TG diet. Footprinting analysis using the swine apo A-IV proximal promoter sequence (+14 to -246 bp) demonstrated three regions protected by the low-TG extracts. Of these three motifs, only ACCTTC showed 100% homology to the human sequence and was further studied. EMSA was performed using probes containing wild-type (WT) and mutant (M) motifs. A shift was noted with the low-TG nuclear extracts with the WT probe but not with the M probe. Excess unlabeled free WT probe competed out the shift, whereas the M probe did not. No significant shift occurred with either probe using high-TG extracts. These results suggest that a repressor protein binds to the ACCTTC motif and becomes unbound during lipid absorption, allowing transcriptional activation of the apo A-IV gene in newborn swine small intestine.

DNAse I DNA protection assay; electrophoretic mobility shift assay; gene transcription; nuclear runoff assay; small intestine


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