Alpha-fetoprotein (AFP) represents a classical model system to study developmental gene regulation in mammalian cells. We previously reported that liver ZBTB20 is developmentally regulated and plays a central role in AFP postnatal repression. Here we show that ZBTB20 is a sequence-specific transcriptional repressor of AFP. By ELISA-based DNA-protein binding assay and conventional gel shift assay, we successfully identified a ZBTB20-binding site at -104/-86 of mouse AFP gene, flanked by two HNF1 sites and two C/EBP sites in the proximal promoter. Importantly, mutation of the core sequence in this site fully abolished its binding to ZBTB20 in vitro, as well as the repression of AFP promoter activity by ZBTB20. The unique ZBTB20 site was highly conserved in rat and human AFP genes, but absent in albumin genes. These help to explain the autonomous regulation of albumin and AFP genes in the liver after birth. Furthermore, we demonstrated that transcriptional repression of AFP gene by ZBTB20 was liver-specific. ZBTB20 was dispensable for AFP silencing in other tissues outside liver. Our data define a cognate ZBTB20 site in AFP promoter which mediates the postnatal repression of AFP gene in the liver.
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This work was supported by National Natural Science Foundation of China grants (81130084, 81100614, 31370754, 31025013), and National “973” Program of China grants (2012CB524904, 2013CB530603).
Zhang, Hai; Cao, Dongmei; Zhou, Luting; Zhang, Ye; Guo, Xiaoqin; Li, Hui; Chen, Yuxia; Spear, Brett T.; Wu, Jia-Wei; Xie, Zhifang; and Zhang, Weiping J, "ZBTB20 Is a Sequence-Specific Transcriptional Repressor of Alpha-Fetoprotein Gene" (2015). Microbiology, Immunology, and Molecular Genetics Faculty Publications. 72.
Figure 1: Identification of ZBTB20-binding sequence in AFP promoter by DNA binding assays.
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Figure 2: Sequence-specific binding of ZBTB20 to the AFP gene.
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Figure 3: Sequence-dependent repression of AFP promoter activity by ZBTB20.
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Figure 4: Analysis of other potential ZBTB20 sites in AFP gene.
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Figure 5: Liver-specific AFP repression by ZBTB20 in adulthood.
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Figure 6: Expression analysis of transcription factors in ZBTB20-null liver.
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Figure 7: AFP −151/−53 confers ZBTB20 repression on albumin core promoter.
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Figure 8: Schematic demonstration of ZBTB20 site and other cis-acting elements in proximal AFP promoter.