Abstract
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.
Highlights
In mammals, alpha-fetoprotein (AFP) and albumin (ALB) genes are closely related in structure, and remain adjacent in genome, with albumin 5′ to AFP gene[1]
To further identify the ZBTB20-binding site in AFP gene, we first established an ELISA-based DNA-protein binding assay (EDBA) system, in which synthetic biotinylated DNA probe was immobilized onto streptavidin-coated plate, and DNA-protein complex was detected by anti-ZBTB20 antibodies colorimetrically (Fig. 1a)
We define a cognate ZBTB20-binding site located between − 104 and − 86 of the AFP gene, which mediates sequence-specific DNA binding to and transcriptional repression by ZBTB20
Summary
Alpha-fetoprotein (AFP) and albumin (ALB) genes are closely related in structure, and remain adjacent in genome, with albumin 5′ to AFP gene[1] They are simultaneously activated during liver specification, but autonomously regulated after birth, with albumin gene active throughout life, and AFP gene dramatically repressed to basal line. Zhx[2] gene, which encodes a zinc finger and homeobox protein, regulates AFP postnatal repression in liver, and its mutation in BALB/cJ mice leads to 5–20 fold higher adult serum AFP levels[24]. Zinc finger protein ZBTB20 is developmentally regulated in liver, and acts as a key repressor of AFP gene transcription in liver, the specific ablation of which in liver leads to thousands-fold increase in AFP mRNA levels in adulthood[25]. We demonstrate that the sequence of − 104/− 86 in mouse AFP gene is a cognate ZBTB20-binding site which mediates sequence-specific binding to and repression by ZBTB20
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