Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 15:5:11979.
doi: 10.1038/srep11979.

ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene

Hai Zhang  1 , Dongmei Cao  1 , Luting Zhou  1 , Ye Zhang  2 , Xiaoqin Guo  2 , Hui Li  2 , Yuxia Chen  2 , Brett T Spear  3 , Jia-Wei Wu  4 , Zhifang Xie  2 , Weiping J Zhang  2
Affiliations

ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene

Hai Zhang et al. Sci Rep. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Identification of ZBTB20-binding sequence in AFP promoter by DNA binding assays.
Different unlabeled probes competed with labeled probe −108/−53 to form the DNA-protein complex. (a) Schematic demonstration of EDBA system to detect ZBTB20 binding to DNA. Biotinylated-DNA probe was incubated with GST fusion protein of ZBTB20, and immobilized onto streptavidin-coated plate. The DNA-protein complex was detected by anti-ZBTB20 antibodies colorimetrically. (b) By EDBA, ZBTB20 bound to mouse AFP gene fragments −108/−53, but rather −170/−104 or −65/−1. The OD450 readout was blanked by GST control. n = 3 experiments. (c) By EDBA, ZBTB20 bound to mouse AFP gene fragment −108/−53 in a dose-dependent manner. n = 3 experiments. (d) Competitive ZBTB20-binding capacity of different fragments of the AFP gene in EDBA. 100-fold excess of the unlabeled DNA fragments were included to bind to ZBTB20 protein in competition with biotinylated probe −108/−53. (e) Competitive ZBTB20-binding capacity of different fragments of the AFP gene in mobility shift assay. 10- or 50-fold excess of unlabeled fragments from the AFP gene competed with 32P-labeled probe −108/−53 for binding to ZBTB20.
Figure 2
Figure 2. Sequence-specific binding of ZBTB20 to the AFP gene.
(a) DNA sequence of wild-type (WT) and mutant fragments −104/−86 of the AFP gene. The mutant sequence was shown in lower case. (b) The competitive ZBTB20-binding capacity of different mutant AFP gene fragments −104/−86 in EDBA. Biotinylated AFP gene fragments −104/−86 bound to ZBTB20 in the presence of 100-fold excess of wt or mutant fragments. Mutant fragments #6 and #7 lost the ability to compete with wt probe −104/−86. (c) In mobility shift assay, ZBTB20 bound to AFP gene fragments −104/−86. The DNA-protein complex formed by 32P-labeled AFP −104/−86 and ZBTB20 was blocked by excessive unlabeled wt probe and supershifted by anti-ZBTB20 antibody. The unlabeled mutant fragment #7 lost the ability to compete with labeled WT probe to form DNA-protein complex.
Figure 3
Figure 3. Sequence-dependent repression of AFP promoter activity by ZBTB20.
(a) Schematic demonstration of the WT and mutant AFP luciferase reporters starting from −837. The mutant nucleotides were shown in lower case. The HNF1 sites at AFP promoter were represented with open ovals. (b) The activity and ZBTB20-responsiveness of different mutant AFP reporters in HepG2 cells. The reporter plasmids were cotransfected into HepG2 cells with mock control vector (gray bar) or ZBTB20-expressing plasmids (black bar). RLU values were normalized to the activity of internal control RL-SV40. n = 3 experiments. *P < 0.05, **P < 0.01 vs mock control. (c) Sequence alignment of mouse AFP gene fragment −104/−86 with mouse albumin gene, and rat and human AFP genes.
Figure 4
Figure 4. Analysis of other potential ZBTB20 sites in AFP gene.
(a) Sequence alignment of AFP gene fragment −104/−86 with −160/−142, −284/−267, and −6474/−6456, respectively. (b) Competitive ZBTB20-binding capacity of different AFP gene fragments in EDBA assay. 100-fold excess of AFP gene fragments competed with biotinylated fragment −104/−86 to bind to ZBTB20. (c) AFP −276/−272 mutant reporter was repressed by ZBTB20 as effectively as WT counterpart. Schematic demonstration of mutant AFP reporter was shown schematically in upper part, with the mutant nucleotides at −276/−272 of AFP gene reporter indicated in lower case. The reporter plasmids were cotransfected into HepG2 cells with mock control vector (gray bar) or ZBTB20-expressing plasmids (black bar). Results are expressed as fold repression of luciferase normalized to the internal control RL-SV40. n = 3 experiments. P > 0.1. (d) ZBTB20 ablation didn’t compromise the inhibitory activity of AFP Enhancer III in liver. Transgenic mice EIII-βgl-Dd were crossed to ZBTB20 global knockout mice, H2-Dd mRNA levels in liver were measured by real-time RT-PCR at the age of day 2 and day 21. ZBTB20+/+Tg− (open bar), ZBTB20−/−Tg− (gray bar), ZBTB20+/+Tg+ (black bar) ZBTB20−/−Tg+ (slashed bar). The transgenic expression did not differ significantly in ZBTB20-null livers. P > 0.05. n = 4 experiments.
Figure 5
Figure 5. Liver-specific AFP repression by ZBTB20 in adulthood.
By real-time RT-PCR, AFP expression levels were measured in liver, gut, brain, and kidney from WT (open bar) or global ZBTB20 KO mice (black bar) at the age of 2 months. * P < 0.01. n = 4 experiments.
Figure 6
Figure 6. Expression analysis of transcription factors in ZBTB20-null liver.
By real-time RT-PCR, liver mRNA levels of AFP and transcription factors were compared between control (gray bar) and liver-specific ZBTB20 KO (LZB20KO, black bar) mice at the age of 6 months, with 36B4 as internal control. AFP mRNA levels in LZB20KO liver were approximately 6,000-fold higher than control (a). The expression levels of transcriptional activators HNF1α, HNF1β, C/EBPα, C/EBPβ, HNF3α, HNF3β, and HNF3γ in LZB20KO livers did not differ significantly from control (b). HNF1β was increased, but did not reach significance. The expression levels of AFP repressors NF1, COUP-TF1, Zhx2, and p53 in LZB20KO livers did not differ significantly from control (c). Zhx2 was decreased, but did not reach significance. The expression levels of cell proliferation regulators c-Myc, c-Jun, and Jun B in LZB20KO livers did not differ significantly from control (d).
Figure 7
Figure 7. AFP −151/−53 confers ZBTB20 repression on albumin core promoter.
(a) Alignment of mouse AFP and albumin promoters. HNF1 sites, ZBTB20-binding site, and TATA box in AFP promoter were shown, with the arrow indicating SpeI site in AFP promoter used for cloning. (b) Schematic demonstration of the chimeric promoters composing of AFP −151/−53 or −178/−53 (in black) and Alb −50/+2 (in gray). Partial sequence adjacent to fusion site was illustrated. The two HNF1 sites of AFP promoter were represented as black ovals. (c) ZBTB20 overexpression repressed AFP/Alb chimeric promoter reporter activity in HepG2 cells. *. P < 0.001 vs Alb-790Luc. n = 4 experiments. Error bar represented s.d.
Figure 8
Figure 8. Schematic demonstration of ZBTB20 site and other cis-acting elements in proximal AFP promoter.
ZBTB20 site (black oval) is located between the two HNF1 sites.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tilghman S. M. The structure and regulation of the alpha-fetoprotein and albumin genes. Oxf Surv Eukaryot Genes 2, 160–206 (1985). - PubMed
    1. Spear B. T. Alpha-fetoprotein gene regulation: lessons from transgenic mice. Semin Cancer Biol 9, 109–116 (1999). - PubMed
    1. Belayew A. & Tilghman S. M. Genetic analysis of alpha-fetoprotein synthesis in mice. Mol Cell Biol 2, 1427–1435 (1982). - PMC - PubMed
    1. Vacher J. & Tilghman S. M. Dominant negative regulation of the mouse alpha-fetoprotein gene in adult liver. Science 250, 1732–1735 (1990). - PubMed
    1. Hammer R. E., Krumlauf R., Camper S.A., Brinster R.L. & Tilghman S.M. Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science 235, 53–58 (1987). - PubMed

Publication types

MeSH terms