Dysregulated AFP expression in LZB20KO liver. (A) AFP mRNA expression in liver from 4-month-old control (gray bar) and LZB20KO (black bar) mice was shown by real-time RT-PCR. n = 3 experiments. (B) Real-time RT-PCR analysis for AFP mRNA expression in liver from control (gray bar) and LZB20KO (black bar) mice during postnatal days 1–28. Each group at individual time points included at least 5–8 mice. (C) Northern blot analysis for AFP and ALB mRNA expression in the livers from 1-day-, 2-week-, and 20-week-old LZB20KO mice (lanes 2, 4, and 6) and their control littermates (lanes 1, 3, and 5). The RNA loading control was demonstrated by EtBr staining of the gel. (D) Western blot analysis of AFP expression in whole liver lysates from 3-week-old control (lanes 1 and 3) and LZB20KO (lanes 2 and 4) mice. The loading control was demonstrated by reprobing with anti-actin antibody. (E and F) In situ β-galactosidase staining showed LacZ expression throughout the liver from 3-week-old ZBTB20^flox/flox/Alb-cre/AFP^−/− compound knockout mice (F) rather than the same-aged ZBTB20^flox/flox/AFP^−/− knockouts (E), which reflected the AFP gene transcriptional activity. Error bar represents SD.

ZBTB20-mediated transcriptional repression of AFP activity. (A) 293T cells were cotransfected with different amounts of Gal4-ZBTB20 fusion expression vector, (GAL)₅-sv40-Luc reporter, and pCMV-β-Gal. Results are expressed as fold repression of luciferase normalized to the internal control. (B–E) HepG2 cells were cotransfected with ZBTB20 expression plasmids, different luciferase reporters, and the internal control pCMV-β-Gal. Results are expressed as fold repression or relative luciferase activity normalized to the internal control. n = 3 experiments. (B) ZBTB20 overexpression repressed AFP-2309Luc reporter (−2309 to +27) activity in HepG2 cells in a dose-dependent manner. n = 4 experiments. (C) ZBTB20 overexpression repressed AFP-2309Luc reporter activity in HepG2 cells but showed no effects on Gck or Alb reporter activity. Gray bar = mock control of empty vector; black bar = ZBTB20 expression vector. n = 3 experiments. (D) ZBTB20 overexpression repressed the activity of the AFP reporter driven by the AFP promoter −2309/+27 or 5′ truncations at −1103, −837, −200, −178, −162, or −151. n = 5 experiments. ∗, P < 0.01; Error bar represents SD.

Developmental activation of ZBTB20 leads to postnatal AFP repression in liver. (A) Immunohistochemical staining of ZBTB20 in the adult liver from normal C57BL/6 mice. ZBTB20 was stained with anti-ZBTB20 antibodies and visualized by Alexa Fluor 594 (red). Nuclei were counterstained with DAPI (blue). CV, central vein. (Scale bar, 50 μm.) (B) Real-time RT-PCR analysis for ZBTB20 mRNA levels in the developing postpartum livers from C57BL/6 mice at different ages. (C) Immunoblot of liver nuclear extracts from C57BL/6 mice at different weeks of age with anti-ZBTB20 (upper row) and anti-Sp1 (lower row) antibodies. n = 2 experiments. (D) The postulated model for AFP repression in postpartum liver. The ZBTB20 gene, which is developmentally activated in postpartum liver with the expression level (red line) increasing gradually, binds to the AFP promoter and leads to the dramatic repression of AFP gene transcription in liver (blue line).

Generation of LZB20KO mice. (A) Schematic demonstration for the gene targeting of exon 6 of the ZBTB20 gene. The diagram of ZBTB20 protein is shown at the top. The probe 5′ is used for Southern blot analysis, which detects a 7.3-kb band for the wild-type (WT) allele and a 5-kb band for the homologously recombined (HR) Neo-flox allele in the EcoRV-digested genomic DNA. The loxP sites are represented as filled triangles. V, EcoRV; X, XhoI; N, NcoI; Neo, neomycin resistance cassette. (B) Southern blot analysis of ES cell clones after EcoRV digestion and hybridization with the 5′ probe. Clone number is indicated above each lane. (C) Schematic demonstration for the removal of Neo and tissue-specific deletion of the floxed exon 6 in liver by Cre/loxP-mediated recombination. ZBTB20^neo-flox mice were mated with protamine-Cre mice to remove the Neo cassette and produce mice with the ZBTB20^flox allele. Repeated mating of ZBTB20^flox mice and ALB-Cre transgenic mice generated LZB20KO (ZBTB20^flox/flox/Alb-cre) mice. Primers P1 and P2 were used for PCR analysis to confirm the presence of exon 6 and its downstream loxP site, and P3 and P4 were used to detect the Neo cassette. (D) PCR analysis of tail genomic DNA distinguishing the ZBTB20^flox allele from the ZBTB20^neo-flox and wild-type alleles. PCR with the primers P1 and P2 gave a 0.8-kb band for the floxed allele and a 0.7-kb band for the WT allele (upper row). PCR with primers P3 and P4 produced a 1.0-kb band for the ZBTB20^neo-flox allele and no specific band from the ZBTB20^flox allele (lower row). Genotypes are indicated above each lane. (E) PCR analysis for Cre-mediated ZBTB20 deletion in liver. Genomic DNA from control (ZBTB20^flox/flox, lane 1) or LZB20KO liver (lane 2–4) was subjected to PCR amplification with primers P1, P2, and P3. Deletion of the floxed exon 6 produced a 1.2-kb band amplified by primers P3 and P2, whereas the 0.8-kb band indicates the presence of floxed exon 6. Efficient deletion of the ZBTB20 target exon in liver was shown by PCR products from increasing DNA template inputs (lane 2–4). (F) By real-time PCR, ZBTB20 mRNA expression was shown in control (ZBTB20^flox/flox) and LZB20KO mice. (G) Western blot analysis for ZBTB20 protein expression in the adult livers from control (ZBTB20^flox/flox) and LZB20KO mice. The nuclear lysate from liver was probed with anti-ZBTB20 (upper row) and anti-Sp1 antibodies (lower row).

Proliferation status of hepatocytes in adult liver. (A and B) Ki67 staining analysis for the livers from ZBTB20^flox/flox (A) and LZB20KO (B) mice at the age of 2 months. The inserts show positive Ki67 staining of intestines from the same animals. (C and D) BrdU staining in the livers from the 2-month-old ZBTB20^flox/flox (C) and LZB20KO (D) mice after 24h of in vivo labeling. The inserts show positive BrdU staining (C and D) of intestines from the same animals. n = 4 experiments. (Scale bar, 50 μm.)

Binding of ZBTB20 to the AFP promoter. (A) ZBTB20 bound to fragment −108/−53 of the Afp gene to form a DNA-protein complex. (B) The DNA-protein complex formed by labeled Afp −108/−53 and ZBTB20 was blocked by excessive unlabeled probe and supershifted by anti-ZBTB20 antibody. (C) ChIP analysis of normal mouse liver at the age of 2 months. Conventional PCR analysis was performed to determine the association of ZBTB20 with the promoter region of AFP in comparison to mouse 18S ORF (ORF). H3 antibody is shown as an internal control for chromatin recovery. IgG antibody is shown as a negative control. (D) Real-time PCR was performed to quantify antibody-bound DNA fragments of multiple liver ChIPs. The graph represents fold enrichment of ZBTB20 bound at AFP promoter region compared with 18S ORF. All values generated by real-time PCR analysis were normalized to control IgG precipitations. Each bar represents the average result from two independent ChIP experiments and six real-time PCR analyses.