Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB/cJ mice is due to a retrovirus insertion in the Zhx2 gene

Abstract

The alpha-fetoprotein (AFP) and H19 genes are transcribed at high levels in the mammalian fetal liver but are rapidly repressed postnatally. This repression in the liver is controlled, at least in part, by the Afr1 gene. Afr1 was defined >25 years ago when BALB/cJ mice were found to have 5- to 20-fold higher adult serum AFP levels compared with all other mouse strains; subsequent studies showed that this elevation was due to higher Afp expression in the liver. H19, which has become a model for genomic imprinting, was identified initially in a screen for Afr1-regulated genes. The BALB/cJ allele (Afr1(b)) is recessive to the wild-type allele (Afr1(a)), consistent with the idea that Afr1 functions as a repressor. By high-resolution mapping, we identified a gene that maps to the Afr1 interval on chromosome 15 and encodes a putative zinc fingers and homeoboxes (ZHX) protein. In BALB/cJ mice, this gene contains a murine endogenous retrovirus within its first intron and produces predominantly an aberrant transcript that no longer encodes a functional protein. Liver-specific overexpression of a Zhx2 transgene restores wild-type H19 repression on a BALB/cJ background, confirming that this gene is responsible for hereditary persistence of Afp and H19 in the livers of BALB/cJ mice. Thus we have identified a genetically defined transcription factor that is involved in developmental gene silencing in mammals. We present a model to explain the liver-specific phenotype in BALB/cJ mice, even though Afr1 is a ubiquitously expressed gene.

Fig. 1.

Persistent H19 expression in adult liver correlates with reduced mRNA of Afr1 candidate gene Zhx2. (A) Determination of Afr1 genotype by Northern analysis. Ten micrograms of total adult liver RNA was used in each lane from the following mice: BALB/cJ (B), C3H (C), and (BALB/cJ × C3H) × BALB/cJ F₂ backcross animals (89, 91, 92, 93, and 95 from litter 1; 218, 219, and 220 from litter 2). Mice 89 and 219 (asterisks) show recombination between D15Mit132 and Afr1, and Afr1 and D15Mit121, respectively. Blots were hybridized with an H19 probe and then reprobed with GAPDH to control for RNA loading. (B) PCR genotyping using tail biopsied DNA from mice described above by using primers for D15Mit132 and D15Mit121 microsatellite markers (designated on right). (C) RT-PCR analysis of total liver RNA from the indicated mice by using primers for RIKEN cDNA clone ID 6320424E06 (labeled a and b in D). Reverse transcription samples were also amplified with primers for the β-actin gene as a control. (D) Structure of the gene encoding RIKEN clone 6320424E06. The gene contains four exons, including an unusually large internal third exon that codes for the entire predicted ZHX2 protein. The first, second, and third introns are 68.4, 58.1, and 14.9 kb in length, respectively.

Fig. 2.

Afr1 is ubiquitously expressed in adult tissues and is more abundant in adult liver than in fetal liver. Northern analysis was performed by using 5 μg of poly(A)⁺ RNA from various BALB/c(Afr1^a) tissues with a probe derived from Afr1 exon 3. FL, fetal day 18.5 liver; B, brain; Th, thymus; H, heart; Lu, lung; L, liver; S, spleen; G, gut; K, kidney; M, muscle; T, testes. The blots were stripped and rehybridized with a GAPDH probe to control for RNA loading.

Fig. 3.

RNA analysis reveals the presence of a correctly initiated but aberrantly sized Afr1 transcript in BALB/cJ mice. (A) Northern analysis was performed with 5 μg of poly(A)⁺ RNA from adult BALB/c (Afr1^a) and BALB/cJ (Afr1^b) liver. In Left, the blot was hybridized with a probe corresponding to the 3′ end of Afr1 exon 1. The arrows denote the 4.4-kb and ≈6.0-kb bands present in BALB/c and BALB/cJ samples, respectively; the blot was overexposed to show the ≈6.0-kb transcript. In Right, the blot was hybridized with a probe corresponding to the 3′ end of Afr1 exon 3. In both Left and Right, the blots were stripped and reprobed with an H19 cDNA probe to confirm the Afr1 phenotype, and stripped again and reprobed for GAPDH to control for RNA loading. (B) S1 nuclease protection assay using RNA derived from the S194 mouse plasmacytoma cell line (S194), Hepa1–6 mouse hepatoma cell line (H-1-6), adult BALB/c liver, and adult BALB/cJ liver. In Upper,5 μg of poly(A)⁺ was hybridized with a probe that spans the 5′ end of Afr1 exon 1 and putative promoter region. In Lower, 100 μg of total RNA was hybridized with a probe that spans the Afr1 exon 4 splice acceptor junction. M, DNA marker lane.

Fig. 4.

Insertion of a mouse endogenous retroviral element into intron 1 of the BALB/cJ Afr1^b allele. (A) Southern analysis of DNA from BALB/c (c) and BALB/cJ (cJ) mice. Ten micrograms of DNA was digested with BamHI or EcoRI, resolved on 0.75% agarose gels, and transferred to nitrocellulose. Blots were probed with a 2.6-kb fragment of intron 1 (shown as a hatched box in B) centered 47 kb downstream of the first exon. (B) Schematic representation of the location of the MERV insertion in the Afr1^b allele. The locations of the BamHI (Bam) and EcoRI (RI) sites are shown (in kb) downstream of the transcription start site. The arrowheads designated c and d represent oligonucleotides used for PCR amplification of DNA from this region. (C) Sequence of the site of MERV integration in the Afr1^b allele and the corresponding Afr1^a allele. BALB/cJ and BALB/c DNA were amplified by PCR using primers c and d in B, cloned, and sequenced. The endogenous sequence is shown in lowercase, with the target site duplications underlined. MERV sequences are in uppercase, with the dashed line representing the intervening MERV region. (D) Sequence of 3′-RACE-walk clone of the chimeric transcript arising from the Afr1^b allele. The sequences from the Afr1 exon 1 are lowercase and MERV sequence is uppercase. Sequence of the exon 1–exon 2 junction of the Afr1^a cDNA from BALB/c is shown below for comparison. (E) RT-PCR confirms the presence of a chimeric transcript in the Afr1^b but not Afr1^a allele. Random hexamer-primed reverse transcription was performed by using 0.5 μg of adult liver poly(A)⁺ RNA from BALB/c and BALB/cJ mice, followed by PCR using a forward primer derived from Afr1 exon 1 and a reverse primer derived from the MERV insertion element. As a control, PCR amplification was performed with the same RT-PCR templates by using β-actin gene primers.

Fig. 5.

Liver-specific expression of a Zhx2 transgene restores H19 repression in an Afr1^b/b background. Three founder mice containing the TTR-Afr1 transgene were backcrossed to BALB/cJ; liver RNA from F₂ offspring was harvested 4 weeks after birth. Data are shown from F₂ littermates from line 1 (high-copy transgene, lanes 1–3) and line 2 (low-copy transgene, lanes 4–7); similar results were seen with the third line. The presence or absence of the TTR-Afr1 transgene (+ or -, respectively) and the endogenous Afr1 genotype (a/b or b/b) were determined by PCR analysis of tail-biopsied DNA from each mouse. RT-PCR (using the same oligonucleotides as those shown in Fig. 1d) confirmed the expression of the endogenous Zhx2 gene in mouse 6. Northern analysis (using an exon 3 probe) confirmed the presence of Zhx2 mRNA from the endogenous locus (4.4-kb band) in mouse 6 and the TTR-Afr1 transgene (Afr1 TG) (3.2-kb band) in mice 1, 3, 6, and 7. H19 levels were determined by Northern analysis and shown to be reduced in the presence of the TTR-Afr1 transgene relative to nontransgenic littermates. GAPDH was used as a control for RNA loading.

Fig. 6.

Zhx2 transcripts are low but detectable in several adult BALB/cJ tissues but are not detectable in the liver. Northern analysis was performed by using 5 μg of poly(A)⁺ RNA from various BALB/cJ (Afr1^b) tissues and BALB/c lung (Afr1^a) with an Afr1 exon 3. The blots were stripped and rehybridized with a GAPDH probe to control for RNA loading. B, brain; Lu, lung; L, liver; K, kidney.