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Mol Cell Biol. Aug 2005; 25(16): 7260–7269.
PMCID: PMC1190267

Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

Abstract

apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf+/ mice were apparently healthy and fertile, no viable acf/ mice were identified. Mutant acf/ embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf/ blastocysts were detectable following implantation at E4.5, and isolated acf/ blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

RNA editing is an important genetic adaptation that permits distinct gene products to be encoded from a single genomic template. One of the most extensively studied examples of this model of mammalian gene regulation is substitutional C-to-U RNA editing of the apolipoprotein B (apoB) transcript, in which a single nucleotide modification results in the translational termination of a truncated protein, apoB48, which is required for intestinal lipid absorption (4). C-to-U editing of the nuclear apoB mRNA is a site-specific deamination reaction that requires a holoenzyme containing a minimal functional core composed of apobec-1, the catalytic deaminase (29), and an RNA binding subunit, apobec-1 complementation factor (ACF) (16, 19). These two proteins interact with one another (16, 19) and also with the substrate apoB RNA (5, 17) in a manner that positions the targeted C residue in an optimal configuration with respect to the active site of the deaminase. Their functional importance and the factors that modulate their respective abundances and subcellular (17) trafficking have been the focus of considerable attention (16, 19, 33).

Studies using recombinant protein and cell extracts have demonstrated that, while necessary for RNA editing, apobec-1 alone is insufficient (23, 26). The requisite importance of apobec-1 was unequivocally revealed through targeted deletion of apobec1, which completely eliminated in vivo C-to-U editing of the murine apoB transcript, demonstrating that apobec-1 is an essential and nonredundant component of the holoenzyme (20, 21). Nevertheless, while essential for C-to-U RNA editing, apobec-1 alone is insufficient. In vitro studies using recombinant apobec-1 and ACF have demonstrated that an optimal stoichiometry exists between these two proteins, each of which interacts with one another and with the apoB RNA, notably at a stretch of AU-rich residues flanking the targeted cytidine. Recent work has identified the major functional domains (including three canonical RNA recognition motifs) within ACF that mediate both protein-protein and protein-RNA interaction and that both facilitate and constrain C-to-U RNA editing of the nuclear apoB transcript (5, 17). In addition, ACF contains a novel nuclear localization sequence that directs the importation of heterologous proteins (6). However, while these findings illustrate the range of biochemical mechanisms underlying the role of ACF in apoB RNA editing in vitro, there is little information concerning the requirement for ACF in in vivo RNA editing or the range of alternative or complementary targets, either protein or RNA or both.

In the current study, we have undertaken the targeted deletion of the murine acf gene in order to establish the requirement for ACF in C-to-U editing of apoB RNA. Our results from this characterization suggest that ACF has a wider range of targets than apoB RNA alone.

MATERIALS AND METHODS

Generation of acf−/− mice.

A genomic bacterial artificial chromosome (BAC) clone was obtained by screening a 129/SvJ genomic BAC library (Incyte Genomics) with a full-length mouse ACF cDNA. To replace the exon 2 containing the initiator ATG of ACF with the coding sequence of green fluorescent protein (GFP) and a phosphoglycerol kinase (PGK)-neomycin (Neo) cassette, a replacement-type targeting vector was engineered. To create the 5′ arm, a fragment containing 2.4 kb of DNA upstream of the translation initiation ATG was amplified by PCR with the BAC clone as the template. The GFP “knock-in” cassette was generated by overlapping PCR with the 5′ arm and a GFP amplicon as the templates. The 3-kb 3′ arm was generated by PCR, also with the BAC clone as the template. Both PCR products, i.e., 5′ arm-GFP and the 3′ arm, were subcloned into pGEMT-easy (Promega). Upon digestion with NotI, ApaI, and XmnI, the 3′ arm was inserted into the NotI/ApaI sites of p1339 (accession number AF335420; a gift from Tim Ley, Washington University) using restriction sites introduced by PCR. The 5′ arm-GFP cassette was added to the KpnI/BamHI sites of the p1339-3′ arm vector upstream of a PGK-Neo cassette. The targeting construct was linearized with ApaI and electroporated into 129/SvJ embryonic stem (ES) cells. Genomic DNA was isolated from G418-resistant colonies, and correctly targeted clones were identified by Southern blot analysis.

Genotyping analysis.

The genotype of ES cells was determined by Southern blotting. Genomic DNA was digested with XbaI, resolved on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a 32P-labeled 793-nucleotide (nt) probe corresponding to the sequence downstream of the 3′ arm. Over 140 clones were analyzed for the homologous recombination event. A single positive clone was identified in which a 6.2-kb band was detected in addition to the 8-kb wild-type band. Targeted clones were expanded in culture, and 200 blastocysts were injected into pseudopregnant recipient C57BL/6 mice. A total of three chimeras were obtained, from which two mice were selected for breeding to establish germ line transmission of the targeted allele. The acf genotypes were determined by PCR with two different sets of three primers (see Fig. Fig.2A).2A). Set 1 included the following: a sense primer (Sse1), FSPmBAC (5′-CTGAACTGGGAATGAGTGGACATG-3′), was designed from intron 1 to amplify the wild-type and targeted alleles; an antisense primer (ASse1a), mACF Ex2 (5′-CCCCGGATTTGTGATTTGATT-3′), based on exon 2, will amplify a 338-bp band; and a third primer (ASse1b), GFP90AS (5′-CACGCTGAACTTGTGGCCGTTTAC3′), corresponding to the GFP sequence, will amplify the targeted allele, producing a 408-bp band. Set 2 included the following: the sense primer (Sse2a), FwdEx2 (5′-ATGGAATCAAATCACAAATCCGGGGATGG-3′), designed from the exon 2, amplifies the wild-type allele producing a 610-bp band; the antisense primer (ASse2), DwnEx2 (5′-CTTTCAAAGGTCTAACATGGGCAGAAGTTG-3′), will amplify both wild-type and targeted alleles; and the third primer (Sse2b), PGK (5′-AGGTGGCGCGAAGGGGCCACCAAAGAACGG-3′), corresponding to the PGK sequence of the Neo cassette, will amplify the targeted allele generating a 230-bp product. acf+/ mice were mated and monitored daily for plugs. Embryos at the indicated timed intervals (days postcoitum [dpc]) were recovered by microdissection for histological analysis or genomic DNA extraction with a Puregene DNA purification kit (Gentra Systems) and genotyping as described above.

FIG. 2.
Targeted disruption of acf gene. (A) Mouse acf gene and targeting vector. Upon homologous recombination of the 5′ and 3′ arms of the targeting vector with the acf locus (top), the exon 2 is replaced by the coding sequence of GFP and a ...

Protein and RNA extraction and analysis.

Tissues were flash frozen in liquid nitrogen and stored at −80°C until used. Protein concentrations were determined (Bio-Rad protein assay) on tissue extracts prior to Western blotting. RNA was isolated using TRIzol (Invitrogen) and 2 μg of DNase-treated RNA used for primer extension analysis of apoB RNA editing and quantitative and semiquantitative reverse transcription (RT)-PCR analysis (7). Real-time quantitative PCRs were performed with an ABI Prism7000 instrument (Applied Biosystems) by use of SYBR Green Master Mix according to the manufacturer's instructions, and mRNA abundance was determined by normalization to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level in each sample. PCR primers were as follows: ACF (5′-GCCAGAATCCTGCAATCCA-3′ and 5′-AGCATACCTCTTCGCTTCATCC-3′) and GAPDH (5′-TGTGTCCGTCGTGGATCTGA-3′ and 5′-CCTGCTTCACCACCTTCTTGA-3′). The distribution of ACF mRNA in mouse tissues was assessed by semiquantitative RT-PCR amplification using the following primers: ACF Fwd Ex2 (5′-ATGGAATCAAATCACAATCCGGGGATGG-3′) and antisenseEx7 (5′-CTGGCAGCAGCCTCCGCCTAGCCATGGCGG-3′). To improve the detection of spliced variants in the tissue survey, PCR was performed using [α-32P]dCTP (3,000 Ci/mmol, 3 μCi/reaction, NEN) as previously described (14). The primers were VBACF17 (5′-CAGCAATTCCCAGTCTTCATTTCC-3′) and VBACF18 (5′-CCCAGGTAAAATGTCATAGAGTTTG-3′). The PCR products were resolved on an 8% polyacrylamide gel electrophoresis (PAGE) gel and analyzed by phosphorimaging. For analysis of promiscuous hyperediting of apoB RNA, a 317-bp sequence of apoB RNA was amplified using primers ND1 (5′-ATCTGACTGGGAGAGACAAGTAGC-3′) and mLiver hypered.rev (5′-CAACAAATAAATAGAGATTATGG-3′). The PCR products were subcloned into PCR-Script (Stratagene) according to the manufacturer's protocol. Ten clones were randomly selected for plasmid DNA isolation and sequencing using an automated sequencer and T3 primer.

siRNA silencing of ACF.

Small interfering (siRNA) oligonucleotides were selected following established parameters (11), and several candidate primers, both murine and human specific, were purchased from Dharmacon Research Inc. Rat hepatoma cells (McArdle7777; American Type Culture Collection), a rodent cell line known to express ACF (7, 32), and human hepatoma cells (HepG2) were grown to 40% confluence on coverslips and transfected with 100 nM ACF or control-scrambled siRNA oligonucleotides according to the manufacturer's instructions (Ambion). Forty-eight hours posttransfection, cells were fixed in 10% formalin (Sigma) and probed with a rabbit anti-caspase 3 (active form) antibody (Cell Signaling) followed by a probe with Cy3 secondary antibody (Jackson Immunoresearch). The nuclei were identified using 4′,6′-diamidino-2-phenylindole (DAPI) (1 mg/ml in phosphate-buffered saline). Silencing of ACF protein expression was also assessed by Western blotting of cell lysates from siRNA or scrambled oligonucleotide-transfected cells grown under identical conditions with a rabbit polyclonal ACF antibody (7).

RESULTS AND DISCUSSION

Murine acf gene structure.

The murine acf gene contains 15 exons with the known functional domains illustrated in Fig. Fig.1A.1A. These findings are generally in agreement with the recent findings of Dur and colleagues (10), with the exception that there are three noncoding exons (exons 5, 6, and 11) reported in the current report (Fig. (Fig.1A).1A). The two major protein products (ACF64 and ACF65) differ, as in humans, by the differential splicing of a single exon (Fig. (Fig.1A),1A), which results in an eight-amino-acid insertion in the larger protein. A similar pattern of alternative splicing was demonstrated in the human ACF gene (14). ACF mRNA expression, as determined by real-time quantitative PCR, was distributed predominantly in the adult liver, small intestine, and kidney (data not shown). This pattern confirms that reported by Dur and colleagues (10). Recombinant human ACF64 and ACF65 reveal indistinguishable biochemical activities (5), but the possibility of an alternative biological function for these isoforms cannot be excluded. ACF43, a splice variant recently described in the rat (26), appears to be a minor transcript (<1%) in murine tissues (data not shown). ACF mRNA and protein were detected at 12 days of mouse embryonic development (Fig. 1B and C). ACF immunostaining was readily detected in the embryonic day 12 (E12) developing heart, spinal cord, and lung tissues (Fig. (Fig.1D),1D), in which there is virtually no detectable ACF mRNA or protein in adults (data not shown).

FIG. 1.FIG. 1.
Genomic organization of the mouse acf gene. (A) Upper panel, schematic representation of the intron-exon junctions. Solid bars represent exons. Solid lines represent introns. The exons encoding the three RNA recognition motifs are indicated by brackets. ...

acf gene targeting and phenotype characterization following heterozygous deletion.

A replacement-type targeting construct (see Materials and Methods) (Fig. (Fig.2A)2A) was electroporated, and ES cells were screened by Southern blotting (Fig. (Fig.2B).2B). acf+/ ES cells were injected into wild-type blastocysts to create germ line acf+/ mice. PCR genotyping (Fig. (Fig.2C)2C) was performed to identify mutant mice. acf+/ mice appeared healthy, grew at a rate similar to wild-type mice, and were fertile. ACF protein content was reduced in the kidney, liver, and small intestine of acf+/ mice relative to that found in wild-type littermates (Fig. (Fig.3A3A).

FIG.3.
Endogenous apoB mRNA editing in wild-type and acf+/−mice. (A) Immunodetection of ACF protein in liver, small intestine (SI), and kidney isolated from wild-type or heterozygous mice. Equal amounts of protein were resolved by sodium dodecyl ...

ACF and apobec-1 interaction yields an enzyme complex whose activity is critically dependent on the stoichiometry of its minimal component subunits (2, 7). To determine the functional consequence of the aforementioned alterations in ACF protein content, we examined the C-to-U editing of endogenous apoB mRNA; the findings demonstrate a consistent and statistically significant increase in the proportions of U6666-containing transcripts in both liver and kidney (Fig. (Fig.3B).3B). apoB mRNA editing was unchanged in the small intestines of these animals, exceeding 95% in both wild-type and acf+/ mice (Fig. (Fig.3B).3B). The demonstration of increased apoB mRNA editing in liver and kidney, by contrast, was somewhat unexpected and suggests that heterozygous deletion of ACF alters the stoichiometry of the core components of the holoenzyme. Among the possible considerations in this scenario are alterations in a metabolically active nuclear ACF pool, changes in nuclear cytoplasmic shuttling of apobec-1 and/or ACF (9), or even changes in ACF interaction with other protein partners. These possibilities will of course require formal evaluation in future studies.

In order to determine whether the increased C-to-U editing activity in acf+/ liver was confined to the canonical site at nt 6666, 10 clones were randomly selected from RT-PCR products, and a total of over 3,100 nt sequences was obtained from a region containing eight C residues, including the canonical site (Fig. (Fig.3C).3C). The results reveal only one clone (Fig. (Fig.3C)3C) in which there was an additional edited C, suggesting that promiscuous editing (25) or hyperediting (31) was not taking place in the setting of increased C-to-U editing at nt 6666. The C-to-U editing of other RNA targets was not examined. Taken together, the functional outcome of the heterozygous targeting of murine acf points to a tissue-specific increase in endogenous apoB RNA editing activity, possibly reflecting a subtle but distinct alteration in the stoichiometry of the core editing complex.

Phenotype associated with homozygous deletion of murine acf.

Three separate lines of acf+/ mice were established from independent founders, two of which were littermates and one of which was from a separate breeding. The progeny were genotyped by PCR (Table (Table1).1). We detected no acf−/− mice from any of these lines after genotyping 42 litters and 332 offspring, indicating that disruption of the acf locus resulted in embryonic lethality. The litter sizes from these heterozygous matings were similar to those from wild-type littermates (range, 7 to 10), and there was no apparent gender bias. In addition to the absence of homozygous acf−/− offspring, we observed a significant increase in heterozygous offspring among all three lines, with the ratio of heterozygous to wild-type mice averaging ~10:1 (range, 8.3 to 12.6) (Table (Table2),2), instead of the 2:1 ratio predicted from Mendelian inheritance. These genotypes were confirmed by two independent primer sets, as detailed in Fig. Fig.2C,2C, suggesting that trivial explanations, such as genotyping errors, are unlikely to account for the apparent increase in the number of heterozygous offspring. In addition, further crosses of the heterozygous acf+/ (mixed background 129/SvJ × C57BL/6) into an acf wild-type C57BL/6 apobec1−/ line resulted in two litters containing a total of 12 mice, all of which were acf+/ (data not shown). The karyotype of the targeted ES cells revealed a normal 40 X,Y distribution, suggesting that there is no chromosomal duplication or major rearrangement occurring in the ES cells used to generate the lines. To our knowledge, there are no other examples of an embryonic lethal phenotype where the distribution of genotypes favors the heterozygosity of the candidate gene, but further dissection of the underlying mechanisms will clearly require additional investigation.

TABLE 1.
Numbers of wild-type, heterozygous, and homozygous null offspring from genotype analysis of progeny from m acf heterozygous intercrosses
TABLE 2.
Details of wild-type and heterozygous acf genotype distribution in each individual line from genotype analysis of progeny from m acf heterozygous intercrosses

To determine the stage at which homozygous mutant mice die, we analyzed embryos at different times of gestation. Genotyping of embryos dissected at E7.5 and E12.5 revealed no acf−/− mice (Table (Table1).1). Furthermore, histological examination of the implantation sites (n = 6) in uterine sections obtained at E6.5 and E7.5 failed to reveal evidence of thrombi at the positions of putative resorbed deciduae (data not shown). In addition and as expected, no empty deciduae were noted under conditions where all implanted embryos (wild-type and heterozygous acf+/) appeared normal (data not shown). Taken together, these observations suggest that premature death of implanted acf−/ embryos was unlikely to account for the lack of acf−/− mice. We also attempted to recover improperly implanted embryos at E4.5 by flushing the uteri of pregnant acf+/ animals but failed to obtain any material. Taken together, these findings point to an early implantation defect in the acf−/− embryos.

To further investigate the possibility that acf−/− embryos fail to implant, we genotyped 42 blastocysts recovered from the uteri of timed pregnant acf+/− mice at E3.5, a period selected to closely precede implantation. The genotype distribution (Table (Table1)1) revealed that 9 were acf−/−, 6 were wild type, and 27 were heterozygous acf+/−. These results indicate that homozygous acf−/− embryos almost certainly fail to implant.

Among the mechanisms we considered for the failure of acf−/− embryos to implant was an inability to develop properly to the stage where implantation can occur. To assess this possibility, blastocysts (E3.5) were harvested from heterozygous acf+/ mice, cultured for 4 to 7 days, and examined for their ability to develop blastocyst outgrowths (27). The outgrowths were then genotyped by PCR. From 62 cultures, 40 blastocysts remained attached and revealed histological evidence of proliferation of both trophoblasts and inner cell masses (8, 28) (data not shown). Genotype analysis of these 40 outgrowths revealed that 22 were wild type and 18 were heterozygous acf+/. However, no acf−/− outgrowths were detected, consistent with the suggestion that acf−/ blastocysts degenerate and undergo cell death within the first 4 days of culture. These findings collectively point to the possibility that ACF is required for preimplantation development.

ACF knockdown in somatic cells results in apoptotic death.

To investigate the possibility that ACF is required for cell survival, we undertook ACF knockdown using siRNA-mediated gene silencing. Rat hepatoma cells were transfected with candidate siRNAs targeting different regions of ACF mRNA (Fig. (Fig.4A).4A). ACF protein abundance was reduced by >70%, with siRNAs targeting regions located 316 and 823 nucleotides downstream of the initiator ATG, whereas no effect was observed when cells were transfected with scrambled siRNAs (Fig. (Fig.4A).4A). In the setting of ACF knockdown, we observed an increased number of apoptotic cells, as indicated by the number of caspase 3-positive nuclei detected in siRNA4-treated cells (Fig. 4B and D). The initiation of apoptosis was supported by the demonstration of cleavage of caspase 3 into its active form (Fig. (Fig.4C).4C). The induction of apoptosis was further analyzed by staining with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. The delivery of siRNA4 to rat hepatoma cells resulted in a significant, approximately threefold increase of the number of nuclei positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (~23%) compared to the number in the scrambled siRNA-treated cells (~8%). The induction of apoptosis in rat hepatoma cells was also replicated in experiments using siRNA knockdown of ACF in primary murine hepatocytes (data not shown).

FIG.4.
Silencing of ACF mRNA in McArdle7777 cells. (A) McArdle7777 cells were transfected with individual siRNAs targeting two distinct regions of ACF mRNA. siRNA2 targets a sequence located 316 nt downstream of the transcription initiation codon. siRNA4 recognizes ...

One potential caveat to the conclusions concerning the effects of siRNA-mediated knockdown of ACF expression is that both rat hepatoma cells and murine hepatocytes express both ACF and apobec-1. Since the unconstrained expression of apobec-1 has been shown to be associated with alterations in cellular mRNA stability (1, 3), DNA mutator activity (13, 22), and even altered growth and malignant transformation (3, 30), it was important to extend the findings of siRNA-mediated knockdown of ACF to cells in which no apobec-1 expression is detected. Accordingly, we turned to the human hepatoma HepG2 cell line, since human liver is a robust source of apoB RNA-editing complementation activity and of ACF expression but does not express apobec-1 (12). siRNA-mediated knockdown of ACF expression in HepG2 cells resulted in an ~76% (four independent assays) decrease in ACF protein expression (Fig. (Fig.5A)5A) and a ~30-fold increase in caspase 3-positive-staining nuclei (Fig. (Fig.5B).5B). These results reinforce the conclusion that ACF knockdown in somatic cells results in apoptosis. In addition, the findings in HepG2 cells strongly imply that this effect is independent of apobec-1 expression.

FIG. 5.
Silencing of ACF in human hepatoma HepG2 cells. (A) Representative Western blot of ACF protein expression in extracts prepared from HepG2 cells transfected with a human ACF-specific siRNA or a scrambled siRNA control. The Hsp40 signal was used to demonstrate ...

To further resolve the observed embryonic lethality associated with homozygous acf deletion in vivo, we endeavored to determine the exact timing and cell type in which apoptosis occurs in E3.5 blastocysts. However, as inferred from immunofluorescent staining, ACF expression was below the level of detection in blastocysts isolated from heterozygous acf matings, precluding a formal conclusion to the question of whether apoptosis occurs exclusively in acf null blastocysts (data not shown). In addition, the transfection of siRNA oligonucleotides into cultured acf+/ ES cells produced inconsistent effects concerning the number of dead and apoptotic cells, which were possibly the result of uncontrolled variations in transfection efficiency (data not shown). We suspect that the resolution of the mechanisms and pathways leading to apoptosis following targeted acf deletion will require the development of a conditional deletor line.

Summary conclusions.

The central finding of this report is that the homozygous deletion of murine acf is associated with embryonic lethality, most likely the result of a developmental defect in blastocyst growth, leading to preimplantation failure. What functional properties of ACF could account for this phenotype? Crucial to its role in catalyzing C-to-U RNA editing is the fact that ACF is an RNA binding protein with high affinity (low kd) for AU-rich RNAs, including apoB RNA, its presumed target (7, 17, 18). In this regard, it is relevant that several AU-rich RNAs, including those for interleukin-6, interleukin-1β, Cox2, and gamma interferon, are all expressed in the early stages of embryonic development, including the blastocyst stage (24). Although the binding of ACF to these targets has yet to be demonstrated, it is not unreasonable to speculate that altered binding of ACF to any of these or other AU-rich targets might interrupt the development of acf/− embryos. Probing these underlying mechanisms will require alternative approaches to bypass the early lethality associated with germ line deletion. A potentially important clue to the mechanisms involved in the early embryonic lethality has emerged from recent findings by Kinnaird and colleagues (15). These workers have demonstrated that RNA interference-mediated silencing of the Caenorhabditis elegans ACF homolog, HRP-2, leads to embryonic arrest, with 48 h of exposure resulting in a failure to progress beyond the 64-cell stage (15). These findings, considered together with the current data, suggest that ACF is an hnRNP family member with a conserved and nonredundant role in early embryonic development.

Acknowledgments

This work was supported by grants (HL-38180, DK-56260, and DK-52574) to N.O.D.

We acknowledge the technical assistance of Karen Hutton in the Morphology Core and Ted Simon and Jennifer Stratman in the Mouse Core of the Washington University DDRCC.

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