The Zinc Finger Transcription Factor 191 Is Required for Early Embryonic Development and Cell Proliferation

EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998

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Research Article

The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

Jianzhong Lia,b, Xia Chene, Hua Yangb, Shuiliang Wangb, Baoyu Guoa, Long Yud, Zhugang Wangc, Jiliang Fub,c,⁎ aDepartment of Biochemical Pharmacy, Second Military Medical University, Shanghai, China bDepartment of Medical Genetics, Second Military Medical University, Shanghai, China cShanghai Nanfang Research Center for Biomodel Organism, Shanghai, China dGenetics Institute, Fudan University, Shanghai, China eShanghai Research Center of Biotechnology, Chinese Academy of Sciences, Shanghai, China

ARTICLE INFORMATION ABSTRACT

Article Chronology: Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN Received 13 March 2006 family member, which shows 94% identity to its mouse homologue zinc finger protein 191 Revised version received (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat 19 August 2006 (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been Accepted 23 August 2006 stated to have a quantitative silencing effect on TH gene expression and to correlate with Available online 30 August 2006 quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate Keywords: the functions of Zfp191 in vivo, we have used homologous recombination to generate mice − Zfp191 that are deficient in Zfp191. Heterozygous Zfp191+/ mice are normal and fertile. − − Gene targeting Homozygous Zfp191 / embryos are severely retarded in development and die at − − − Embryonic lethality approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191 / and Zfp191+/ Cell proliferation embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable − − Zfp191 / cell lines were obtained by derivation of embryonic stem (ES) cells from − blastocysts of Zfp191+/ intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation. © 2006 Elsevier Inc. All rights reserved.

Introduction sequences and subsequently modulate gene transcription [1]. A significant number of transcription factors use a conserved The regulation of gene expression in response to intrinsic and zinc finger domain to bind their target DNAs. Zinc finger extrinsic cues is a fundamental cellular process in the growth factors participate in a variety of cellular activities, such as and development of organisms. Critical to the control of gene development and differentiation, and play a role in human expression are transcription factors that bind to specific DNA disease. In fact, the human genome encompasses approxi-

⁎ Corresponding author. Department of Medical Genetics, Second Military Medical University, Shanghai, China. Fax: +86 21 65980521. E-mail address: [email protected] (J. Fu).

mately 600 to700 genes that contain a particular C2H2-type of lacking the TH gene (homozygous mutation) die at a late zinc finger, which employs two cysteine and two histidine stage of embryonic development or soon after birth [33,38]. amino acid residues to coordinate the single zinc atom in the The mouse Zfp191 has been previously isolated from – finger-like structure [2 4]. In the C2H2-type of zinc finger chondrocytic and mesenchymal precursor cell lines using proteins, there is a highly conserved consensus sequence a subtractive hybridization screening. Zfp191 mRNA is TGEKP(F/Y)X (X representing any amino acid) between adja- expressed during embryonic development and in different cent zinc finger motifs. The zinc finger proteins containing organs in adult, including rib cartilage, suggesting that this specific structure are termed Krüppel-like zinc finger Zfp191 may have a role in cartilage differentiation and in proteins because the structure was first found in Drosophila basic cellular processes [39]. To investigate the physiologi- Krüppel protein [5,6]. Many Krüppel-like factors exhibit cal functions of Zfp191 in vivo, we have mutated Zfp191 using − diverse regulatory functions in cell growth, proliferation, targeted mutagenesis in embryonic stem cells. Zfp191+/ mice − − differentiation, and embryogenesis [7–10]. The SCAN (SRE- did not display an overt phenotype, while Zfp191 / embryos ZBP, Ctfin 51, AW-1, and Number 18) [11,12], which is also were severely retarded in development and died at around − − known as the leucine rich region (LeR) [12], KRAB (Krüppel- 7.5 days post-fertilization. Unexpectedly, in Zfp191 / and − associated box) [13,14], and POZ (poxvirus and zinc finger) Zfp191+/ embryos, TH gene expression is not affected. [15] domains are highly conserved modules found in the Blastocyst outgrowth experiments and the RNA inter-

N-terminus of C2H2 zinc finger transcription factors that have ference (RNAi)-mediated knockdown of ZNF191 in cul- been demonstrated to mediate specific protein–protein inter- tured cells confirmed the essential role of Zfp191 in cell actions [16–19]. The SCAN domain is a conserved motif that proliferation. appears to control the association of SCAN-containing proteins into non-covalent complexes, and may be the primary mechanism underlying partner choice in the dimerization of Materials and methods these transcription factors [20–24]. The genes encoding SCAN domains are clustered, often in tandem arrays, in both the Creation of targeting vector human and mouse genomes and are capable of generating isoforms that may affect the function of family members. The targeting construct used for the homologous recombina- Although the function of most of the family members is not tion experiments was produced utilizing the pSSC-9 plasmid known, some of the SCAN domain family members play roles [40]. A genomic clone containing the entire coding region of in cell survival and differentiation or development [17,25–27]. the Zfp191 gene was isolated from a 129/SvJ genomic lambda

Like the KRAB motif, the SCAN domain-containing C2H2 zinc library (Stratagene) using the probe of human ZNF191 cDNA. A finger proteins are unique to the vertebrate lineage [2,3]. 2.3 kb BamHI–BamHI genomic fragment containing intron 1 Human zinc finger protein 191 (ZNF191, also known as and part of the exon 2 was subcloned into the BamHI site ZNF24 [28,29]) is a member of the SCAN domain family of downstream of the TK-neo cassette. Additionally, a 8.2 kb SacI– Krüppel-like zinc finger transcription factors [16]. This gene XhoI genomic fragment containing part of the exon 4 and the is initially named as RSG-A (for retinoic acid suppressed flanking region was subcloned between the XbaI and SalI sites gene-A) because its mRNA can be amplified by homologous upstream of the TK-neo cassette, yielding the final targeting RT-PCR only in retinoic acid-untreated but not in retinoic vector (Fig. 1A), having 2.3-kb 5′ and 8.2-kb 3′ homology arms. acid-treated acute promyelocytic leukemia NB4 cells [30]. All cloning steps followed standard methodology [41]. For ZNF191 is located on chromosome 18q12.1 [28,30] and electroporation, the vector was linearized with the restriction contains four exons. ZNF191 shows 94% identity to its enzyme SfiI. mouse homologue zinc finger protein 191 (Zfp191, also called ZF-12), which is the most highly conserved among the Targeting of the Zfp191 gene in mouse embryonic stem cells human–mouse SCAN family member orthologues pairs [20].

ZNF191 contains four continuous typical C2H2 zinc fingers in Forty micrograms of linearized plasmid DNA was electropo- its C-terminus, and one SCAN domain in its N-terminus rated into 129/SvJ embryonic stem (ES) cells, and cells were [28,30]. The SCAN domain of ZNF191 displays a suppressive selected for resistance to 400μg/ml G418 and 2 μmol/l effect on the transcription in CHO and NIH3T3 cells [30]. gancyclovir. All cell manipulations were performed as Biochemical binding study shows the SCAN domain of ZNF191 described [42]. To screen for the correctly targeted Zfp191 as a selective oligomerization domain [22]. Tissue mRNA gene, the drug-resistant ES cell clones were analyzed by PCR analysis showed that ZNF191 gene was ubiquitously expressed under standard conditions using a forward primer within the [30,31]. Recently, a study demonstrated that ZNF191 can 5′-region outside of the targeting vector (Zfp191KO-F1, 5′- specifically bind to the TCAT repeats (HUMTH01) in the first CGCACCACCTACTGGGAT-3′) and a reverse primer within the intron of the human tyrosine hydroxylase (TH) gene, which neo cassette (Neo-R1, 5′-CGGGTGTTGGGTCGTTTG-3′), produ- encodes the rate-limiting enzyme in the synthesis of cing a 2463 bp product, as indicated in Fig. 1A. Amplification catecholamines [31,32]. Allelic variations of HUMTH01 have conditions were: denaturation at 94°C for 45 s, annealing at a quantitative silencing effect on TH gene expression in vitro, 56°C for 1 min and extension at 72°C for 2 min and 30 s for 35 and correlate with quantitative and qualitative changes in cycles, followed by a final 5 min incubation at 72°C for chain the binding by ZNF191 [31]. Alteration in TH activity is elongation. LA Taq polymerase and buffers were obtained involved in the pathogenesis of certain disorders derived from TaKaRa. To verify the correct targeting, 10 μg aliquots of from catecholaminergic dysfunction [33–37]. Knockout mice genomic DNA from PCR-positive clones were digested either 3992 EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998

Fig. 1 – Disruption of the mouse Zfp191 gene by homologous recombination. (A) Schematically presented structures of the wild-type Zfp191 allele, targeting vector, and the disrupted Zfp191 allele. Exons (black boxes) and selected restriction sites (B, BamHI and X, XhoI) the selection of homologous recombination events and to disrupt the most coding region of the Zfp191 gene. The 15.7-kb SfiI fragment electroporated into embryonic stem cells is indicated under the targeting vector. Southern analysis probes (black bars) a and b are 0.4-kb XhoI–BamHI and 1.2-kb XhoI–BamHI genomic fragments, respectively, located upstream of the 5′ homology arm and downstream of the 3′ homology arm of the targeting vector. Wild-type and disrupted genomic XhoI fragments (2.7 and 3.9 kb, respectively), and BamHI fragments (13.2 and 10.6 kb, respectively) recognized by probes a and b are indicated. PCR primers for identification of the embryonic stem cell clones with targeted Zfp191 allele were Zfp191KO-F1 (solid forward arrowhead) and Neo-R1 (solid reverse arrowhead). The embryos and the mice were genotyped by allele discriminating PCR with primers Zfp191KO-F2 (open forward arrowhead), Neo-R1, and Zfp191KO-R1 (open reverse arrowhead). (B) Southern blot analysis of embryonic stem cell clones. Genomic DNA was digested with XhoIorBamHI and hybridized to probes a or b described above, respectively. Expected banding pattern was observed on a neomycin/gancyclovir selected and PCR positive clone. (C) Allele discriminating PCR used for genotyping of the embryos and post-partum mice. Primers − − used are described above. From the left are shown products from the PCRs of Zfp191+/+ (1166 base pairs), Zfp191 / − (556 base pairs), and Zfp191+/ (1166 and 556 base pairs) blastocysts. The molecular weight marker as bp is indicated on the right. with XhoI or with BamHI and subjected to Southern blot GGTAATCCCTGAGCATCTTCTT-3′)(Fig. 1A). This allele discri- analysis using the 420 bp XhoI–BamHI (Fig. 1A, probe a) or minating PCR scheme allowed convenient detection of both 1221 bp XhoI–BamHI (Fig. 1A, probe b) fragments of the mouse wild-type (1166 bp product) and disrupted Zfp191 alleles (552 bp Zfp191 gene, respectively, as probes. DNA labeling was product) in one PCR reaction. performed by random primer extension using [a-32P]dCTP. Characterization of embryonic lethality Generation of chimeric mice and germline transmission of the − disrupted allele Zfp191+/ mice were mated and the offspring was analyzed by allele discriminating PCR, and by Southern blotting, as − − ES cells carrying the correctly disrupted Zfp191 gene were described above. Since no Zfp191 / offspring was detected, injected into C57BL/6J blastocysts and then were transferred the embryos at selected developmental stages were analyzed. into pseudopregnant C57BL/6J×CBA/J foster mothers to produce To analyze blastocysts, preimplantation embryos were flushed chimeric animals. The resulting chimeric males, identified by from the uterus at the forth day of the pregnancy and the presence of agouti coat pigmentation, were bred to C57BL/6J transferred individually to 10 μl of 1× PCR buffer containing females to establish germline transmission of the disrupted 50 μg/ml proteinase K, lysed for 2 h at 55°C, and subjected to allele. The offspring carrying disrupted Zfp191 alleles was allele discriminating PCR as described above. To analyze identified by PCR as described for ES cells. Further verification gastrulae, embryos were dissected at 7.5 and 8.5 days post- of correct targeting was accomplished by PCR amplification with coitum from pregnant mothers in 1× phosphate-buffered forward primer Zfp191KO-F2 (5′-CTTTCGCTTATGTACT- saline, photographed, and analyzed by allele discriminating TGTTGG-3′) and reverse primers Neo-R1 and Zfp191KO-R1 (5′- PCR as described above. EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998 3993

Histological analysis of embryos (forward, 5′-GAAGATGCTCAGGGATTACCG-3′;reverse,5′- ATTCAAGGCATTTGTAGGGTT-3′), which yielded 348 and Deciduae were dissected free of uterine tissues, fixed over- 460 bp products, respectively. To serve as an internal control, night in freshly prepared 1% paraformaldehyde–phosphate- a 570 bp fragment of the β-actin cDNA was also amplified with buffered saline (PBS), washed in PBS, dehydrated with primers (forward, 5′-CCATCTACGAGGGCTATGCT-3′; reverse, increasing concentrations of ethanol, cleared in xylene, and 5′-GCTGGAAGGTGGACAGTGAG-3′). For cDNAs from HEK293 embedded in paraffin. The paraffin-embedded blocks were cells, a 481 bp fragment was amplified using ZNF191 specific then sectioned serially in a sagittal plane at a thickness of primers (forward: 5′-ATCAGCGGTGGCCACATCAA-3′ and 7 μm. The first series of the equally spaced sections was reverse: 5′-GATGGGCCCAACCCAATATAT-3′). To serve as an stained with Harris hematoxylin and eosin. internal control, a 381 bp fragment of the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA was co-amplified In vitro culture of preimplantation embryo with primers (forward, 5′-AACTTTGGTATCGTGGAAGGA-3′; reverse, 5′-GGAGGAGTGGGTGTCGCTGT-3′). The PCRs were Blastocysts were isolated at embryonic day 3.5 (E3.5) from carried out for 30–45 cycles at 94°C for 1 min, 55°C for 1 min, and − intercrosses between Zfp191+/ heterozygotes and then cul- 72°C for 1 min with a final extension of 5 min at 72°C. All tured individually in gelatin-coated microwells in Dulbecco's RT-PCR experiments were repeated at least twice and the modified Eagle medium (DMEM) supplemented with 15% fetal products were electrophoresed on 1.5% agarose gels. The gel calf serum (FCS). After 7 days in culture, outgrowths were images were obtained by exposure of the gels to an ultraviolet photographed and harvested for genotyping by PCR. (UV) transilluminator and recorded with Fluor-S Multimager (Bio-Rad). Signals were quantified by density analysis of the Construction of hairpin siRNA expression vector digital images using Quantity One image software (Bio-Rad). For semi-quantitative analysis, the density ratios of TH/ The ZNF191 mRNA (GenBank accession number NM_006965) β-actin, Zfp191/β-actin, and ZNF191/GAPDH were calculated was searched for target RNA interference sites using the online and used as an indication for the relative expression. The software (http://www.ambion.com/techlib/misc/siRNA_finder. average value for the mock treatment was taken as 100. html). Three sequences of the type AA (N19) from the ZNF191 mRNA 624–642, 1163–1181, and 1190–1208bp were selected to construct hairpin siRNA expression vector. The sequences Results and discussion chosen were checked for significant homology to other genes in the human genome database and none was found. To design Disruption of the Zfp191 gene results in early embryonic the hairpin siRNA insert, we specified a 19-nt sequence derived lethality from the transcript, separated by a 9-nt spacer from the reverse complement of the same 19-nt sequence. The hairpin siRNA One genomic clone, ∼18 kb, comprising the complete Zfp191 sequences were synthesized as two complementary DNA gene (GenBank accession number AY052495) was isolated oligonucleotides, annealed and ligated into ApaI/SalIsitesof from a lambda 129/SvJ genomic library using a human ZNF191 pSilencer1.0-U6 (Ambion) and sequenced. Name designation of cDNA probe. The Zfp191 gene is composed of four exons, and the resulting plasmids was pSZNF191-1, -2, or -3 based on the the entire open reading frame (ORF) for Zfp191 resides in exons target protein and region of mRNA downstream from the AUG 2–4 in the mouse genome [43]. A replacement-type vector codon (1 being the closest to the AUG codon). The resulting containing 10.5 kb of isogenic DNA, which allowed the transcript was predicted to form a 19-bp stem–loop structure deletion of exon 2–4, was constructed and used to generate − (Fig. 5A). heterozygous Zfp191 knockout mice (Zfp191+/ )(Fig. 1A). The targeting construct was electroporated into 129/SvJ embryonic Cell culture and transfection stem cells. 510 stable G418/gancyclovir clones were first analyzed by PCR with primers Zfp191KO-F1 and Neo-R1 (Fig. HEK293 cell was grown in DMDM supplemented with 10% of 1A). To verify the correct targeting of the Zfp191 gene, the PCR- heat-inactivated fetal calf serum. The cells were transfected positive clones were further analyzed by Southern blotting of with recombinant plasmids or empty vector (4 μg/well) in genomic DNA using probes a or b (Fig. 1A). Five clones 6-well plates using Lipofectamine 2000 (Invitrogen), according displayed the expected DNA restriction fragments. A Southern to the manufacturer's protocol. blot of XhoIorBamHI-digested embryonic stem cell DNA probed with probes a or b, respectively, is presented in Fig. 1B. RT–PCR analysis Cells from three correctly targeted clones (ES376, ES452 and ES493) were injected into C57BL/6J blastocysts. Injected Total RNAs were isolated from E6.5 embryos or cultured blastocysts were placed into the uteri of pseudopregnant HEK293 cells by use of Trizol reagent (Invitrogen) following C57BL/6J×CBA/J females resulting in a total of 10 mice with the instructions of the manufacturer. cDNAs were prepared chimeric coat color pattern. Upon mating to C57BL/6J mice, from 1 μg of total RNA using AMV reverse transcriptase germline transmission was achieved with two chimeric (Promega) and random primers. For cDNAs from embryos, (∼70%, ∼90%) male mice derived from two different mutant semi-quantitative PCRs were performed with TH specific ES clones (ES452 and ES493). No phenotypic differences primers (forward, 5′-GTTTGACCCTGACCTGGACC-3′; reverse, between animals derived from the two independent clones 5′-TCACGGGCAGACAGTAGACC-3′), Zfp191 specific primers were observed. The resulting Zfp191 heterozygous mutant 3994 EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998 mice were indistinguishable from their wild-type littermates in viability, growth, development, and fertility. Both male − and female Zfp191+/ mice were fertile and produced viable offspring with C57BL/6J at approximately 1:1 ratio of females to males. Eight newborn mice, five heterozygous and three wild-type, were analyzed for morphological and histological changes without any significant effects of heterozygosity for Zfp191 being found (data not shown). However, the matings − − of Zfp191+/ males with Zfp191+/ females produced no − − Zfp191 / offspring. Of the 103 born mice, 31 were Zfp191+/+ − − − (30.1%) and 72 were Zfp191+/ (69.9%), but no Zfp191 / mice − − were found (Table 1). Hence the Zfp191 / genotype was assumed to be embryonic lethal. This was confirmed by the nonsignificant difference (p=0.530) between the observed − Zfp191+/+:Zfp191+/ ratio and the expected 1:2 ratio in the Chi-square test. As an initial step to investigate the stage of prenatal death, blastocysts were flushed 3.5 days post-coitum (d.p.c.) − from the uteri of pregnant Zfp191+/ females (mated with − Zfp191+/ males). Resulting 53 viable blastocysts were − genotyped as Zfp191+/+ (28.3%), Zfp191+/ (52.8%), and − − Zfp191 / (18.9%) (Table 1). A typical PCR result is shown in Fig. 1C. Viable blastocysts showed a Mendelian distribution − − of wild-type and targeted genotypes by the Chi-square test Fig. 2 – (A) Growth retardation of Zfp191 / embryos at −/− (p=0.595). Zfp191 blastocysts were morphologically indis- 8.5 days of development. Embryos resulting from the +/− +/+ − tinguishable from Zfp191 or Zfp191 embryos. Therefore, matings of Zfp191+/ mice were removed from pregnant −/− at the blastocyst stage, Zfp191 embryos were viable and mothers at various time points to determine the temporal − − identified at the expected Mendelian frequency. Embryos window of prenatal death of Zfp191 / embryos. The figures − were dissected 7.5 and 8.5 days post-coitum from the uteri show a normally developed E8.5 Zfp191+/ embryo (+/−) and +/− +/− − − of pregnant Zfp191 females mated with Zfp191 males three retarded E8.5 Zfp191 / embryos (−/−) from the same and genotyped. Among the 38 E7.5 embryos, 31.6% were mother. Magnification is 25-fold. (B) Histological sections of +/+ +/− −/− − − Zfp191 , 52.6% were Zfp191 , and 15.8% were Zfp191 wild-type and Zfp191 / mutant embryos grown in utero. The −/− (Table 1). However, 4 out of 6 Zfp191 embryos at this embryos inside the uterus were fixed, sectioned, and stained stage were visibly retarded in size (Fig. 2B). Among the 28 with H&E. Left panels, wild-type mouse embryos at E7.5 −/− − − E8.5 embryos, all 5 Zfp191 embryos were severely retarded stage; right panels Zfp191 / embryos at E7.5 stage. The − − in growth. Three of them are shown in Fig. 2A along with a Zfp191 / embryo is mostly resorbed. − Zfp191+/ E8.5 embryo that had developed normally. The high proportion of abnormal embryos indicated that these mutants were homozygous for Zfp191 deletion, and they Zfp191 is required for the expansion of the ICM in vitro died at around 7.5 days post-coitum due to impaired cell proliferation. To address whether the abortive development of Zfp191- deficient embryos was due to a general cell growth defect or lineage specific defects, blastocysts isolated at E3.5 were individually cultured in vitro for 7 days on gelatin-coated Table 1 – Genotypes of offspring from intercrosses of plates (Fig. 3). During the early phases of these cultures, wild- +/− Zfp191 mice type or mutant blastocysts, attached to the substrate, hatched − − − Age Zfp191+/+ Zfp191+/ Zfp191 / Total P value from the zona pellucida, and started to expand their inner cell masses (ICMs, pluripotent cells that produce all embryonic E3.5 15 (28.3%) 28 (52.8%) 10 (18.9%) 53 0.595 components) and to form outgrowths having the classical E7.5 12 (31.6%) 20 (52.6%) 6 (15.8%) a 38 0.358 E8.5 9 (32.1%) 14 (50.0%) 5 (17.9%) b 28 0.565 appearance of migrating trophoblastic cells (outer cells that − 4 weeks 31 (30.1%) 72 (69.9%) 0 (0%) 103 2.98×10 8 produce placenta and extraembryonic tissues) (Fig. 3A). However, by day 3, the ICMs of the Zfp191 null blastocysts Embryos were isolated at the indicated time of gestation or at 4 stopped proliferating, and they degenerated soon after. After 5 weeks postnatal. Zfp191 genotype was determined by PCR. Data are presented as a number of embryos followed by percentage in days in vitro, the ICM-derived cells were not visible, and parentheses. χ2 analysis was used to determine the probability that Zfp191-deficient trophoblastic giant cells failed to proliferate the genotype frequency differed from the expected Mendelian ratio but persisted in 7 days cultures (Fig. 3B). These results indicate due simply to chance. that neither ICM nor trophoblasts of Zfp191 homozygous a −/− Four out of the 6 E7.5 Zfp191 embryos were retarded in growth. mutant embryos are capable of proliferating in vitro. The b −/− All of the E8.5 Zfp191 embryos were severely retarded in continued existence of trophectoderm cells in the absence of growth. Zfp191 indicates that Zfp191 is not essential for cell survival. EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998 3995

mouse embryos with RT-PCR. β-actin was used as a control, since Zfp191has not been implicated in the regulation of this gene. The expression level of the TH gene is indistinguishable between the wild-type, heterozygous, and homozygous mice (Fig. 4), suggesting that other genes may compensate at least in part for the loss of Zfp191 activity, ameliorating the effect of the Zfp191 knockout. HBP1, an HMG box transcription factor, which binds the TACT motif, was also cloned [31].Wealso analyzed the TH gene expression in human ZNF191 transgenic − − Fig. 3 – Defective growth of Zfp191 / blastocysts in vitro. mice [45] with RT-PCR. No significant difference in the TH gene Heterozygous (+/−) and homozygous mutant (−/−) blastocysts expression was observed between the wild-type and trans- were cultured on gelatin-coated dishes for 7 days. (A) The genic mice (data not shown), suggesting that Zfp191 may inner cell mass (ICM) grew normally and was surrounded by regulate the expression of novel target molecules. − trophoblast giant cells (TG) in Zfp191+/ blastocysts. (B) The The Krüppel-like zinc finger transcription factor Zfp191 is − − ICM was completely absent in the Zfp191 / blastocysts. highly expressed in multiple tissues throughout embryonic Magnification is 10-fold. development. This study demonstrates, for the first time, that Zfp191 is essential for normal mouse embryogenesis and cell proliferation. The data presented here show that complete The RNA interference-mediated knockdown of ZNF191 deletion of the Zfp191 gene has a severe impact on embryonic inhibits cell proliferation development at an earlier stage. Although embryos are able to implant, they appear to be strongly delayed in development To examine whether Zfp191 is also required for the prolifera- and die at approximately E7.5 (Fig. 3). Consistent with these − − − − tion of cultured cells, we first attempted to generate Zfp191 / findings, in vitro outgrowth studies showed that Zfp191 / − ES cells. A single clone was subjected to selection with an blastocysts compared to either Zfp191+/ or WT blastocysts are increased G418 concentration (2 mg/ml [44]), but among the 89 severely impaired in their ability to form trophoblasts and − − surviving ES subclones, not a single viable Zfp191 / line was proliferate (Fig. 4 and data not shown). The Zfp191 message is − − identified. Our failure to obtain Zfp191 / ES clones suggests present in pre-implantation embryo (data not shown). We that Zfp191 is also essential for the proliferation of ES cells. conclude that differentiation and cleavage can proceed in the To further explore the requirement of Zfp191 for the absence of embryonic Zfp191 transcription and lead to the − − proliferation of cultured cells, we examined the effect of the formation of normal Zfp191 / blastocysts. Embryonic death RNA-immediated knockdown of human ZNF191 in HEK293 was observed after the blastocyst stage but before organo- cells (Fig. 5). For that purpose, three hairpin siRNA expres- genesis, during a period of rapid cellular proliferation and sion vectors were constructed and transfected into the cells. differentiation. This implies a requirement for Zfp191 Cells were harvested at 48 h, total RNA was isolated, and the protein in a specific function, related to developmental expression of ZNF191 was analyzed by semi-quantitative stage, differentiation, or proliferation. Outgrowths from − − RT-PCR. Forty-eight hours post-transfection, the expression Zfp191 / blastocysts also failed to generate proliferating levels of ZNF191 were significantly suppressed by up to cell types of embryonic or extraembryonic lineages. The RNA 80%–90% in both pSZNF191-1 and pSZNF191-3 transfected HEK293 cells (Fig. 5B, lanes 2 and 4). These results indicated that pSZNF191-1 or pSZNF191-3 siRNA expression plasmids could effectively mediate the suppression of the expression of ZNF191. These inhibitory effects were shown to be specific because no non-specific down-regulation of gene expression was observed, as demonstrated by the GAPDH control (Fig. 5B). Three days after the transfection, the cell proliferation was significantly inhibited in pSZNF191-1 (Fig. 5D.b) or pSZNF191-3(data not shown) transfection as compared with empty vector (Fig. 5D.a) or pSZNF191-2 (data not shown). After 5 days, some of the ZNF191 siRNA-treated cells began to recover and divide. Resumption of growth Fig. 4 – Expression of a putative Zfp191 target gene. RNA was corresponded to an increase in ZNF191 levels observed by isolated from individual E6.5 embryos, cDNA synthesized, using RT-PCR (data not shown) after the transient knock- and the TH gene expression was analyzed by PCR. β-actin down of ZNF191. was used to standardize the PCR assay. Samples were taken after variable numbers of cycles and analyzed by Zfp191 and TH mRNA expression in mouse embryos electrophoresis on 1.5% agarose gels. cDNAs were analyzed in duplicate for three different embryos of each genotype Human ZNF191 specifically bind to the microsatellite HUMTH01 (wild-type, +/−,or−/−). The TH gene expression is not affected − − − and has a quantitative silencing effect on TH gene expression in Zfp191+/ or Zfp191 / embryos, while the Zfp191 gene in vitro [31]. TH gene has been suggested to be regulated by expression is reduced in +/− and undetectable in Zfp191. Therefore, we analyzed TH gene expression of in E6.5 −/− embryos. 3996 EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998

Fig. 5 – Effects of the RNAi-mediated knockdown of ZNF191 on the proliferation of HEK293 cells. (A) Sequence and expected structure of the hairpin siRNA against ZNF191 in vivo. (B) RT-PCR and gel electrophoresis of amplified products. The sizes of the amplified products are 481 bp (ZNF191) and 381 bp (GAPDH). Lane 1, the control cells (transfected with empty vector). Lane 2, lane 3, or lane 4, HEK293 cells transfected with pSZNF191-1, -2, or -3, respectively. Lane 5, DNA size marker. (C) Semi-quantitative analysis as described in Materials and methods. (D) Number and morphology of HEK293 cells 72 h after empty vector transfection (a) or after transfection with pSZNF191-1 (b). The RNAi-induced knockdown of ZNF191 results in a lower number of cells, indicating slower growth.

interference-mediated knockdown of ZNF191 inhibits HEK293 In summary, our study demonstrates that Zfp191 has a cell proliferation. The knockout embryos are severely retarded vital and unique role in early mouse development and cell in development. This might reflect a general function for proliferation. However, disruption of one allele of the Zfp191 Zfp191 in every cell type, consistent with its ubiquitous expres- gene did not lead to significant physiological and pathological − − sion pattern [30,31]. changes. TH gene expression is not affected in Zfp191 / and − − − An intriguing question is why Zfp191 / embryos survive Zfp191+/ embryos. How Zfp191 regulates genes important for until E7.5 while the knockdown of Zfp191 in cultured cells is early mouse development is the subject of future studies. associated with a block of proliferation within 3 days (Fig. 5D). One possible explanation is that the need for Zfp191 increases with the speed of cell cycle progression. This could explain − − Acknowledgments why Zfp191 / embryos die in a period associated with an enhanced proliferation rate and why a loss of Zfp191 results in We thank XuDong Zhao and Xia Sun for technical assistance. an immediate block of cell proliferation in the very rapidly This work was supported in part by grants from the National dividing HEK293 cells. Another possible explanation for the Natural Science Foundation of China (No.39830360 and relatively late effect of a loss of Zfp191 on embryonic 30400269). development is that the first days of embryonic development are maintained by maternally supplied Zfp191. In addition, it is possible that this pool of Zfp191 has a slower turnover than REFERENCES that in cultured cells. The early embryonic lethality of the mice described here, before organogenesis has been completed, precluded an [1] A.M. Ghaleb, M.O. Nandan, S. Chanchevalap, W.B. Dalton, I.M. exhaustive survey of these target genes. Conditional inactiva- Hisamuddin, V.W. Yang, Kruppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation, Cell Res. 15 tion of the Zfp191 gene will be required to address the role of (2005) 92–96. Zfp191 in fully developed tissues. In addition, such mice will [2] J.C. Venter, M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural, G.G. be a valuable tool to establish the role of Zfp191 in the Sutton, H.O. Smith, M. Yandell, C.A. Evans, R.A. Holt, J.D. maintenance of terminally differentiated cells. Gocayne, P. Amanatides, R.M. Ballew, D.H. Huson, J.R. EXPERIMENTAL CELL RESEARCH 312 (2006) 3990– 3998 3997

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