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Human Molecular Genetics Pages 847-854  

Zim1, a maternally expressed mouse Kruppel-type zinc-finger gene located in proximal chromosome 7
Introduction
Results
   Isolation of the Zim1 gene
   Zim1 is maternally expressed
   Zim1 expression during embryonic development
   Spatial expression pattern of Zim1 during development
Discussion
Materials And Methods
   Mouse BAC clone isolation and analysis
   Imprinting tests
   cDNA isolation and 5[prime]- and 3[prime]-RACE
   Sequencing and sequence analysis
   Northern blot analysis
   Whole mount in situ hybridization
Acknowledgements
References

Zim1, a maternally expressed mouse Kruppel-type zinc-finger gene located in proximal chromosome 7

Zim1, a maternally expressed mouse Kruppel-type zinc-finger gene located in proximal chromosome 7

Joomyeong Kim, Xiaochen Lu and Lisa Stubbs

Human Genome Center, Biology and Biotechnology Research Program, L-452, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94551, USA

Received December 14, 1998; Revised and Accepted February 2, 1999 DDBJ/EMBL/GenBank accession no. AF111101

In analysis of a conserved region of proximal mouse chromosome 7 and human chromosome 19q, we have isolated a novel mouse gene, Zim1 (imprinted zinc-finger gene 1), encoding a typical Kruppel-type (C2H2) zinc-finger protein, located within 30 kb of a known imprinted gene, Peg3 (paternally expressed gene 3). Our studies demonstrate that Zim1 is also imprinted; the gene is expressed mainly from the maternal allele and at high levels only during embryonic and neonatal stages. In contrast to most tissues, Zim1 is expressed biallelically in neonatal and adult brain with slightly more input from the maternal allele. Zim1 produces multiple transcripts that range in size from 7.5 to 15 kb. The 7.5 kb transcript is expressed at highest levels and appears to be embryo specific. Whole mount in situ hybridization analysis indicates that Zim1 is expressed at significant levels in the apical ectodermal ridge of the limb buds during embryogenesis, suggesting a potential role of Zim1 in limb formation. We have identified the potential human ortholog of Zim1 near PEG3 in a conserved, gene-rich region of human chromosome 19q13.4. The close juxtaposition of reciprocally imprinted genes has also been seen in other imprinted regions, such as human 11p15.5/Mmu7 (H19/Igf2) and suggests that the two genes may be co-regulated. These and other data suggest the presence of an unexplored, conserved imprinted domain in human chromosome 19q13.4 and proximal Mmu7.

INTRODUCTION

Genomic imprinting is an epigenetic process in mammals in which two parental alleles are marked during gametogenesis, resulting in parent-of-origin-dependent monoallelic expression or repression of certain genes during development. Approximately 30 different imprinted genes have been identified in human and mouse (1) and as many as 100 such genes are estimated to exist in the mammalian genome (2). Most of the known imprinted genes are clustered in chromosomal domains, indicating that genomic imprinting is a long-range phenomenon that affects relatively large chromosomal regions. Several studies have provided evidence to suggest that relatively small sequence elements, called imprinting centers (3,4), may regulate the imprinting of a whole domain and orchestrate coordinated expression of individual imprinted genes in each region (5,6).

Phenotypic studies of mice bred to carry partial uniparental disomies have permitted the identification of nine major imprinted domains located on seven different chromosomes (7). Similar observations drawn from surveys of human uniparental disomies (8) indicated that imprinting is conserved within mammals (9). Mouse chromosome 7 (Mmu7) contains at least three different imprinted domains, located in the proximal, central and distal portions of the chromosome, respectively. Two of the three known imprinted Mmu7 domains are syntenically homologous to human regions that are also associated with imprinted genetic disorders. The central Mmu7 domain is related to human chromosome 15q11-q13 (H15q11-q13), which contains genes associated with Prader-Willi and Angelman syndromes (3). The distal Mmu7 imprinted domain is related to H11p15.5 and contains genes related to those associated with Beckwith-Wiedemann syndrome (10). Although no clear human imprinted disorder has been mapped to chromosome 19q, maternal disomy (paternal deficiency) of the related proximal Mmu7 region is associated with late embryonic lethality in mice (11). Since animals disomic only for the proximal region have not been studied, the effects of paternal disomy of this region are uncertain. No imprinted genes had been identified within the proximal Mmu7 imprinted region until recently, when Peg3 (paternally expressed gene 3) was isolated and mapped (12). The human homolog of Peg3 is located in the telomeric portion of H19q13.4, a gene-rich segment that harbors several families of clustered Kruppel-type zinc-finger (ZNF) genes (13,14)

Since imprinted genes are often clustered, we predicted that additional imprinted loci would be found in the region of conserved homology of H19q13.4 and proximal Mmu7. Using the well-defined physical map of human chromosome 19 as a guide (13), we have localized several known and novel genes within the interval surrounding human and mouse PEG3/Peg3 (14; J. Kim, unpublished data). In this paper, we describe the isolation and characterization of one gene, Zim1 (imprinted zinc-finger gene 1), a novel ZNF gene located within 30 kb of Peg3. Zim1 is expressed mainly from the maternal allele during the embryonic and neonatal stages and is expressed at high levels only in embryonic tissues. These studies indicate that an uncharacterized imprinted domain surrounds Peg3 in proximal Mmu7 and suggests that conserved genes in H19q13.4 may also be imprinted.

RESULTS

Isolation of the Zim1 gene

Previous studies had positioned PEG3/Peg3 within human and mouse regions also known to contain large numbers of tightly clustered ZNF genes (14-16). To test whether genes adjacent to human PEG3 are ZNF genes we hybridized a conserved Kruppel-associated box (KRAB)-positive genomic fragment (25670Krab), isolated from the ZNF134 gene cluster [located 1 Mb distal to human PEG3 (14)], to restriction digests of cosmid 14378 containing human PEG3. Subsequently, we detected and isolated a positively hybridizing fragment in cosmid 14378 (herein referred to as 14378Krab). To search for potential Kruppel-type ZNF genes near Peg3 in mouse, a series of bacterial artificial chromosome (BAC) clones containing mouse Peg3 (Fig. 1) were hybridized with the human 14738Krab probe. The human KRAB sequence detected a 2.0 kb fragment in EcoRI-digested BAC DNA, indicating the presence of related ZNF gene sequences near mouse Peg3. We screened a BAC 588F20 subgenomic library with the human 14378Krab sequence and also with the ZNF consensus oligonucleotide probe to isolate these potential ZNF sequences. Sequence analyses of positively hybridizing fragments indicated the presence of a novel, uncharacterized Kruppel-type ZNF gene. To obtain a full-length cDNA for the predicted gene, several rounds of RT-PCR and 5[prime]- and 3[prime]-RACE were performed with 14.5 day embryo and adult brain cDNA templates, using primers derived from the sequence of positively hybridizing BAC fragments. These experiments yielded a 3.0 kb nucleotide sequence containing one ORF, 579 amino acids in length (Fig. 2A). The predicted ORF of this gene, called Zim1, is composed of the KRAB domain (A and B box) at the N-terminus, 11 ZNF units at the C-terminus and a spacer domain positioned between the KRAB and finger domains. This organization, KRAB, spacer and finger region, represents the typical structure of KRAB-containing, Kruppel-type ZNF genes (16,17). Eleven finger units of Zim1 are typical C2H2 type and some of these finger units are also connected by a conserved linker sequence (HTGEKPY).


Figure 1. Comparative physical map of the human and mouse Peg3/Zim1 regions. (A) The relative physical locations of PEG3 and previously identified ZNF genes are shown in the diagram of human chromosome 19qtel. (B) A physical map of a 200 kb region of proximal Mmu7 gives the relative locations and transcription direction of Zim1 and Peg3, as well as the restriction enzyme sites used in map construction, indicated by E (EagI), C (ClaI) and S (SalI). Mouse BAC coverage for this region is also shown.

   A
   B

Figure 2. (A) The amino acid sequence of Zim1. The 5[prime]-UTR, KRAB A and B, spacer and finger domains are shown and the two single-letter codes, C (cysteine) and H (histidine), in the finger domain are in bold. (B) The genomic organization of Zim1. The relative positions of four exons are shown on the map. E, position of the EagI restriction enzyme sites. The positions of primers used for the 5[prime]- and 3[prime]-RACE are indicated and the minor forms of the Zim1 cDNAs are also depicted.

To determine the exon-intron structure of Zim1, we employed a long-distance PCR strategy using oligonucleotides derived from the Zim1 cDNA sequence (Fig. 2B). The cDNA sequence of Zim1 is comprised of four different exons which are distributed over a genomic interval of ~13 kb. Most exon-intron boundary sequences of Zim1 are in good agreement with the consensus sequence (AG/GT) of the exon-intron joining region. 5[prime]-RACE experiments also yielded two other minor forms of Zim1 (Fig. 2B). One alternative 5[prime]-RACE clone obtained from adult brain contained the intron located between the KRAB A and B exons as a part of its transcript. Other 5[prime]-RACE clones contained a different alternative exon-intron boundary; the intron located immediately 5[prime] of the KRAB A sequence was included in the cDNA products. This region is composed largely of a simple dinucleotide repeat, (CACA)n, and does not contain an ORF. Although both unusual transcript forms may simply represent unusually stable splicing intermediates, imprinted genes such as Igf2, Igf2r and Ube3a are known to be transcribed in both the sense and antisense directions. The antisense products are believed to serve important functions despite containing no obvious ORF (18-20). Therefore, although the significance of these observations remains uncertain, the minor forms of Zim1 cDNA we have detected may also represent functional forms of RNA produced from this locus. Inspection of 3[prime]-RACE clones also indicated that Zim1 may utilize multiple alternative polyadenylation sites (Fig. 2).

Physical mapping experiments demonstrated that Zim1 is located within 30 kb of Peg3 and that Zim1 and Peg3 are transcribed in opposite directions (Fig. 1B). To find the potential human homolog of mouse Zim1, the KRAB and finger regions of Zim1 were hybridized to high density filter arrays of the human chromosome 19-specific library (13,21). The Zim1 finger probe detected a single strong positive cosmid, located next to PEG3, and the same cosmid was also strongly positive with the KRAB probe. These hybridization experiments indicate that the human homolog of mouse Zim1 or a related gene is a near neighbor of human PEG3 and also suggest conservation of the Peg3/Zim1 genomic interval in both human and mouse (Fig. 1).

Zim1 is maternally expressed

To test the imprinting status of Zim1, we analyzed mRNA isolated from tissues of Mus musculus (C3Hf)×M.spretus hybrid animals. Comparison of the DNA sequence of ZNF regions of Zim1 transcripts identified the presence of a single base substitution that distinguishes M.spretus from C3Hf mRNA. The result of this single base change is that cDNA produced from C3Hf transcripts contains a recognition site for the restriction enzyme MspI, whereas the Zim1 cDNA from M.spretus does not. Since ZNF sequences of Zim1 are encoded by a single exon, amplification of contaminating genomic DNA presented a potential complication to these studies. To decrease and monitor the presence of genomic DNA, all templates were treated with RNase-free DNase I before cDNA generation and each PCR reaction was conducted alongside a parallel reaction containing RNA that had not been treated with reverse transcriptase.

Analysis of Zim1 transcripts produced in seven different tissues isolated from M.spretus×C3Hf hybrid neonates indicated that Zim1 is expressed monoallelically in most tissues (Fig. 3A). Zim1 transcripts are derived mainly from the maternal allele in neonatal liver, lung, kidney, testis, heart and spleen. In contrast, Zim1 appears to be expressed from both maternal and paternal alleles in neonatal brain, with slightly more input appearing to be derived from the maternal chromosome. To confirm this result, a reciprocal analysis was conducted using interspecific backcross progeny (the offspring of an interspecific hybrid female and a C3Hf male; Fig. 3B). The results of this reciprocal analysis were consistent with those obtained with hybrid mice, except that brain expression appeared to be monoallelic in the backcross animals. Although this difference was consistent throughout several independent trials, it is uncertain whether the reason for this discrepancy might be due to differences in genome imprinting between different subspecies, as shown in another case, Kvlqt1 (22). It is also possible that this discrepancy might be caused simply by an unknown artifact of our imprinting test.


Figure 3. Monoallelic expression of Zim1. The schematic diagrams depict interspecific crosses used to determine parental alleles of imprinted loci. Rectangles represent parental alleles, with MspI restriction enzyme site polymorphism indicated by an internal line. (A) Imprinting test result using RNAs derived from F1 neonatal tissues of an interspecific cross, male M.spretus (SPT)×female M.musculus (C3H). (B) Result of reciprocal imprinting test using neonatal tissues derived from F2 offspring of the backcross, male M.musculus (C3H)×female F1. (C) Imprinting test result using day 9.5 and 14.5 embryos. Products of RT-PCR were digested with MspI and separated on 1.8% agarose gels to differentiate paternal (P) and maternal (M) transcripts.

Analysis of Zim1 transcripts produced by mouse embryos at different stages of gestation confirmed monoallelic expression. In fact, Zim1 imprinting appears to be more pronounced during embryonic stages, with no trace of paternal Zim1 transcripts detected in 9.5 and 14.5 d.p.c. embryos (Fig. 3C). Zim1 expression was also examined in tissues isolated from 1-month-old hybrid mice, but these studies were not conclusive since Zim1 transcripts were present at very low levels in tissues isolated from these older mice (data not shown).

Zim1 expression during embryonic development

To examine the tissue-specific expression of Zim1, we hybridized the Zim1 cDNA probe to a northern blot carrying mRNA isolated from 16.5 d.p.c. whole mouse embryos and a selection of adult tissues (Fig. 4A). High levels of Zim1 expression were detected in embryos; in agreement with RT-PCR results, very low levels of the transcript were detected in tissues of 2-month-old mice. The major Zim1 transcript detected in embryonic tissues is ~7.5 kb in length. Long exposure (1 week) of the same northern blot indicated that the 9 kb Zim1 transcript, but not the 7.5 kb species that predominates in embryos, is present at low levels in adult brain. Additional northern analyses indicated that other adult tissues, including heart and fat, also contain low levels of the 9 kb transcript (data not shown). To obtain more detailed information about the embryonic expression of Zim1, we hybridized two different Zim1 probes, derived from the KRAB and 3[prime]-UTR regions of the gene, to the embryo northern blot (Fig. 4B). Two different probes produced exactly the same result, as shown in Figure 4B; there are four different Zim1 transcripts detected in embryos ranging in size from 7.5 to 15 kb and two major transcripts, 7.5 and 9 kb in size, show the highest expression levels in 14.5 d.p.c. embryos. The collection of different Zim1 transcripts may be products of alternative splicing or, as suggested by their tissue-specific expression, may be produced through the use of alternative Zim1 promoters. Alternative use of different polyadenylation sites, suggested by RACE studies (see above), may also contribute to length differences in transcripts produced from the Zim1 gene.


Figure 4. Northern blot analyses of Zim1. Each lane contains 2 µg of poly(A)+ RNA. The 3[prime]-UTR of Zim1 was used as a probe. (A) Comparison of adult organ-specific expression with day 16.5 whole embryo expression. (B) Embryonic expression profile. RNAs were isolated from embryos at 10.5-16.5 d.p.c.


Spatial expression pattern of Zim1 during development

To study the spatial expression pattern of Zim1 during development, we performed whole mount in situ hybridization on embryos isolated at different embryonic stages (Fig. 5). Significant levels of Zim1 expression were detected in the apical ectodermal ridge (AER) of limb buds throughout the 9.5-14.5 d.p.c. period (Fig. 5A). As shown in Figure 5A, Zim1 expression is detected in the AER region of forelimb buds and also the future AER region of hindlimb buds, indicating that Zim1 expression in AER precedes limb bud formation. The dark signals observed in the otic vesicles of these embryos were also observed with sense probes, although the AER signals were not (data not shown). The coloration of the otic vesicles is therefore due to non-specific trapping of the probe, as confirmed by several control experiments. The Zim1 expression patterns in AER were compared with those of fibroblast growth factor 8 (Fgf8) (Fig. 5B and C), which has been shown to be expressed in the AER and is thought to be involved in limb formation (23). Zim1 expression in the AER appears to be much more restricted spatially than does that of Fgf8; Zim1 is expressed at the tip of the AER whereas Fgf8 is expressed in the whole AER region. Zim1 expression is also consistently observed to be greater in the anterior region of the AER than in the posterior region while Fgf8 is expressed throughout the anterior-posterior axis of the AER. We also detected lower levels of Zim1 expression in other tissues, including somites and primordial gut, but the significance of these low levels of expression is unclear at present and needs to be studied more thoroughly before conclusions can be drawn.


Figure 5. Zim1 expression in embryos by whole mount in situ hybridization. (A) Day 9.5 embryo, lateral view. Zim1 expression is detected in the forelimb buds (FL) and hindlimb buds (HL). Note that the hindlimb buds have not fully formed in this stage embryo but Zim1 expression is still detected in the future hindlimb bud region.(B) Forelimb buds of day 9.5 embryos, lateral view. (C) Forelimb buds of day 9.5 embryos, frontal view. Comparison of Zim1 and Fgf8 expression patterns in the AER.


DISCUSSION

We have identified and characterized a novel imprinted gene, Zim1, located near Peg3 in the proximal Mmu7, and have confirmed the presence of a closely related human gene in the syntenically homologous region of H19q13.4. Like Peg3, Zim1 contains regions that are predicted to encode Kruppel-type (C2H2) ZNF domains. However, the two genes differ markedly in structure. In addition to the ZNF segment, Peg3 contains additional protein coding motifs, including proline-rich domains that are not commonly found in genes of the Kruppel-type (12). In contrast, the sequence and organization of Zim1 are that of a typical Kruppel-type gene, predicted to encode a protein with N-terminal KRAB domains linked through a spacer sequence to C-terminal finger regions. Most KRAB-containing Kruppel-type ZNF genes are expected to act as transcriptional repressors based on the evidence that the KRAB domain shows a repressing activity when joined to other DNA-binding modules (24,25) and also that the ZNF domains of C2H2-type ZNFs exhibit DNA-binding capability (26).

Zim1 is expressed at high levels only in embryonic tissues and is transcribed primarily from the maternal allele during embryonic and neonatal stages. The gene is expressed at significant levels in the AER of the developing limb buds. The AER is known to be a major signaling center for the developing limb; the AER controls the growth and differentiation of cells located underneath the AER to form proper limbs (27). The high expression levels in AER might suggest a potential role of Zim1 in AER and also a possible link to other genes that are involved in limb formation, such as Fgf8, Sonic hedgehog (Shh) and Wnt7a.

In contrast to the function of other known imprinted genes, Zim1 is unique in that the predicted function of this Kruppel-type ZNF gene is transcriptional control of other downstream genes. Developing embryos are expected to require multiple layers of transcriptional regulation to coordinate precise timing of expression for many genes and it is interesting to speculate that Zim1 might play a role in regulating genes which must be repressed during normal development. Many imprinted genes, including Igf2, Ins2, Igf2r, Grf1 and Gnas1, are known to be involved in cell growth and signal transduction. Therefore, the imprinting effects of these genes on growth and development is easily envisioned. However, the effects of silencing or overexpressing a transcription factor gene would be more complicated and indirect and manifested in the function of unknown downstream genes. Considering the hypothesis that parent-offspring conflict is one of the major reasons for imprinting in placental mammals (28,29), it is possible that Zim1 might be part of a genetic cascade involved in fetal and/or neonatal growth. Although the function of Zim1 remains to be proven, imprinting of a potential transcription factor adds a potentially interesting twist to our understanding of this complicated genetic phenomenon.

Animals carrying partial paternal disomy (uniparental disomy) including proximal Mmu7 exhibit reduced postnatal growth rates and viability (30). Since the breakpoint of the translocation used to produce these partial disomies is located below the central imprinted domain, these animals are disomic not only for the Zim1-Peg3 but also for the Snrpn-Ube3a region (31). The phenotype associated with partial disomy of proximal Mmu7 may therefore be associated with the absence of transcripts from more than one maternally expressed gene. The imprinting status of Ube3a, which represents the only maternally expressed gene discovered so far in the central Mmu7 imprinted region (4), is restricted spatially and temporally, with imprinted expression documented only in a specific region of the adult brain (32). Therefore, the extent to which Ube3a influences neonatal growth may be limited. With the discovery of Zim1, it is possible to imagine that the loss of regulatory control of a downstream gene(s) may in fact be a contributing factor to the retardation of postnatal growth in disomic mice.

The close proximity of Zim1 (maternally expressed) and Peg3 (paternally expressed) is reminiscent of gene organization observed in other imprinted domains, such as those containing Igf2 and H19 (Beckwith-Wiedemann syndrome region) and Snrpn and Ube3a (Prader-Willi and Angelman syndrome region). Although this close juxtaposition of pairs of reciprocally imprinted genes cannot be generalized as a common feature of imprinted domains, this trend is compatible with one recent hypothesis. The enhancer competition model (6) hypothesizes that two reciprocally imprinted genes compete in cis for a single, shared enhancer, with the result that only one gene can be expressed at a given time from one parental allele. In support of this model, Igf2 and H19 have been shown to rely upon the shared endoderm-specific enhancer. Although it remains to be studied in future whether Peg3 and Zim1 share unidentified enhancers and also whether the imprinting of Peg3/Zim1 can be explained by the enhancer competition model, the identification of a second imprinted gene near a known imprinted gene in proximal Mmu7 suggests that the imprinting of Peg3 and Zim1 might be related to each other and also that this region might have more imprinted genes.

MATERIALS AND METHODS

Mouse BAC clone isolation and analysis

To obtain large-insert clones containing mouse Peg3, we screened high density mouse 129/Sv BAC library filters (Research Genetics, Huntsville, AL) with a pool of three mouse genomic fragments corresponding to the transcribed regions of Peg3 (12,14). BAC DNA samples were prepared with the alkaline lysis protocol (33), digested with several rare-cutting enzymes, including ClaI, EagI, SalI and NotI, and separated on pulsed-field gels (run in a Chef Mapper instrument; Bio-Rad, Hercules, CA). DNA was transferred to nylon membranes (Hybond; Amersham, Uppsala, Sweden) and hybridized according to standard protocols (34). BAC end clones were isolated using a single primer PCR approach (35). Subgenomic libraries were generated from one Peg3-containing BAC clone (588F20) by ligating Sau3AI digests of BAC DNA into [lambda]ZAP-II (Stratagene, La Jolla, CA) and M13 phage vectors.

Imprinting tests

To test the expression of Zim1 from maternal and paternal alleles, we isolated tissues from hybrid offspring produced by crossing M.musculus (C3Hf) females with M.spretus male mice. To confirm monoallelic expression of Zim1, we analyzed tissues from offspring of M.musculus×M.spretus hybrid females, tested to carry M.spretus alleles of Zim1, which were backcrossed to C3Hf males. Embryos at two different stages (E9.5 and E14.5) and tissues from 7-day-old and 1-month-old animals were collected from each cross. RNA was isolated using a commercially available kit (rapid total RNA isolation kit; 5[prime]-3[prime], Boulder, CO). RNA samples were treated with RNase-free DNase I (Stratagene) for 30 min at 37°C and 50 µg were used for the synthesis of cDNA (using the cDNA synthesis module; Amersham). The final volume of each reverse transcription reaction was 40 µl, and 1 µl of this material was taken for each PCR reaction. For imprinting tests of Zim1, two oligonucleotides were used, Zim1 F5 (5[prime]-GAGAAGCCGTACTGCTGTCA-3[prime]) and Zim1 F2 (5[prime]-CTTGCACCGGTACCTGGAGT-3[prime]). PCR amplification of the Zim1 finger region was performed using the following program in a Perkin Elmer-Cetus (Foster City, CA) GeneAmp 9600 instrument: 95°C for 30 s, 60°C for 1 min, 72°C for 1 min for 30 cycles; 72°C for 5 min for 1 cycle. PCR reactions were carried out in a 50 µl reaction mixture containing 300 ng of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1 mM dNTPs, 1% Triton X-100 and 1.25 U Taq DNA polymerase. The quality of PCR reactions was checked on 1.8% agarose gels. cDNA templates were checked for genomic DNA contamination by performing parallel PCR reactions using templates that had not been treated with reverse transcriptase. Ten microliters of each PCR reaction mixture was digested with 10 U MspI in a 30 µl reaction and the digests examined after separation on 1.8% agarose gels.

cDNA isolation and 5[prime]- and 3[prime]-RACE

Hybridization of a human cosmid containing PEG3 sequences (cosmid 14378) with a probe corresponding to the conserved KRAB motif associated with many ZNF genes (36) indicated that a Kruppel-type ZNF gene was located near PEG3. The KRAB-containing fragment was subcloned from the cosmid and used to screen the subgenomic library generated with BAC 588F20. Fragments isolated from the BAC contained a conserved KRAB sequence representing portions of a novel mouse gene. ZNF encoding sequences were isolated from the same mouse BAC by screening the subgenomic library with a degenerate ZNF oligonucleotide probe [CA(C/T)AC(A/T)GG(A/T/G)GA- (A/G)AA(A/G)CC(T/C/A)TA (37)]; this probe corresponds to the conserved amino acid sequence motif HTGEKPY, which is unique to Kruppel-type ZNF genes. The nucleotide sequence linking the KRAB A and finger regions of Zim1 was obtained by analyzing RT-PCR products generated from a 14.5 day embryo cDNA template (mouse 14.5 day embryo Marathon cDNA template; Clontech, Palo Alto, CA) with two oligonucleotide primers, KRAB A (5[prime]-AGGAACCAGTGATCTTCAAA-3[prime]) and F2 (5[prime]-CTTGCACCGGTACCTGGAGT-3[prime]). To obtain the 5[prime]- and 3[prime]-ends of Zim1, we employed the RACE technique (38). Two different cDNA templates derived from 14.5 day embryo and adult brain RNA were used. The sequences of oligonucleotides used for the 5[prime]- and 3[prime]-RACE of Zim1 are as follows: F6 (5[prime]-GCTTTGGATTTGTGGAACTG-3[prime]) and F7 (5[prime]-TTGAGATGGCCTGTCATCAC-3[prime]) for 5[prime]-RACE; UTR1 (5[prime]-CCCTACTTGGAGCATTTGTG-3[prime]) and UTR2 (5[prime]-CTTGCCTTCCAATAACTAAG-3[prime]) for 3[prime]-RACE. Amplified RACE products were separated on 0.8% agarose gels and the major fragments in terms of mass were isolated from the gels using a gel extraction column (QIAquick gel extraction kit; Qiagen, Valencia, CA). The fragments were subcloned into the TA cloning vector (TA cloning kit; Invitrogen, Carlsbad, CA).

Sequencing and sequence analysis

Subcloned genomic and cDNA fragments were sequenced from both directions using a fluorescence-based cycle sequencing DNA sequencing kit (dye terminator sequencing core kit; PE Applied Biosystems, Warrington, UK) and reactions analyzed on an ABI 373 automated sequencer. Sequence alignments and database searches were analyzed using GCG software v.8 (Genetics Computer Group, Madison, WI).

Northern blot analysis

Poly(A)+ RNA was isolated from embryos and from adult tissues using oligo(dT) columns [rapid poly(A)+ mRNA isolation kit; 5[prime]-3[prime]]. Two micrograms of purified poly(A)+ RNAs were separated on a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane (Hybond; Amersham) and cross-linked to the blot by UV irradiation. Northern blots were hybridized with probes representing different Zim1 transcribed regions: (i) the KRAB A region or (ii) the 3[prime]-UTR region of the gene. Procedures and conditions for generating probes and for performing hybridizations were as previously described (34).

Whole mount in situ hybridization

To generate an antisense and a sense riboprobe for the Zim1 in situ hybridization, one EcoRI fragment of Zim1 corresponding to the finger region of Zim1 (Genbank accession no. AF111101, nt 1354-2495) was subcloned into predigested pBluescript and used as a template DNA for in vitro transcription reactions. The template DNA for an antisense probe was generated by PCR using two oligonucleotide primers, T3 (5[prime]-ATTAACCCTCACTAAAG-3[prime]) and F3 (5[prime]-GGTTTCACTGACAAAACTAC-3[prime]), and the template DNA for a sense probe with T7 (5[prime]-TAATACGACTCACTATAG-3[prime]) and F2 (5[prime]-CTTGCACCGGTACCTGGAGT-3[prime]). Amplified PCR products were treated once with phenol-chloroform, washed with TE on a microcon-100 (Amicon, Beverly, MA) and concentrated to 1 µg/µl concentration. One microgram of each template DNA was used for each in vitro transcription reaction with T7 and T3 RNA polymerases. To generate an antisense probe for mouse Fgf8, two different oligonucleotides, Fgf8a (5[prime]-GCTGGGCAGGGAGCCCACTT-3[prime]) and Fgf8b (5[prime]-CTTCTGCCATGGCGTTGATG-3[prime]), were used to amplify exons 2 and 3 of Fgf8 (GenBank accession no. Z46883; 39) and then this PCR product was used for the second round PCR to prepare the template DNA for in vitro transcription with two primers, Fgf8a and Fgf8c (5[prime]-TAATACGACTCACTATAGGGCTTCTGCCATGGCGTTGATG-3[prime]). For the detection of probes, antisense and sense probes were labeled with digoxigenin-UTP with an RNA labeling kit (DIG RNA labeling kit; Boehringer Mannheim, Indianapolis, IN) and anti-DIG-alkaline phosphatase from sheep and BM Purple AP substrate (Boehringer Mannheim) were used according to the manufacturer's instructions. Whole mount in situ hybridization experiments were performed according to a standard protocol (40) with minimum modification.

ACKNOWLEDGEMENTS

We would like to thank Dr Xin Wei and Jihwan Song for their technical advice and thorough discussions of whole mount in situ hybridization. We also thank Dr Elbert Branscomb for his encouragement and comments throughout the experiments and Xiojia Ren, Anne Bergmann, Loren Hauser and Don Carpenter for their technical support. This work was performed under the auspices of the US Department of Energy by the LLNL under contract no. W-7405-ENG-48.

REFERENCES

1. Morrison, I.M. and Reeve, A.E. (1998) A catalogue of imprinted genes and parent-of-origin effects in humans and animals. Hum. Mol. Genet., 7, 1599-1609. MEDLINE Abstract

2. Barlow, D.P. (1997) Competition-a common motif for the imprinting mechanism? EMBO J., 16, 6899-6905. MEDLINE Abstract

3. Nicholls, R.D. (1994) New insights reveal complex mechanisms involved in genomic imprinting. Am. J. Hum. Genet., 54, 733-740. MEDLINE Abstract

4. Nicholls, R.D., Saitoh, S. and Horsthemke, B. (1998) Imprinting in Prader-Willi and Angelman syndromes. Trends Genet., 14, 194-200. MEDLINE Abstract

5. Dittrich. B., Buiting, K., Korn, B., Rickard, S., Buxton, J., Saitoh, S., Nicholls, R.D., Poustka, A., Winterpacht, A., Zabel, B. and Horsthemke, B. (1996) Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene. Nature Genet., 14, 163-170. MEDLINE Abstract

6. Tilghman, S.M., Caspary, T. and Ingram, R.S. (1998) Competitive edge at the imprinted Prader-Willi/Angelman region? Nature Genet., 18, 206-208. MEDLINE Abstract

7. Beechey, C.V. and Cattanach, B.M. (1996) Genetic imprinting map. Mouse Genet., 94, 96-99.

8. Ledbetter, D.H. and Engel, E. (1995) Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum. Mol. Genet., 4, 1757-1764. MEDLINE Abstract

9. Barlow, D.P. (1995) Gametic imprinting in mammals. Science, 270, 1610-1613. MEDLINE Abstract

10. Reik, W. and Maher, E.R. (1997) Imprinting in clusters: lessons from Beckwith-Wiedemann syndrome. Trends Genet., 13, 330-334. MEDLINE Abstract

11. Searle, A.G. and Beechey, C.V. (1990) Genome imprinting phenomena on mouse chromosome 7. Genet. Res., 56, 237-244. MEDLINE Abstract

12. Kuroiwa, Y., Kaneko-Ishino, T., Kagitani, F., Kohda, T., Li, L.-L., Tada, M., Suzuki, R., Yokoyama, M., Shiroishi, T., Wakana, S., Barton, S.C., Ishino, F. and Surani, M.A. (1996) Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein. Nature Genet., 12, 186-190. MEDLINE Abstract

13. Ashworth, L.K., Batzer, M.A., Brandriff, B., Branscomb, E., deJong, P., Garcia, E., Garnes, J., Gordon, L., Lamerdin, J.E., Lennon, G., Mohrenweiser, H., Olsen, A., Slezak, T. and Carrano, A.V. (1995) A metric physical map of human chromosome 19. Nature Genet., 11, 422-427. MEDLINE Abstract

14. Kim, J., Ashworth, L., Branscomb, E. and Stubbs, L. (1997) The human homolog of a mouse-imprinted gene, Peg3, maps to a zinc finger gene-rich region of human chromosome 19q13.4. Genome Res., 7, 532-540. MEDLINE Abstract

15. Stubbs, L.J., Carver, E.A., Shannon, M.E., Kim, J., Geisler, J., Generoso, E.E., Stanford, B.G., Dunn, W.C., Mohrenweiser, H., Zimmermann, W., Watt, S.M. and Ashworth, L.K. (1996) Detailed comparative map of human chromosome 19q and related regions of the mouse genome. Genomics, 35, 499-508. MEDLINE Abstract

16. Shannon, M., Kim, J., Ashworth, L., Branscomb, E. and Stubbs, L. (1998) Tandem zinc-finger gene families in mammals: insights and unanswered questions. DNA Seq., 8, 303-315.

17. Bellefroid, E.J., Marine, J.-C., Ried, T., Lecocq, P.J., Riviere, M., Amemiya, C., Poncelet, D.A., Coulie, P.J., de Jong, P., Szpirer, C., Ward, D.C. and Martial, J.A. (1993) Clustered organization of homologous KRAB zinc-finger genes with enhanced expression in human T lymphoid cells. EMBO J., 12, 329-335.

18. Moore, T., Constancia, M., Zubair, M., Bailleul, B., Feil, R., Sasaki, H. and Reik, W. (1997) Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2. Proc. Natl Acad. Sci. USA, 94, 12509-12514. MEDLINE Abstract

19. Wutz, A., Smrzka, O., Schweifer, N., Schellander, K., Wagner, E.F. and Barlow, D.P. (1997) Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature, 389, 745-749. MEDLINE Abstract

20. Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. and Lalande, M. (1998) An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nature Genet., 19, 15-16. MEDLINE Abstract

21. Olsen, A., Conbs, J., Garcia, E., Elliot, J., Amemiya, C., deJong, P. and Treadgill, G. (1993) Automated production of high density cosmid and YAC colony filters using a robotic workstation. BioTechniques, 14, 116-123. MEDLINE Abstract

22. Jiang, S., Hemann, M.A., Lee, M.P. and Feinberg, A.P. (1998) Strain-dependent developmental relaxation of imprinting of an endogenous mouse gene, kvlqt1. Genomics, 53, 395-399. MEDLINE Abstract

23. Crossley, P.H., Monowada, G., MacArthur, C.A. and Martin, G.R. (1996) Roles for FGF8 in the induction, initiation and maintenance of chick development of the tetrapod limb. Cell, 84, 127-136. MEDLINE Abstract

24. Margolin, J.F., Friedman, J.R., Meyer, W.K.H., Vissing, H., Thiesen, H.-J. and Rauscher, F.J. (1994) Kruppel associated boxes are potent transcriptional repression domains. Proc. Natl Acad. Sci. USA, 91, 4509-4513. MEDLINE Abstract

25. Witzgall, R., O'Leary, E., Leaf, A., Onaldi, K. and Bonventre, J.V. (1994) The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl Acad. Sci. USA, 91, 4514-4518. MEDLINE Abstract

26. Choo, Y. and Klug, A. (1994) Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc. Natl Acad. Sci. USA, 91, 11168-11172. MEDLINE Abstract

27. Gilbert, S.F. (1997) Developmental Biology, 5th Edn. Sinauer Associates, Sunderland, MA.

28. Moore, T. and Haig, D. (1991) Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet., 7, 45-49. MEDLINE Abstract

29. Haig, D. (1993) Genetic conflicts in human pregnancy. Q. Rev. Biol., 68, 495-532. MEDLINE Abstract

30. Cattanach, B.M., Barr, J.A., Evans, E.P., Burtenshaw, M., Beechey, C.V., Leff, S.E., Brannan, C.I., Copeland, N.G., Jenkins, N.A. and Jones, J. (1992) A candidate mouse model for Prader-Willi syndrome which shows an absence of Snrpn expression. Nature Genet., 2, 270-274. MEDLINE Abstract

31. Cattanach, B.M., Barr, J.A., Beechey, C.V., Martin, J., Noebels, J. and Jones, J. (1997) A candidate model for Angelman syndrome in the mouse. Mamm. Genome, 8, 472-478. MEDLINE Abstract

32. Albrecht, U., Sutcliffe, J.S., Cattanach, B.M., Beechy, C.V., Armstrong, D., Eichele, G. and Beaudet, A.L. (1997) Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nature Genet., 17, 75-78. MEDLINE Abstract

33. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

34. Stubbs, L., Huxley, C., Hogan, B., Evans, T., Fried, M., Duboule, D. and Lehrach, H. (1990) The hox-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2. Genomics, 6, 645-650. MEDLINE Abstract

35. Kim, J., Carver, E.A. and Stubbs, L. (1997) Amplification and sequencing of end fragments from bacterial artificial chromosome clones by single-primer polymerase chain reaction. Anal. Biochem., 253, 272-275. MEDLINE Abstract

36. Bellefroid, E.J., Poncelet, D.A., Lecocq, P.J., Revelent, O. and Martial, J.A. (1991) The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc. Natl Acad. Sci. USA, 88, 3608-3612. MEDLINE Abstract

37. Hoovers, J.M., Mannens, M., John, R., Bliek, J., Van Heyningen, V., Porteus, D.J., Leschot, N.J., Weterveld, A. and Little, P.F.R. (1992) High-resolution localization of 69 potential human zinc finger protein genes: a number are clustered. Genomics, 12, 254-263. MEDLINE Abstract

38. Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl Acad. Sci. USA, 85, 8998-9002. MEDLINE Abstract

39. Crossley, P.H. and Martin, G.R. (1995) The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development, 121, 439-451. MEDLINE Abstract

40. Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

*To whom correspondence should be addressed. Tel: +1 925 423 2274; Fax: +1 925 422 2282; Email: kim16@llnl.gov

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