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Human Molecular Genetics Pages 795-803  

Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region
Introduction
Results
   Characterization of the mouse Zfp127 gene
   Comparison of the mouse Zfp127 and human ZNF127 genes
   Tissue and germline expression of the mouse Zfp127 gene
   Functional imprinting of the mouse Zfp127 gene
Discussion
Materials And Methods
   Mice
   Clones and DNA sequence analyses
   RNA techniques
Abbreviations
Acknowledgements
References

Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region

Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region

Michelle T. C. Jong1,2, Alisoun H. Carey3,+, Kim A. Caldwell4,§, Michel H. Lau3, Mary Ann Handel4, Daniel J. Driscoll2, Colin L. Stewart3,¶, Eugene M. Rinchik5 and Robert D. Nicholls1,*

1Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics, University Hospitals of Cleveland, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA, 2R. C. Philips Unit, Division of Pediatric Genetics and Center for Mammalian Genetics, University of Florida College of Medicine, Gainesville, FL 32610, USA, 3Roche Institute of Molecular Biology, Nutley, NJ 07110, USA, 4Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA and 5Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

Received January 25, 1999; Accepted January 26, 1999

DDBJ/EMBL/GenBank accession no. U19106

A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C3HC4) and three C3H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculus×M.spretus F1 interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS.

INTRODUCTION

Genomic imprinting results in the expression of certain genes from only one of the two parental chromosomes as a result of differential modification of the paternal and maternal alleles during gametogenesis (1). In the mouse, nearly 30 genes from 12 chromosomal locations show maternal or paternal allele-specific expression (2). Most of these, and several additional chromosome regions, show imprinted phenotypes dependent upon the parental origin of uniparental disomy (UPD) for the region (3). However, only certain types of prominent phenotypes are readily observable in mouse with the techniques used and some imprinted phenotypes may go undetected. Imprinted genes in several of these chromosomal regions are clustered, which may reflect coordinate regulatory mechanisms operating on such genes (4,5). In distal mouse chromosome 7, the H19, Mash2, p57KIP2, Kvlqt1 and Cd81 (the latter only prior to embryonic day 8.5) genes are expressed from the maternal chromosome only (1,6), while the Igf2, Igf2as and Ins2 genes are paternally expressed only (1,7). Another imprinted domain, in chromosome 17, contains the maternally expressed Igf2r gene and the paternally expressed Igf2rAS transcript and Mas proto-oncogene (1,8,9). This clustering of imprinted genes complicates the definition of which genes are responsible for specific imprinted phenotypes (4,5).

One of the most intriguing imprinted domains is represented by the human chromosome 15q11-q13 and homologous mouse chromosome 7C regions, in which abnormalities of imprinted gene inheritance are associated with two different neurobehavioral disorders. The presence of a 15q11-q13 deletion, UPD or an imprinting defect leads to loss of paternal- or maternal-only gene expression and Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively (5,10). Similarly, each of these genotypes for the homologous mouse chromosome region is associated with differential imprinted phenotypes (11-13; J.M. Gabriel, M. Merchant, T. Ohta, Y. Ji, R.G. Caldwell, M.J. Ramsey, J.D. Tucker, R. Longnecker and R.D. Nicholls, submitted for publication). This region contains a cluster of imprinted genes, with paternal expression of Snrpn, Ipw and Ndn and maternal expression of Ube3a in brain (5,11,14-18). PWS is thought to be a contiguous gene syndrome, arising from the loss of expression of multiple paternally expressed genes, while intragenic mutations in UBE3A lead to AS (reviewed in refs 5,19). The contribution of each identified paternally expressed gene to PWS is unknown and, since the imprinted domain appears to span an ~2 Mb region, it is likely that multiple additional imprinted genes exist in the homologous human 15q11-q13 and mouse 7C regions.

Previously, we identified a novel imprinted human gene, ZNF127, and an associated antisense gene, ZNF127AS (20). We report here that mouse Zfp127 and Zfp127as are evolutionarily conserved and that Zfp127 is imprinted in various mouse tissues, where it is expressed exclusively from the paternal chromosome. Zfp127 is expressed in pre- and post-meiotic spermatogenic cells, and we propose that transcriptional activity of this gene may play a role in the imprinting process. The novel imprinted Zfp127 and antisense genes may play a role in the etiology of the PWS mouse model phenotype.

RESULTS

Characterization of the mouse Zfp127 gene

A mouse partial cDNA clone, p34-1-111, was previously isolated from a mouse brain library using the human DN34 cDNA as a probe (21). This mouse cDNA is now identified as encoding the 3[prime]-end of the Zfp127 gene (Fig. 1). Three [lambda] clones spanning 28 kb of the Zfp127 genomic locus were isolated from a 129/Sv mouse genomic library using this cDNA probe (A.H. Carey, M.T.C. Jong, R.D. Nicholls and C.L. Stewart, unpublished data). Subcloning and DNA sequence analysis revealed that the mouse Zfp127 gene spans 2.6 kb and is intronless (Fig. 1b, d and e), with a typical mammalian CpG island at the 5[prime]-end (Fig. 1b). The transcription start site for Zfp127 was mapped to a single A nucleotide by 5[prime]-rapid amplification of cDNA ends (RACE) (Fig. 1a and e, and data not shown). There are three potential polyadenylation signals, with the third of these used in the isolated 34-1-111 cDNA (Fig. 1d and e). Two mouse Zfp127 3[prime] ESTs occur in the public EST database (AA387483 and AA119257).

   (a) - (d)
   (e)

Figure 1. Genomic structure of the mouse Zfp127 locus. (a) Schematic of the Zfp127 transcript (hatched bar, coding sequence). (b) Restriction map of the genomic Zfp127 locus, illustrating the 5[prime] CpG island (black box) and colinearity of the genomic region with the mRNA. Arrows, direct repeats; Bs, BssHII; C, ClaI; E, EcoRI; Eg, EagI; Hh, HhaI; M, MspI/HpaII; N, NotI; Na, NaeI; Sm, SmaI; R, RsrII; T, TaqI. (c) PCR primers (arrows) referred to in the text. (d) Subclones used for analysis of the Zfp127 locus. pEN40, pEN13 and pE26 are genomic subclones, p34-1-111 is a cDNA (21) and p5[prime]-Zfp is a 5[prime]-RACE clone. (e) Nucleotide sequence of mouse Zfp127 and deduced amino acid sequence (GenBank accession no. U19106). Nucleotide features include, in respective order, the 5[prime] transcription start site (bold, nt 601), initiation codon (underlined), conserved 3[prime]-UTR motif (bold), three polyadenylation signals (bold underlined) and site of polyadenylation (vertical arrow), including an 8 nt flanking sequence conserved in the human. Coding features include three C3H (bold) and one RING (bold italic) zinc-finger motifs and two classes of direct repeats (arrows underneath the corresponding amino acid and nucleotide sequences).

The mouse Zfp127 gene encodes a putative polypeptide of 544 amino acids, with five putative zinc-finger motifs (Fig. 1e). This includes a centrally located C3HC4 (C, Cys; H, His), or RING, zinc-finger motif (20,22), as well as three widely spaced C3H motifs and a unique pattern of conserved Cys-His residues between the second C3H motif and the RING zinc-finger motif (see also ref. 20). Unlike human ZNF127, the mouse Zfp127 gene contains two locally repetitive sequences, a 54 bp partially diverged direct repeat and 10 tandem copies of a hexanucleotide repeat (Fig. 1b and e). Since each repeat is in-frame and a multiple of three, these repeat sequences do not abolish the coding capacity of the gene.

Comparison of the mouse Zfp127 and human ZNF127 genes

The mouse and human ZNF127 orthologous polypeptide sequences share similar structural motifs, including all five putative zinc-finger motifs, and have an overall identity of 69% (82% similarity, considering conservative substitutions) at the amino acid level (Fig. 2a). The RING zinc-finger motif has the highest degree of conservation (85% identical, 92% similar), with the N- and C-terminal regions of the putative polypeptides less well conserved. An acidic region of five Glu residues between the RING finger and the third C3H motif and a putative basic nuclear localization sequence within the RING zinc-finger motif are conserved (Fig. 2a). However, the subcellular localization is not known at this time. The tandem repeat of the mouse 129/Sv Zfp127 gene encodes a region of 20 amino acids with alternating Met/Val/Leu and Pro, not present in the human, although the function of this sequence is unknown.


Figure 2. Comparison of (a) polypeptide and (b) 3[prime]-UTR nucleotide sequences of the mouse Zfp127 and human ZNF127 genes. (a) Identical amino acid residues are boxed and similar residues are shaded. Sequences encoded by direct and tandem repeats are indicated by arrows. H, human; M, mouse. (b) Identical nucleotides are shaded, while motifs identical or similar to the `unstable mRNA' AUUUA element are boxed or underlined, respectively.

As expected, the mouse Zfp127 and human ZNF127 5[prime]-untranslated region (UTR) and 3[prime]-UTR sequences are generally less conserved than the putative coding sequences. Intriguingly, however, the 3[prime]-UTR contains a 99 bp sequence that has a composition of 76% A-T in mouse (79% A-T in human) but surprisingly shows 90% identity between the two species (Fig. 2b). The conserved sequence includes two ATTTA motifs characteristic of unstable mRNAs (23,24) and multiple A-T-rich sequences differing from the ATTTA motif by a single nucleotide. This observation suggests that the mouse Zfp127 and human ZNF127 mRNA molecules are likely to have rapid turnover in the cell and the likely functional significance is highlighted by the unexpected sequence conservation in this region.

Tissue and germline expression of the mouse Zfp127 gene

The mouse Zfp127 gene is ubiquitously expressed as a major transcript of ~2.9 kb, with the highest steady-state level present in testis, among the adult tissues examined by northern blot analysis (Fig. 3a). Given the previous finding of a conserved motif likely to confer mRNA instability on the Zfp127 transcript, the high level of detectable transcripts in testis may represent a higher level of transcription or the absence of factors mediating mRNA instability. The smaller 1.8 kb transcript may represent an alternative spliced transcript from this locus as it is present at highest levels in testis and this type of event commonly occurs in post-meiotic germ cells (see below). While we cannot at present rule out that the 1.8 kb transcript may represent a highly expressed Zfp127-related sequence, related sequences are not detected by Southern blot (21; data not shown) or PCR analyses with the reagents and conditions used within this report.


Figure 3. Expression of Zfp127 in somatic tissues and during spermatogenesis. (a) Adult tissue expression. h, heart; b, brain; s, spleen; lu, lung; l, liver; sm, skeletal muscle; k, kidney; t, testis. (b) Developmental stages of mouse spermatogenesis. Testis samples have somatic cells and, on a given postnatal day of development, include: developing germ cells that are mitotic spermatogonia (8 days); then addition of early spermatocytes, pre-leptotene through zygotene (12 and 15 days); appearance of pachytene spermatocytes (17 days); then the first post-meiotic haploid round spermatids (20 days); and more mature haploid cells, including elongating spermatids (28, 35 and 42 days). Other samples are: XX, XX,Sxr testis (no germ cells); T, normal adult testis. (c) Cells from highly enriched stages of spermatogenesis. Symbols as for (b) and: G, spermatogonia; LZ, leptotene and zygotene spermatocytes; P, pachytene spermatocytes; RS, round spermatids; RB, residual bodies. (d) Antisense Zpf127as and Zfp127 genes detected by RT-PCR. Primers used for RT and PCR are shown.

We have investigated further the expression of Zfp127 during spermatogenesis, since this gene: (i) shows a high level of expression in testis; (ii) shows imprinting with paternal-only expression (see below); and (iii) encodes a putative zinc-finger protein that could function in organization or regulation of the paternal genome in germ cells. Analysis of expression in XX sex reversed (Sxr) mouse testis (Fig. 3b and c), which is devoid of germ cells, shows that only the 2.9 kb Zfp127 transcript is expressed in somatic cells of the testis, including the Sertoli cells. Analysis of expression in testis RNA from mice of 8-42 days post natal age, which span progressive stages of spermatogenesis from mitotic spermatogonial cells through meiosis to post-meiotic cells, demonstrates an increase in the level of both the 2.9 and 1.8 kb Zfp127 transcripts at more mature stages (Fig. 3b), suggesting expression in post-meiotic germ cells. Therefore, enriched germ cell populations isolated by gradient sedimentation were further examined (Fig. 3c). No expression was detected in mitotic spermatogonia or early meiotic stages, but both Zfp127 transcripts were found in pachytene spermatocytes, round spermatids and lower levels in residual bodies, the cytoplasmic droplets that are shed from maturing spermatids. The increase in level of the Zfp127 1.8 kb transcript in post-meiotic germ cells, in particular round spermatids, is most striking since it was not detected in somatic cells of the testis (Fig. 3b and c). As expected, the 1.5 kb germ cell-specific[beta]-actin mRNA increased in post-meiotic germ cells, including residual bodies, where the somatic 2.1 kb [beta]-actin transcripts are not seen (Fig. 3b and c).

To examine whether the mouse Zfp127 gene may have an orthologous antisense gene corresponding to the ZNF127AS transcript, we performed RT-PCR using strand-specific primers to initiate reverse transcription. This analysis identified expression of both Zfp127 and a putative antisense transcript (Fig. 3d), the latter termed Zfp127as. One intron has been identified to date in human ZNF127AS and the one characterized exon-intron boundary in mouse is conserved (20). By northern analysis and RT-PCR of adult tissues, we could not detect the Zfp127as transcript, as also found for human adult tissues (20). Due to the difficulty in detection of Zfp127as, we were not able to examine the imprinting status. Subsequently, expression of the paternal allele only for Zfp127as was observed by in situ (12) or RT-PCR expression analyses (J. Jones, M.T.C. Jong, R.D. Nicholls and B.M. Cattanach, unpublished data) using tissues from embryos with maternal or paternal UPD for mouse chromosome 7C.

Functional imprinting of the mouse Zfp127 gene

The in-frame hexanucleotide tandem repeat in the coding region of the mouse Zfp127 gene shows length (9-11 copies) and sequence polymorphism among the four strains and subspecies of mice examined (Fig. 4a). This feature was used to test functional imprinting of Zfp127 in an F1 cross between two mouse strains that differ in copy number of the variable number of tandem repeat (VNTR) locus. RT-PCR analysis of mRNA from different tissues of 129/Rl (mother) or Mus spretus (father), using a strand-specific (RN104) or oligo(dT) primer for reverse transcription and PCR primers spanning the VNTR, shows a product 12 bp larger in 129/Rl (Fig. 4b). Genomic DNA contamination in the RNA samples was ruled out by showing the absence of PCR products in the -RT controls. Adult brain, heart and kidney mRNA from two F1 animals each show an RT-PCR band identical in size to the M.spretus parent whereas the maternal 129/Rl allele was not present (Fig. 4b). These data establish that mouse Zfp127 is expressed only from the paternal chromosome.


Figure 4. Functional imprinting of the mouse Zfp127 gene. (a) Hexanucleotide VNTR sequence in mouse Zfp127. Dots indicate unchanged sequence and gaps represent the absence of a specific VNTR unit. Nine, 10 or 11 copies of the VNTR are found in M.spretus, 129/Sv and CD-1 or 129/Rl subspecies or strains, respectively. (b) Imprinting analysis. Expression of Zfp127 was examined by RT-PCR using primers spanning the VNTR and RNA from various tissues of parental and F1 animals from a 129/Rl (Rl)×M.spretus (Ms) cross. The F1 RNA was a combined sample from male and female offspring. +RT, PCR following RT; -RT, PCR with no RT to demonstrate lack of DNA contamination, since Zfp127 is intronless. (c) Zfp127 is not expressed in parthenogenetic derived cells. Northern analysis of two normal (N), three parthenogenetic (P) and one androgenetic (A) primary embryonic fibroblast cell lines. The control gene (Rpl19) is expressed in all cell lines.

To confirm the authenticity of the F1 RNA samples, we analyzed the allele specificity for expression of the Xist gene using an expressed polymorphism between the two parental subspecies (25). Xist transcripts representing both parental alleles were detected in the F1 sample, as would be expected from the random nature of X inactivation in females (data not shown). Interestingly, two Zfp127 RT-PCR bands representing both parental alleles were observed in liver mRNA from the F1 animals, suggesting that the expression was biparental in this tissue (Fig. 4b). Nevertheless, the signal representing the paternal allele is stronger than the maternal allele, suggesting retention of preferential paternal Zfp127 expression even in this tissue. Similar results for liver were obtained using RNA from embryos with maternal or paternal UPD (J. Jones, M.T.C. Jong, R.D. Nicholls and B.

M. Cattanach, unpublished data).

The mouse Zfp127 gene is expressed at the blastocyst stage and embryonic days 8-17, as well as in undifferentiated and differentiated embryonic stem (ES) cells (A.H. Carey and C.L. Stewart, unpublished data). Therefore, imprinting of Zfp127 was also tested by northern analysis using fibroblasts derived from parthenogenetic (maternal chromosome only) and androgenetic (paternal chromosome only) ES cells (26,27). The results indicate that Zfp127 is expressed abundantly in the androgenetic and normal cell lines but is not expressed in the parthenogenetic derived cells (Fig. 4c) and, therefore, is functionally imprinted in early mouse development. As controls, other imprinted genes behaved as expected in these same cells (C.L. Stewart, unpublished data) and non-imprinted genes are expressed in all three cell types (Fig. 4c).

DISCUSSION

The Zfp127 gene is an intronless, imprinted gene that is expressed from the paternal allele only in the majority of mouse tissues. Its structure suggests an origin by retrotransposition via a reverse transcribed intermediate from an ancestral intron-containing gene (28,29), a hypothesis which is supported by our recent identification of the ancestral gene (T.A. Gray and R.D. Nicholls, unpublished data). This event must have occurred at least 80 million years ago, since a well-conserved human ortholog (ZNF127) is also known (20). The high evolutionary conservation, tissue-specific expression patterns and demonstration of in vitro translation for the human gene (20) strongly suggest that Zfp127 and ZNF127 encode functional proteins. Similarly, two other intronless imprinted genes, U2af1-rs1 and Ins1, are also functional retroposons and it has been suggested that establishment of imprinting at these two genes may have occurred by a mechanism similar to those transgenes that show allele-specific expression and methylation patterns dependent on the sex of the parental lineage (30). Genes flanking the neomorphic imprinted U2af1-rs1 and Ins1 genes do not appear to be imprinted, consistent with this idea. However, we propose an independent pathway in which evolutionary insertion of the ancestral Zfp127/ZNF127 gene into a large imprinted domain regulated by a common cis-acting mechanism during gametogenesis (see below) led to the acquisition of imprinting for this gene.

It may be that the ancestral Zfp127/ZNF127 gene inserted into the 3[prime]-UTR of an existing gene, since both human (20) and mouse demonstrate the presence at this locus of an antisense gene (ZNF127AS and Zfp127as, respectively). The mouse Zfp127as gene is expressed at highest levels during early embryonic development where it is imprinted and expressed from the paternal allele only (12; J. Jones, M.T.C. Jong, R.D. Nicholls and B.M. Cattanach, unpublished data). In the human, the ZNF127AS gene is expressed weakly during fetal development and at very low levels in adult brain regions (20), but has not been studied in the earlier embryo. Nevertheless, this gene remains only partially cloned and it will be necessary to isolate further 5[prime] sequences to determine whether it encodes a protein or represents a regulatory RNA, similar to several other non-coding imprinted transcripts (1,7,9). The potential antisense regulation of these and similar genetic loci are discussed in the accompanying paper (20).

The mouse Zfp127 and human ZNF127 genes represent the first identified members of a novel small family of closely related genes in the mammalian genome. This includes at least seven loci in the human and five in the mouse, each species with multiple functional members (T.A. Gray and R.D. Nicholls, unpublished data). Each active gene family member is predicted to encode a ribonucleoprotein, on the basis of the classes of zinc-finger motifs found within the encoded coding sequences (20). This includes a RING (C3HC4) zinc-finger motif with likely function in protein-protein interactions (22) and multiple C3H zinc-finger motifs that are expected to function in RNA binding (reviewed in ref. 20). Therefore, mouse Zfp127 is likely to function in gene or RNA regulation and the high levels of expression in post-meiotic stages of spermatogenesis may be indicative of such a role. Although mice lacking a paternally transmitted, active copy of this gene are viable and fertile and appear normal (A.H. Carey, M.T.C. Jong, R.D. Nicholls and C.L. Stewart, unpublished data; see ref. 20 for further phenotype discussion), it is likely that expressed members of this new gene family have overlapping and perhaps partially redundant function. Thus, gene targeting of each expressed member is likely to be necessary to determine the function of each. Interestingly, the human ZNF127 and mouse Zfp127 mRNAs share an AU-rich segment in the 3[prime]-UTR, characterized by AUUUA elements, which are found in mRNAs with post-transcriptional regulation that is mediated by mRNA instability. Indeed, the 3[prime]-AU-rich element identified in Zfp127/ZNF127 is more highly conserved than the sequence in the protein coding regions, as for similar elements found in mammalian lymphokines, cytokines and proto-oncogenes (23,24,31). This observation is consistent with a role for Zfp127/ZNF127 in cell regulation, as for the early response `unstable mRNAs' that comprise this class of transcripts (24).

Each paternally expressed gene in the 2 Mb imprinted domain of human 15q11-q13 or mouse chromosome 7C is a candidate to play a role in the imprinted phenotype of PWS (5,32) or PWS mouse models (11,13; J.M. Gabriel, M. Merchant, T. Ohta, Y. Ji, R.G. Caldwell, M.J. Ramsey, J.D. Tucker, R. Longnecker and R.D. Nicholls, submitted for publication), respectively. PWS is thought to be a contiguous gene syndrome, although the number and identity of genes contributing to the disorder is unknown (5). The potential role of each relevant imprinted gene will need to be assessed by the identification of specific gene mutations in patients with single phenotypic components of PWS, by transgenic rescue of PWS mouse models or by specific gene knockout or deletion, although to date these latter methodologies have not found a significant role for Zfp127 (see above), Snrpn (13) or Ipw (K. Goss, J. Schryver, M. Dhar, R.D. Nicholls, E.M. Rinchik and D.K. Johnson, unpublished data) in the PWS phenotype.

All imprinted genes within human 15q11-q13 are under a regional mechanism that controls imprint switching in the germline (5). Studies of PWS and AS syndrome patients with an imprinting mutation have led to definition of a genetic element, the imprinting center (IC), that localizes to the 5[prime]-end of the SNRPN gene (33-35). A deletion of the IC at the SNRPN promoter blocks in cis the maternal to paternal imprint switch during parental spermatogenesis, leading to paternal transmission of a maternal epigenotype and hence to PWS in those offspring inheriting the mutation. As a consequence, imprinted genes in 15q11-q13 that are normally expressed from the paternal allele are silenced, including ZNF127 (36). Similar results were recently obtained by knocking out the equivalent region of the Snrpn gene in the mouse (13), indicating conservation of the gametogenic imprint switch mechanism. Since the ZNF127 and Zfp127 genes map at one boundary of this imprinted domain in the human (20) and in the mouse, respectively, they will be important in determining the mechanism by which the IC signal is propagated to and recognized by each imprinted gene within the germline.

Although these genes are clearly under a primary IC-mediated regional imprint switch mechanism (5), the local regulatory mechanism(s) by which each gene receives and responds to the IC signal is unknown. Local repeat structures have been found in many imprinted genes and are implicated in establishment of the methylation imprint (1,37,38), although the molecular basis by which such an element might be associated with imprinting is not yet known. While repetitive sequences are found within the mouse Zfp127 gene, such sequences do not occur within the vicinity of the human ZNF127 gene, which does retain imprinting (20). Therefore, another mechanism may be involved in the local regulation of the human ZNF127 and orthologous mouse Zfp127 genes.

Since both human ZNF127 and mouse Zfp127 are expressed at high levels in testis, in meiotic and post-meiotic germ cells, it is tempting to speculate that expression in the male germline contributes to the setting of the paternal-specific gametic imprint at this locus. A similar idea has been proposed for expression of the paternal allele of Xist in early development (39). The gametic imprint is likely to involve specific marking of the parental alleles by DNA methylation, as for other imprinted genes (1,8,40,41). Interestingly, study of genomic DNA from sperm and ovary for human ZNF127 suggests that only the maternal allele is at least partially methylated (20), consistent with a role for paternal gene expression protecting the paternal allele from methylation. In keeping with these observations, mouse Zfp127 shows imprinted expression in the preimplantation embryo (J. Jones, M.T.C. Jong, R.D. Nicholls and B.M. Cattanach, unpublished data), similar to that observed for mouse Snrpn (42). This may be explained as a consequence of the gametic DNA methylation being located in 5[prime] sequences directly controlling expression (20,43) or if methylation is established as an immediate post-zygotic event (R. Shemer and A. Razin, personal communication). In contrast, the larger Igf2r gene carries the gametic DNA methylation within the body of the gene and requires post-implantation mechanisms to direct allele-specific events to the promoter (8,9,44). Finally, maintenance of the methylation imprint of ZNF127 only in the brain (20), as in the mouse (R. Shemer and A. Razin, personal communication), may also result from high activity of the gene in this tissue with protection of the paternal allele from undergoing methylation. Further work will be important to determine the exact mechanisms involved in the establishment and resetting of the genomic imprinting pattern for each gene or chromosomal domain at each stage during germ cell and post-zygotic somatic development. Studies of the large imprinted domain from the homologous human 15q11-q13 and mouse 7C chromosomal regions will continue to play a significant role in advances to our understanding of this complex gene regulatory phenomenon.

MATERIALS AND METHODS

Mice

129/Rl × M.spretus F1 mice were bred at Oak Ridge National Laboratory (Oak Ridge, TN) by crossing p cch/p cch females from the segregating inbred strain 129/Rl- p cch/p c to males from a non-inbred M.spretus stock (a gift from the late V. Chapman). Differentiated fibroblasts of androgenetic and parthenogenetic origin were derived from andogenetic and parthenogenetic chimeras. Chimeras were produced from androgenetic or parthenogenetic ES cells that constitutively expressed the NeoR gene, using standard procedures (45). Androgenetic and parthenogenetic fibroblasts were derived by explanting the chimeras in culture, followed by selection in G418 to remove the wild-type cells.

Clones and DNA sequence analyses

Isolation of mouse Zfp127 partial 3[prime] cDNA (21) and bacteriophage genomic [lambda] clones are described elsewhere (A.H. Carey, M.T.C. Jong, R.D. Nicholls and C.L. Stewart, unpublished data). All subclones were in pBS-SK(+) (Stratagene, La Jolla, CA). DNA sequencing and DNA preparation were as described (20). DNA and amino acid sequences were analyzed using BLAST (http://www3.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast ) and PROSITE (http://expasy.hcuge.ch/prosite/prosite.html ) and alignments were performed using the Megalign program (DNAStar, Madison, WI).

RNA techniques

Total RNA from various mouse tissues was prepared by a standard CsCl gradient method (46). Poly(A)+ RNAs were prepared using the PolyATract System 1000 (Promega, Madison, WI). To remove genomic DNA contamination in RNA samples, 200 ng of poly(A)+ RNA or 2 µg of total RNA were digested with 1 µl DNase I (1 U/µl, RNase-free; Gibco BRL, Gaithersburg, MD) for 15 min at room temperature. DNase I was inactivated by adding 1 µl of 20 mM EDTA and heating for 10 min at 65°C. The RNA samples were used following one phenol-chloroform extraction and isopropanol precipitation.

To map the transcription start site of the mouse Zfp127 gene, we used 5[prime]-RACE with a 5[prime] AmpliFINDER kit (Clontech, Palo Alto, CA). First strand cDNA was synthesized from poly(A)+ RNA extracted from the tsA1s9 cell line, a temperature-sensitive mutant of mouse L cells, using a gene-specific primer, RN121 (5[prime]-TCTCTCTCAATTGCCGGGCTCTGC-3[prime]). The upstream nested primer RN119 (5[prime]-CCGCTGGGCTCAATGGGAGCTGTAGAC-3[prime]) was subsequently used for PCR amplification with the anchor primer provided in the kit. All PCR products were cloned using the TA Cloning kit (Invitrogen, Carlsbad, CA), transformants screened with radioactive probes, and positive clones were sequenced.

First strand synthesis for RT-PCR was performed using 1 µg of total RNA in the Superscript Preamplification System (Gibco BRL) with either oligo(dT), random hexamers or gene-specific primers. The strand-specific primer for Zfp127 mRNA was RN104 (5[prime]-CATGGGGGTATGCACAC-3[prime]) or RN65 (5[prime]-GACAACAGAGCTTTAGC-3[prime]) and for Zfp127as was RN43 (5[prime]-GGTATATATACATGGGCAGTGCAAGG-3[prime]). A second aliquot of RNA was not reverse transcribed and used as the -RT control. Both aliquots were used as templates in subsequent PCR reactions, using Taq DNA polymerase and a Perkin Elmer Cetus (Foster City, CA) DNA Thermal Cycler 480. The Zfp127 VNTR in different mouse strains was analyzed by RT-PCR or genomic PCR using PCR primers RN120 (5[prime]-ACAGCCTTACCGAGGTCGCATGG-3[prime]) and RN104. The PCR conditions were 30 cycles of 1 min at 94°C, 2 min at 65°C and 2 min at 72°C. For Xist RT-PCR and HindIII digestion, primers MX23b and MIX20 were used and conditions were as described (25). PCR products were resolved on an 8% polyacrylamide gel, stained with ethidium bromide and photographed.

Northern blot filters for adult tissues were purchased from Clontech and hybridized and washed under standard high stringency conditions (21). Autoradiograms were exposed for at least 1 week for Zfp127; in contrast, use of probes from ancestral Zfp127-like gene sequences only require 1 day exposures (T.A. Gray and R.D. Nicholls, unpublished data).

Highly enriched fractions of germ cells were prepared by enzymatic digestion of testes and sedimentation of germ cells on bovine serum albumin gradients by methods previously described (47,48). Pachytene spermatocytes, round spermatids and residual bodies were obtained from adult mice, a mixed population of leptotene and zygotene spermatocytes was obtained from 18-day-old mice and a mixed population of types A and B spermatogonia was obtained from 8-day-old mice. Cell fractions were assessed for purity using differential interference contrast optics; purities of the cell fractions were typically >85%. Total RNA was isolated by the guanidinium isothiocyanate method (49) from fresh germ cell preparations. Equivalent amounts (15 µg) of total RNA were separated by electrophoresis in 1% agarose gels with 2.2 M formaldehyde, transferred by capillary blotting to nylon membrane (Hybond; Amersham, Arlington Heights, IL) and UV crosslinked at 1280 µJ/cm2 with a Stratalinker (Stratagene).

ABBREVIATIONS

AS, Angelman syndrome; EST, expressed sequence tag; IC, imprinting center; PCR, polymerase chain reaction; PWS, Prader-Willi syndrome; RACE, rapid amplification of cDNA ends; RT, reverse transcription; UPD, uniparental disomy; UTR, untranslated region; VNTR, variable number of tandem repeats.

ACKNOWLEDGEMENTS

We thank Dr Todd A. Gray for critical comments on the manuscript, Drs Bruce Cattanach, Jan Jones, Ruth Shemer and Aharon Razin for discussions during the work and for sharing pre-publication data, and Wayne Gottlieb, Michael F. Waters, James Amos-Landgraf and Nancy Rebert for expert technical assistance. This work was supported by grants from the National Institutes of Health (HD34191 to R.D.N. and D.J.D.; HD36079 to R.D.N.; HD33816 to M.A.H.), American Cancer Society (R.D.N.), International Human Frontiers of Science Program Organization (R.D.N.), the Pew Scholars Program in Biomedical Sciences (R.D.N.) and the US Department of Energy (under contract to Lockheed-Martin Energy Systems Inc., to E.M.R.). During this work, M.T.C.J. was the recipient of a Development travel fellowship.

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*To whom correspondence should be addressed. Tel: +1 216 368 3331; Fax: +1 216 368 3432; Email: rxn19@po.cwru.edu
Present addresses: +Oxagen Ltd, 91 Milton Park, Abingdon, Oxon OX14 4RY, UK; §Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Cancer and Developmental Biology Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA

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