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Lara A. Underkoffler,2,
Andrew J. Wood,1
Joelle N. Collins,2
Patrick T. Williams,2
Jeffrey A. Golden,4
Eugene F. Schuster Jr.,1
Kathleen M. Loomes,3 and
Rebecca J. Oakey1*
Department of Medical and Molecular Genetics, Guy's, King's and St. Thomas' School of Medicine, King's College London, 8th Floor, Guy's Tower, London SE1 9RT, United Kingdom,1 Division of Human Genetics,2 Division of Gastroenterology,3 Department of Pathology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 191044
Received 20 January 2005/ Returned for modification 19 February 2005/ Accepted 23 March 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression profiling has been used to identify novel imprinted transcripts by using chromosome anomalies in the mouse (7) and by using parthenogenotes versus normally fertilized embryos or androgenotes (16, 20, 23). We describe here an imprinted gene identified on mouse chromosome 7 (7), along with the genomic and epigenetic features characteristic of imprinting.
Through the identification and characterization of imprinted loci such as the Igf2/H19 region (4, 30), the Dlk-Gtl2 region (34), and the BWS region (24, 31), it has emerged that imprinted genes are frequently clustered in domains (28) and are associated with differentially methylated regions (DMRs) (9), often coinciding with imprinting control regions (35, 40). Other notable features include their frequent association with alternatively spliced transcripts (26), CTCF binding sites (14), CpG islands (40), and direct repeats (27, 42). Alternative splicing and antisense transcripts have been particularly well studied at the Gnas locus on mouse chromosome 2, which elicits a complex pattern of parent of origin- and promoter-dependent expression (39). Alternative splice forms of the imprinted Grb10 gene are expressed from either or both alleles in a tissue-specific manner (5), and whether alternative splicing has a mechanistic relationship to imprinting will be better understood by further study.
The novel imprinted gene identified from this microarray screen is a variant of Inpp5f, an inositol phosphatase gene that maps to distal mouse chromosome 7 at 116,088,744 to 116,174,591 bp, ca. 14 Mb proximal to the H19/Igf2 cluster, in a region that shares conserved linkage with human chromosome 10q26. We have cloned and characterized the Inpp5f_v2 transcript, which is expressed only in the brain, and allele-specific assays have shown that it is expressed only from the paternal allele. Differential methylation of a CpG island has been found between the maternal (methylated) and paternal (unmethylated) alleles consistent with allele-specific expression. The related longer transcript, Inpp5f is biallelically expressed in all tested tissues and is associated with a different hypomethylated CpG island.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Allele-specific assays in intersubspecies hybrids. Newborn brain and kidney RNA from C57BL/6/J (B6), Mus mus castaneus (cast), cast x B6, and B6 x cast intersubspecies hybrids was reverse transcribed into cDNA. Polymorphisms were identified by sequencing Inpp5f_v2 in the B6 and cast strains. The Inpp5f_v2 exon 1 polymorphism is at 689 bp of exon 1 and was assayed by amplifying with primers 5'-ACCTAAGTCCGATGGCGTTCTC-3' and 5'-TTTCTATTCTTTCCAGGTCTTCTAGG-3' to distinguish a G in B6 and a T in cast sequence. Inpp5f was assayed for imprinting by a polymorphism unique to this longer transcript. This polymorphism was located in exon 5 at 252 bp, is an A in B6 and a G in cast, and is assayed by using the primers 5'-CATACTGCCTGCTGATGGAGTCAC-3' and 3'-AGAAAAGGAGAAACTGGAGCGG-3'.
Northern blot analysis. A MessageMap Northern blot (Stratagene) of adult BALB/c mouse tissues was probed, stripped, and rehybridized with probes specific for different regions of Inpp5f_v2. The probe for exon 6 was generated with primers 5'-TTCAGAAGAGTCCAGCAGAACCC-3' and 5'-CCATATTCCAGGATGACTGCCTG-3'. The probe for exons 2 to 4 was generated with primers 5'-AAGTGCTGCTGCTGCTGTCTAAC-3' and 5'-TTGCCATCTTCTTCAGGACTACG-3'. An actin probe was used as a loading control. The probe for exons 11, 12, and 13 of Inpp5f (long transcript) was generated from primers 5'-AACCCTCACGGATGCCATTC-3' and 5'-TCACCCTTTAGAGCAGCAGTCC-3'. The probe for exons 13 and 14 was generated from primers 5'-TGACTCCATCAGCAGGCAGTATG-3' and 5'-CTGTAGGCATCCTTGAACCGAC-3'. A Northern blot with mouse brain and kidney RNA was probed with an exon 1-specific probe generated from primers 5'-ATGCGACCATTGTCTCCGTG-3' and 5'-CATTCTGAAAACTGCTGCTTGAGC-3'.
Bioinformatics. Expressed sequence tags (ESTs) were identified from the NCBI database, the TIGR database, and the UCSC genome browser. The mouse genomic sequence for Inpp5f_v2 analyzed was 116,165,000 to 116,175,000 nucleotides (UCSC May 2004 assembly, NCBI build 33). The human genomic sequence obtained for chromosome 10 was nucleotides 121,566,025 to 121,579,332 (NCBI build 35) determined by the Vistaplot program. The mouse sequence was assembled from RACE product sequencing and from the ESTs with accession numbers BB639524, BB646689, AW561896, and BE305393. The mouse genomic sequence was obtained by sequencing a BAC clone and from the UCSC genome browser sequences in the region of the ESTs listed above.
Programs used for sequence analysis. CpG plot (http://bioweb.pasteur.fr/seqanal/interfaces/cpgplot.html) was used to report the incidence of CpG islands. The parameters used are a CG content of >50%, an observed/expected ratio of >0.6, and a window of 200 bp with a minimum length of 250 bp. A Vista plot (http://www.gsd.lbl.gov/vista/index.shtml) was used for visualizing global DNA sequence alignments (10). The genome assemblies and regions used were 116,165,000 to 116,175,000 bp for chromosome 7 from the May 2004 (mm 5) NCBI build 33 of the mouse genome and 121,566,025 to 121,579,332 bp for chromosome 10 from the human May 2004 (hg 17) NCBI build 35 of the human genome prealigned with SLAGAN (6). Exon 1 was shaded manually because Vistaplot autoshading does not currently contain the novel murine Inpp5f_v2 gene. The sequence used for the CTCF binding site search was CCGCNNGGNGNC (25, 41), which was analyzed with the nucleic acid pattern search tool FUZZNUC in EMBOSS (http://ngfnblast.gbf.de/EMBOSS). Direct repeats were detected by using the Repeatmasker2 software from the University of Washington (http://repeatmasker.genome.washington.edu/).
Section in situ hybridization. Section in situ hybridization was performed on fixed CD1 mouse embryos harvested from 12.5 to 16.5 days postcoitum (dpc). The mouse embryos were fixed in 4% paraformaldehyde and embedded in paraffin wax, and sections were cut 7 µm thick. Radioactive riboprobes were transcribed in the presence of 35[S]UTP. Sense (control) and antisense (test) probes were generated for the test gene. The EST IMAGE:575575 was used as the template for the probe cloned into the EcoRI and NotI sites of a modified pT7T3 vector. The region of probe was exon 6 of Inpp5f_v2, and thus the signal represents both long and short transcripts. The antisense template was linearized with EcoRI and transcribed with T3 polymerase. The sense template was linearized with NotI and transcribed with T7 polymerase. The protocol for radioactive section in situ hybridization that was followed was essentially as described in references 29 and 38. The slides were counterstained with Hoechst and the signal, visualized by using fluorescence microscopy, was photographed.
Bisulfite mutagenesis and sequencing. The methylation status of CpG dinucleotides within the two CpG islands associated with the promoters of Inpp5f (CpG1) and Inpp5f_v2 (CpG2) (Fig. 1) were investigated in tissues from the F1 progeny of C57BL/6J-Ei (B6) mothers and Mus mus castaneus (cast) fathers at 8 weeks of age. A total of 0.5 µg of B6 x cast genomic DNA was digested with EcoRI and converted with 3.25 M sodium meta-bisulfite in 0.93 M hydroxyquinone essentially as described in reference 8. Desulfonated converted DNA was amplified with primers specific for CpG1 and CpG2 (Fig. 1). A region from CpG1 associated with the nonimprinted Inpp5f transcript was amplified in a nested PCR amplification with the primers forward 5'-TATAGTTTTAYGTTGGGGAGG (where Y is a mix of C and T) and reverse 5'-AAAAAAATACACTAAAAAAAATAACC-3' for 35 cycles at 55°C and 1.5 mM MgCl2, followed by a second round with forward primer 5'-GGTATGGAGTTTTTTTAGGTTAAGGAT-3' and reverse primer 5'-CCTAAAACAAAAAAACTCCCC-3' for 35 cycles at 55°C and 1.5 mM MgCl2. The product size is 269 bp with two C/A polymorphisms between B6 and cast at positions 162 and 165, respectively. The primers used for the CpG2 island upstream of the imprinted Inpp5f_v2 transcript were also nested and were forward primer 5'-TTAGGATTTAGAGTATTTGTAGAAA-3' and reverse primer TTTACAAAAAAAATACAACCCCACTA-3' for 35 cycles at 55°C and 1.5 mM MgCl2, followed by forward primer 5'-TTTGGTAGTTTTTTGTTTATTAAGT-3' and reverse primer 5'-ACCCCACTAACACTTTAACCATAAAT-3' for 35 cycles at 55°C and 1.5 mM MgCl2. This gives a 367-bp product with a T/G (B6/cast) polymorphism at position 36. The PCR products were gel purified by using the QiaEXII (Qiagen) kit and cloned by using the TOPO TA cloning system (Invitrogen). Individual clones were isolated by using a Qiaprep minispin kit and sequenced from T3 and T7 primers by using standard ABI sequencing technology (Big Dye v3.1) to determine the methylation status of the CpG dinucleotides and the parental origin of each strand. Only DNA strands >95% converted were used for analysis. All strands were known to be derived from unique template strands on the basis of either epigenotype or an unconverted cytosine occurring outside a CpG dinucleotide (data not shown).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3.2-kb transcript was identified by using probes containing exons 2 to 4 and exon 6 of Inpp5f_v2 (Fig. 4a and b). An additional, larger transcript (4.7 kb) was detected on Northern blots in all tissues, including brain (Fig. 4a, b, d, and e). Probes specific to the larger transcript covering exons 11 to 14 detect the larger transcript in all tissues tested but do not detect the shorter transcript, as expected (Fig. 4d and e). Thus, the larger transcript represents full-length Inpp5f. Figure 4f shows that exon 1 is specific to brain and absent in kidney. The UCSC genome browser reveals two additional putative brain-specific transcripts; these were not detected by Northern blot analysis.
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Comparative sequence analysis identified a gene in humans with sequence similarity to Inpp5f called hSac2 or INPP5F (19). No mouse orthologues of hSac2/INPP5F have been previously described. A human-mouse conservation analysis of Inpp5f_v2 was performed by using VistaPlot. Five exons are highly conserved (Fig. 3c) in a region of conserved linkage between mouse chromosome 7 and human chromosome 10. Exon 1 of Inpp5f_v2 is unique and begins between exons 15 to 16 of the full-length Inpp5f gene (Fig. 1). Although exon 1 shows a slightly lower degree of conservation between mice and humans (Fig. 3c), a high degree of conservation is maintained in parts of the CpG island.
Imprinted genes are frequently associated with CTCF binding sites, and multiple putative CTCF binding site sequences were detected within the CpG island associated with Inpp5f_v2 by sequence searching (Fig. 1). In addition, a consensus CTCF direct repeat (n = 2) is located further 3' between exons 4 and 5 in an intronic sequence. The human INPP5F_v2 CpG island showed no exact matches to the CTCF consensus but, allowing for two mismatches, 13 consensus sequences were detected in the forward direction and 14 in the reverse orientation. The structure of the human INPP5F_v2 transcript predicted from Refseq clone NM_198330 (the orthologue of the mouse Inpp5f_v2 gene) is shown in Fig. 3d and is similar to the mouse.
Spatial and temporal expression. A low-magnification 14.5-dpc embryo parasagittal section (lateral to the midline) illustrates the brain specific expression (Fig. 5a). A higher-power sagittal image (Fig. 5b) shows expression in the lateral cortex. There is strong labeling primarily outside the ventricular (proliferative) zone (VZ) dorsally and ventrally, although some VZ labeling is also seen dorsally and frontally. A high-power view of a 12.5-dpc embryo highlighting the reduced expression in the VZ and strong expression in the maturing regions of the midbrain and hindbrain is shown in Fig. 5c. There is an obvious ventral gap in the expression extending from the VZ that could represent the isthmus. Expression is seen in the hindbrain and in a small part of the tectum. The area that will become the brainstem is strongly labeled, whereas the rhombic lip (more dorsally located precursor of the cerebellum) shows less labeling as does the more rostral portion of the tectum (Fig. 5d). At 14.5dpc, expression is seen throughout the nervous system with slightly less expression in the ventricular zones. Figure 5e shows expression in the dorsal root ganglia lateral to the spinal cord and separated by vertebral bodies. Expression is seen in the tectum with a gradient of expression from the rostral to the caudal region (Fig. 5f). Expression is again primarily outside the ventricular zone, although the decreased expression caudally may be due to less developmental progress. Strong expression is also seen in the ventral midbrain and hindbrain, primarily outside the VZ. At this stage, relatively strong expression is seen in the rhombic lip, adjacent to the choroid plexus. At 16.5-dpc expression is seen in the cerebral cortex, the olfactory bulb, the ganglionic eminence, the thalamus, the hippocampus, and the retina. Slightly less expression is seen in the inferior colliculus compared to the superior colliculus (data not shown). The probe used was from exon 6 of Inpp5f_v2 so it also reflects expression of Inpp5f.
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2 of 1.341 and a P value of 0.2469. Similarly, the Fisher’s exact test for count data, for the small sample size, returns a P value of 0.22, a finding consistent with the 2 result.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inpp5f_v2, Inpp5f, and hSac2/INPP5F. Inpp5f_v2 is located on distal mouse chromosome 7, band position 7F3 at 116.1 Mb (UCSC Genome Browser using the Mouse May 2004 assembly), ca. 14 Mb proximal of the H19/Igf2 cluster. This region of the genome shares conserved linkage with human chromosome 10q26.13 in the region of the human gene hSac2/INPP5F. Comparative mouse and human sequence analysis has identified sequence similarity between Inpp5f_v2 and the hSac2/INPP5F gene. No mouse orthologue of hSac2/INPP5F has previously been described. Inpp5f_v2 is a transcriptional variant of Inpp5f. Inositol phosphatases are involved in endocytosis and exocytosis at nerve terminals, a process essential for clathrin coat shedding and synaptic vesicle recycling (1), which is important in the developing brain and in postnatal survival. It has been shown that full-length human INPP5F has inositol 4,5-bisphosphatase activity in vitro (19). However, unlike hSac2/INPP5F, Inpp5f_v2 does not contain a Sac domain but rather is similar to the variable C-terminal domain of hSac2/INPP5F (Fig. 1). It may be that Inpp5f_v2 originated independently of the Sac domain of Inpp5f and that the larger transcript coopted the exons of Inpp5f_v2 as its C-terminal domain, the function of which is unknown.
Transcript size, sequence features, and tissue distribution of Inpp5f_v2. Northern analyses reveal a brain-specific 3.2-kb transcript and a larger 4.7-kb transcript present in all tested tissues (Fig. 4). The 3.2-kb Inpp5f_v2 transcript is imprinted in brain (Fig. 2a) and the longer Inpp5f transcript is biallelically expressed in both brain and kidney (Fig. 2b and c). In situ hybridization studies illustrate that Inpp5f_v2 and Inpp5f are expressed in brain structures and the developing nervous system over a range of developmental time points in the mouse embryo (Fig. 5).
Approximately 40 to 60% of the multiexon genes in the mouse and human transcriptomes exhibit alternative splicing (21, 43). This facilitates increased complexity of the mammalian proteome and altered protein function. In many cases, the alternative use of a first exon is regulated by alternative promoters, conferring tissue specificity to the different isoforms of a gene. A recent genome survey reports that >2,000 genes in the mouse and >3,000 genes in humans have alternative first exons spliced to a common 3' transcript (44) and, in many cases, conserved CpG islands are associated with the variable exon. Inpp5f_v2 uses an alternative transcriptional start site between exons 15 and 16 of Inpp5f and thus is the first example we are aware of in which the unique first exon is embedded within an intron near the 3' end of the gene rather than the selection among first exons seen at the Gnas locus (26). At the Gnas locus, three CpG islands are associated with three alternative start sites sharing common 3' exons. The transcription of Gnas is dependent on the methylation state of the CpG islands, as can be seen in the alternative use of the first exon of Nesp, Gnasxl (26), or Gnas1A (18). In humans, the alternative WT1 transcript AWT1 is similarly organized, is paternally expressed, and consists of exons 2 to 10 of the WT1 gene. However, like at the Gnas locus, this transcript uses a novel 5' exon located in the first intron of the nonimprinted WT1 gene (11).
Differential methylation. Differential methylation has been associated with most of the characterized imprinted genes, such as those in the H19/Igf2 region (36) and at the Dlk1/Gtl2 locus (34). In both of these domains, CpGs are hypermethylated on the silent paternal allele and hypomethylated on the expressing maternal allele. The methylation status of two CpG islands associated with Inpp5f and Inpp5f_v2 was determined. The CpG2 is differentially methylated in brain, where the expressing paternal allele is unmethylated and the silent maternal allele is methylated (Fig. 6b). This differentially methylated region is likely to be involved in the imprinted expression of Inpp5f_v2 in brain. However, three maternal alleles out of thirteen were hypomethylated (Fig. 6b), but no corresponding maternally derived transcription was detected in the allele-specific RT-PCR assay. Although the in situ hybridization data suggest that expression of the Inpp5fv2 transcript varies in different regions of the brain (Fig. 5), this is unlikely to account for these findings since differential methylation is also present in nonexpressing kidney tissue (Fig. 6c), albeit to a lesser extent. One possible explanation is that this DMR exerts its role in the imprinting of Inpp5f_v2 at an early stage of embryonic development and is not absolutely required for maintenance of the imprint in neonatal and adult tissues. A similar situation has been found in the Angelman/Prader-Willi Syndrome region, where differential methylation established in the germ line and maintained at 7.5 dpc is lost by adulthood (15). Bisulfite sequencing analysis of CpG2 in gametes and early-stage embryos would determine whether this DMR is germ line derived and progressively lost during pre- and postnatal development.
CpG1 is hypomethylated on both parental alleles (Fig. 6a), and Inpp5f is biallelically expressed in both kidney and brain. The differential methylation pattern at CpG2 is maintained in tissues where Inpp5f_v2 is not expressed, suggesting that a brain-specific transcriptional regulation mechanism other than methylation may be involved and possibly imposed in addition to the epigenetic silencing of the maternal allele. For Inpp5f_v2, methylation is related to imprinted gene expression but not to tissue specificity; this is observed in the human NDN gene, where a promoter-associated CpG island exhibits differential methylation independently of tissue-specific transcriptional status (17).
One of the elements involved in the control of the reciprocal imprinting of the H19 and Igf2 genes is the vertebrate enhancer blocking protein, CTCF, which binds to sites in DNA to block promoter-enhancer interactions (4, 14, 33). Multiple CTCF binding site sequences were detected within the Inpp5f_v2 CpG2 in mice (Fig. 1). However, functional CTCF binding sites cannot be predicted from sequence alone and provide only an indication of potential binding in vivo.
Many imprinted genes are clustered, suggesting that they reside in domains that are epigenetically modified, resulting in the coordinate regulation of more than one imprinted gene. Although the identification of a DMR near Inpp5f_v2 is consistent with an imprinted domain, one might expect one or more additional imprinted genes to be present. Inspection of flanking sequence in combination with microarray differential expression data in this region revealed a cluster of differentially expressed transcripts 3' to this gene. Further study of this flanking cluster of imprinted genes will shed light on whether Inpp5f_v2 resides alone or is coordinately regulated as part of a larger region.
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ACKNOWLEDGMENTS |
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We thank Colin V. Beechey for the T65H mouse tissues and careful reading of the manuscript. We thank Kathryn Woodfine for Affymetrix GeneChip hybridizations and Trevelyan Menheniott and Reiner Schulz for analysis of Affymetrix microarray data and statistical analysis.
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FOOTNOTES |
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J.D.C. and L.A.U. contributed equally to this study.
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