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. 1997 Nov 11;94(23):12509-14.
doi: 10.1073/pnas.94.23.12509.

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2

T Moore  1 , M Constancia, M Zubair, B Bailleul, R Feil, H Sasaki, W Reik
Affiliations

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2

T Moore et al. Proc Natl Acad Sci U S A. .

Abstract

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

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Figures

Figure 1
Figure 1
(A) Genomic map of mouse Igf2 and upstream transcripts. Igf2 promoters (horizontal arrows): P0, P1, P2, and P3. Ins2; Igf2 exons (black boxes): u1, u2, E1-E6. DNA repeats (shaded boxes): B2, CG-rich. DNase I HS (vertical arrows); ∗, embryo-specific HS. P0 transcript: exons (thick line), introns (thin line) includes exons u1, u2, E4-E6. A 1591-bp PCR product of P0 (primers UP2 and LP1) was cloned and sequenced. P0 poly(A)+ addition sites were not mapped; however, on Northern blots P0 is identical in size to the 5-kb P2 transcript (data not shown). The identical size suggests similar inclusion of E6 sequences downstream of LP1. Transcribed AS regions were named according to the nearest associated poly(A)+ addition sites—a, b, c: Igf2as-a, Igf2as-b, Igf2as-c. Putative transcription start sites, a′, b′, and c′. Open boxes represent sequenced AS RT-PCR products from primer pairs UP5 and LP7: b transcript, UP6 and LP6: c transcript, UP3 and LP9 or UP4 and LP5: a or a′ transcript. We were unable to clone or sequence a fragment from primer pair UP3 and LP5, and therefore cannot propose a final structure for a putative a-a′ AS transcript. Three hundred base pair regions upstream of putative transcription start sites were examined for regulatory sequences using the tfd software program at the Medical Research Council, Human Genome Mapping Project. Only the b′ site was associated with major promoter element motifs. (B) DNA sequence data for upstream transcripts. Nucleotide positions are numbered as per ref. . Putative transcription starts (vertical arrows): P0: 8,512; b′: 17,587; c′: 11,943. P0 acceptor splice sites (ASS) and donor splice sites (DSS): u1 putative ASS (underlined): 8,584–8,597; u1 DSS: 8,825–8,832; u2 ASS: 10,120–10,134; u2 DSS: 10,952–10,959. AS transcript splice sites and (underlined) poly (A+) addition signal nucleotide positions numbered for the complementary, upper strand, as follows: b1 DSS: 17,406–17,399; b2 ASS, DSS: 12,932–12,918, 12,862–12,855; b3 ASS, DSS: 12,200–12,186, 11,976–11,969; b4 ASS: 11,273–11,259. Poly(A)+ signals: a: 8,120–8,115; b: 9,651–9,646; c: 9,808–9,803.
Figure 2
Figure 2
Northern blot analysis of embryonic and adult RNA. (A) Age-matched, late gestation normal and matdi7 mouse fetal and placental RNA probed with the 2.2-kb BE DNA probe. Blot was autoradiographed for 1 week. Lack of expression of the 5-kb placenta-specific transcript and the 4-kb transcript in matdi7 samples is consistent with imprinted (paternal) expression. Hybridization with control H19 probe confirms integrity of matdi7 RNA. (B) Identical aliquots of midgestation mouse fetal and placental poly(A)+ enriched RNA were blotted and probed with sense and AS riboprobes spanning the 2.2-kb BE region. The sense probe detects multiple (3–4 kb) transcripts in both fetus and placenta; the AS probe detects a single placenta-specific 5-kb transcript. Following RNase treatment, blot was autoradiographed for 5 days. Relative band intensities between T3 and T7 probes do not reflect relative abundance of sense and AS transcripts. (C) Transcripts of widely different sizes were detected using the 2.2-kb BE DNA probe on Northern blots of adult mouse tissue total RNAs. Strongest expression was in kidney, which contains a predominant transcript of ≈3 kb. The blot was autoradiographed for 1 week.
Figure 3
Figure 3
RT-PCR analysis of parental allelic expression of Igf2 upstream transcripts. Three M. domesticus/M. spretus polymorphisms were used to detect imprinted expression: M. spretus specific BsaAI (primers UP7 and LP8) in exon 6 was used for P0 transcript. (This transcript is detectable in fetus using RT-PCR.) M. spretus-specific BamHI (primers UP10 and LP9) and XbaI (primers UP9 and LP6) in the upstream region (see Results for approximate positions of polymorphic sites) were used for AS transcripts. d, s, d × s, s × d: cDNA samples from midgestation M. domesticus, M. spretus, (M. domesticus × SD7)F1, and (SD7 × M. domesticus)F1, respectively. cDNA samples 1 and 2: (+/+; +/SD7) and (ΔH19/+; +/SD7) littermates; 3 and 4: (+/SD7; +/+) and (+/SD7; ΔH19/+) littermates. F, fetus; P, placenta. Primer pairs UP7, LP8, and UP10, LP9 preferentially amplified M. domesticus alleles from (M. domesticus × M.spretus)F1 genomic DNA (data not shown), which probably accounts for stronger M. domesticus bands in F1 samples with biallelic expression. Results indicate paternal allelic expression of all transcripts and biallelic expression following maternal, but not paternal, inheritance of the ΔH19 null allele.
Figure 4
Figure 4
DNase I sensitivity analysis of the region spanning the B2 repeat using a XhoI–EcoRI 0.78-kb fragment as a probe. Embryo-specific (1.9 kb) site was the only nonconstitutive HS detected in the Igf2 upstream region. B2 repeat (open box); HS sites (vertical arrows); *, embryo-specific site.
Figure 5
Figure 5
DNA methylation analysis of region spanning exons u1 and u2. (A) Probe B on a Southern blot of BamHI/SmaI digested DNA from samples as indicated. Polymorphic BamHI sites and hybridization signals denoted by subscripts in parentheses: d, M. domesticus; s, M. spretus. Normal vs. disomy analysis: 2.4-kb and 0.9-kb fragments in normal, but not matDi7 placenta, indicates relative demethylation of paternal allele. Maternal deletion of H19 in (M. domesticus × M. spretus)F1 samples: relative demethylation of paternal (M. spretus) allele in placenta indicated by reduced intensity of 3.7-kb, and appearance of 3.5-kb, bands. Demethylation of maternal (M. domesticus) allele in placenta in response to H19 deletion indicated by appearance of 2.4-kb and 0.9-kb bands. (B) Summary of analysis of fetus and placenta from normal and maternal H19-deficient midgestation embryos. Probes: A, XbaI–EcoRI; B, XbaI–XbaI; C, BamHI–EcoRI fragments. H1–H5, HpaII clusters; Sm, SmaI. Mat, maternal allele; Pat, paternal allele. Filled or partially filled circles indicate degree of methylation of sites. H19 deletion has no effect on fetus. H4 is fully methylated in all samples.
Figure 6
Figure 6
Mouse-human DNA sequence comparisons. (A) DNA sequence comparison of 5 kb of mouse Igf2 spanning exon u1 to CpG island, and homologous human region, using GCG sequence analysis program. B, BamHI; u1, u2, mouse upstream exons 1 and 2; E2, E3, human exons 2 and 3; human sequence EMBL accession: Y13633. (B) Self-comparison of 1,200 bp of human and mouse Igf2 sequences spanning human exon 3 and mouse exon u2 regions using strider program. In contrast to human, the mouse sequence exhibits a large repeat block.
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