Imprinted silencing of Slc22a2 and Slc22a3 does not need transcriptional overlap between Igf2r and Air

Abstract

Silencing of the paternal allele of three imprinted genes (Igf2r, Slc22a2 and Slc22a3) requires cis expression of the Air RNA that overlaps the promoter of one of them (Igf2r). Air is a non-coding RNA whose mode of action is unknown. We tested the role of the Igf2r promoter and the role of transcriptional overlap between Igf2r and Air in silencing in this cluster. We analyzed imprinted expression in mice in which the Igf2r promoter is replaced by a thymidine kinase promoter that preserves a transcription overlap with Air, and in mice with a deleted Igf2r promoter that lack any transcriptional overlap with Air. Imprinted silencing of Air, Slc22a2 and Slc22a3 is maintained by the replacement promoter and also in the absence of transcriptional overlap with Air. These results exclude a role for the Igf2r promoter and for transcriptional overlap between Igf2r and Air in silencing Air, Slc22a2 and Slc22a3. Although these results do not completely exclude a role for a double-stranded RNA silencing mechanism, they do allow the possibility that the Air RNA has intrinsic cis silencing properties.

Fig. 1. Four Igf2r promoter replacement/deletion alleles. (A) A map of the Igf2r, Air, Slc22a2 and Slc22a3 imprinted cluster with arrows marking transcriptional orientation. Black box, genes with bi-allelic expression; gray cross-hatched box, imprinted protein-coding genes with maternal-specific expression; gray checked box, imprinted non-coding Air RNA with paternal-specific expression. (B) The V1 and V2 alleles in which, respectively, 444 and 29 bp of the Igf2r allele were replaced by a thymidine kinase promoter–neomycin (TKneo) cassette (Ludwig et al., 1996) are drawn above the wild-type allele. The targeting construct for the Igf2r-TKneo allele is shown below and contains a 12.4 kb EcoRV fragment (bp 89 965–102 345; AJ249895) from the Igf2r locus, from which a 4682 bp SnaBI–SfuI fragment including the entire Igf2r CpG-island promoter and exon 1 was replaced by a 1200 bp loxP (open triangles) flanked cassette containing a TKneo resistance gene and polyadenylation signal (box labeled TKneo). Homologous recombination in embryonic stem (ES) cells yielded the Igf2r-TKneo allele, and in vivo Cre-mediated deletion of the TKneo cassette generated the Igf2r-del allele. Fragments: EcoRV (EV); EcoRI (E); BglII (B); SnaBI (Sn); SmaI (Sm); NotI (N); SalI (S); SfuI (Sf); and MluI (M). The probes (tai, kodel and msi) used for the methylation analyses are shown as black bars above the wild-type allele. (C) Correctly targeted Igf2r-TKneo ES clones were identified by DNA blot of BglII-digested DNA and probe kodel, yielding a 6 kb fragment instead of the 9.5 kb wild-type fragment. (D) The Igf2r-del allele was generated by crossing Igf2r-TKneo mice with mice carrying a CMV-Cre transgene (Schwenk et al., 1995), identified by DNA blot of BglII-digested DNA and probe kodel. Cre-mediated deletion of the TKneo cassette changes the 6.0 kb fragment from the Igf2r-TKneo allele to 4.9 kb, generating the Igf2r-del allele. (E) Absence of Igf2r mRNA from the Igf2r-TKneo and Igf2r-del alleles. RNA blot of 11.5 d.p.c. embryo RNA hybridized with cDNA probes detecting Igf2r exons 3–6 or Gapd used as loading control.

Fig. 2. Parental-specific expression in three Igf2r promoter replacement alleles. (A and B) The V1 and V2 alleles have imprinted paternal expression of Air (gray solid bar) and imprinted maternal expression of neomycin (neo, gray dotted bar). RNase protection analysis (RPA) on 16.5 d.p.c. embryonic RNA with probe Airneo that detects the Air RNA at the position of the thymidine kinase promoter–neomycin (TKneo) cassette (A) or with probe tkneo that detects the neomycin RNA produced from the TK promoter (B). The probes Airneo and tkneo do not recognize endogenous Air RNA. Aprt exon 3: RNA loading control. Lane P, input probes; lane C, tRNA hybridization control. (C and D) The Igf2r-neo allele has imprinted paternal expression of Air (gray solid bar) and imprinted maternal expression of neomycin (neo, gray dotted bar). RPA on 11.5 d.p.c. embryo RNA from T^hp/Igf2r-neo reciprocal heterozygous crosses with probe Airneo that detects the Air RNA at the position of the TKneo cassette (C) or with probe tkneo that detects the neomycin RNA produced from the TK promoter (D). Controls as above. (E) The Igf2r-neo allele shows imprinted maternal expression of Slc22a2 and Slc22a3. RNA blot of 11.5 d.p.c. placenta RNA from T^hp/Igf2r-neo reciprocal heterozygous crosses hybridized with a cDNA probes detecting Slc22a2 (top), Slc22a3 (middle) or Gapd (bottom) used as loading control.

Fig. 3. Parental-specific methylation of three Igf2r promoter replacement alleles. (A) Thymidine kinase promoter–neomycin (TKneo) methylation analysis in V1 and V2 alleles. Heterozygous 16.5 d.p.c. embryonic DNA carrying either paternally derived (gray solid bars) or maternally derived (gray dotted bars) V1 and V2 replacement alleles was digested with EcoRI and hybridized with probe tai (Figure 1B). Note that EcoRI is methyl sensitive when G flanks the GAATTC recognition site and the CG dinucleotide is methylated (Ludwig et al., 1996). The V1 allele generates 4.0 kb when the EcoRI is unmethylated and 5.8 kb for a methylated site. V1 paternal transmission (gray solid bar) shows full methylation at this site, and V1 maternal transmission (gray dotted bar) shows partial (∼40%) methylation at this site. The V2 allele yields 4.4 or 6.2 kb for an unmethylated and methylated sites, respectively. V2 paternal transmission (gray solid bar) shows partial (∼80%) methylation at this site, and V2 maternal transmission (gray dotted bar) shows no methylation at this site. The Igf2r wild-type (wt) promoter fragment is 5.0 kb and does not contain a methyl-sensitive EcoRI site. (B) TKneo promoter methylation analysis in the Igf2r-TKneo allele. Heterozygous 13.5 d.p.c. embryonic DNA carrying either a paternally derived (gray solid bar) or maternally derived (gray dotted bar) Igf2r-TKneo replacement allele was digested with BglII (–) or BglII and MluI (+) and hybridized with probe kodel (Figure 1B). Note that, in crosses with the T^hp allele, only bands from the opposite allele are seen. The Igf2r-TKneo allele yields 5.0 or 6.0 kb for an unmethylated and methylated sites, respectively. Igf2r-TKneo paternal transmission (gray solid bar) shows almost complete methylation at this site (a faint 5.0 kb unmethylated can be seen on the original image), and Igf2r-TKneo maternal transmission (gray dotted bar) shows partial (∼50%) methylation at this site. The wild-type Igf2r promoter fragment is 9.5 kb and does not contain an MluI site. (C) Igf2r promoter (NotI) methylation analysis in the wild-type Igf2r promoter. T^hp/+ heterozygous 13.5 d.p.c. embryonic DNA carrying either a paternally derived (gray solid bar) or maternally derived (gray dotted bar) wild-type Igf2r promoter was digested with BglII (–) or BglII and NotI (+) and hybridized with probe kodel (Figure 1B). The wild-type Igf2r promoter yields 5.5 or 9.5 kb for an unmethylated and methylated sites, respectively. Igf2r wild-type promoter paternal transmission (gray solid bar) shows partial (∼50%) methylation at this site, and Igf2r wild-type promoter maternal transmission (gray dotted bar) shows no methylation at this site. The Igf2r-TKneo promoter fragment is 6.0 kb and lacks this NotI site (it was deleted in the targeting event). (D) Air promoter (MluI) methylation analysis in the Igf2r-TKneo and wild-type Igf2r alleles. Heterozygous 13.5 d.p.c. embryonic DNA carrying either a paternally derived (gray solid bar) or maternally derived (gray dotted bar) Igf2r-TKneo or a wild-type Igf2r allele was digested with BglII (–) or BglII and MluI (+) and hybridized with probe msi (Figure 1B). The Air promoter yields 8.2 or 9.7 kb for an unmethylated and methylated sites, respectively. Both the Igf2r-TKneo and wild-type Igf2r alleles show no methylation at this site on paternal transmission (gray solid bar) and full methylation on maternal transmission (gray dotted bar).

Fig. 4. Imprinted expression and methylation in the Igf2r-del allele. (A) Slc22a2 and Slc22a3 imprinted expression from the Igf2r-del allele is maternal specific (gray dotted bars), identical to the wild-type situation. RNA blot of 11.5 d.p.c. placenta RNA from T^hp/Igf2r-del and T^hp/wild-type reciprocal crosses hybridized with cDNA probes detecting Slc22a2, Slc22a3 or Gapd used as loading control. Note that imprinted expression of Slc22a2 and Slc22a3 is restricted to the embryonic placenta (Zwart et al., 2001). (B) Air RNA imprinted expression from the Igf2r-del allele is paternal specific (gray solid bars), identical to the wild-type situation. RNase protection analysis using probe MlMs1 on 11.5 d.p.c. embryo RNA from T^hp/Igf2r-del and from T^hp/wild-type reciprocal crosses. This probe detects multiple fragments (asterisk) as it overlaps multiple Air transcription start sites. The open triangle indicates non-specific protected bands. Controls as in Figure 3. (C) Air promoter methylation on the paternal (gray solid bar) and maternal (gray dotted bar) Igf2r-del allele is identical to the wild-type Air promoter. Heterozygous 13.5 d.p.c. embryonic DNA from T^hp/Igf2r-del and from T^hp/wild-type reciprocal crosses was cut with BglII (–) or BglII and MluI (+) and hybridized with probe msi (Figure 1B).The unmethylated and methylated alleles are 8.2 and 9.7 kb, respectively.