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. 2006 May 15;20(10):1268-82.
doi: 10.1101/gad.1416906.

Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes

Debora Mancini-Dinardo  1 , Scott J S Steele, John M Levorse, Robert S Ingram, Shirley M Tilghman
Affiliations

Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes

Debora Mancini-Dinardo et al. Genes Dev. .

Abstract

The imprinted gene cluster at the telomeric end of mouse chromosome 7 contains a differentially methylated CpG island, KvDMR, that is required for the imprinting of multiple genes, including the genes encoding the maternally expressed placental-specific transcription factor ASCL2, the cyclin-dependent kinase CDKN1C, and the potassium channel KCNQ1. The KvDMR, which maps within intron 10 of Kcnq1, contains the promoter for a paternally expressed, noncoding, antisense transcript, Kcnq1ot1. A 244-base-pair deletion of the promoter on the paternal allele leads to the derepression of all silent genes tested. To distinguish between the loss of silencing as the consequence of the absence of transcription or the transcript itself, we prematurely truncated the Kcnq1ot1 transcript by inserting a transcriptional stop signal downstream of the promoter. We show that the lack of a full-length Kcnq1ot1 transcript on the paternal chromosome leads to the expression of genes that are normally paternally repressed. Finally, we demonstrate that five highly conserved repeats residing at the 5' end of the Kcnq1ot1 transcript are not required for imprinting at this locus.

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Figures

Figure 1.
Figure 1.
Deletion of the KvDMR. (A) The top line represents a physical map of the imprinted domain in mouse distal chromosome 7, with maternally expressed genes represented by red boxes and paternally expressed genes shown as blue boxes. Depicted below is an enlargement of the KvDMR (blue box), illustrating the three DNase I hypersensitivity sites (HS; vertical arrows), the Kcnq1ot1 transcription start site (horizontal arrow), and the five repeats (vertical bars). The location of the KvDMR deletion described by Fitzpatrick et al. (2002) is depicted by the green rectangle. The targeting vector is shown below, in which the neomycin resistance gene (neo) flanked by loxP sites (closed triangles) replaces 3.6 kb of the DMR. The herpes simplex virus thymidine kinase gene (HSV-TK) was included to provide negative selection. The structures of the targeted loci, before and after the deletion of the neo gene, are indicated. (B) Southern blot analysis of DNAs prepared from wild-type (WT), heterozygous DMRΔ +neo, and heterozygous DMRΔ (−neo) mice after digestion with restriction enzymes and hybridization with the probes 1–3 as indicated in the diagrams. The diagrams of the endogenous and targeted loci depict the positions and sizes of the fragments detected by the probes. (C) RNA protection analysis of RNAs isolated from wild type and mice inheriting DMRΔ paternally using probe 3. (P) PstI; (X) XhoI; (H) HindIII; (B) BamHI; (RV) EcoRV; (Bg) BglI.
Figure 2.
Figure 2.
Effect of the KvDMR deletion on imprinting. Allele-specific RT–PCR assays were used to analyze the expression of the genes listed on the left using primers specific for each transcript (Table 2). Analysis was performed on E13.5 placental RNA isolated from offspring of reciprocal crosses between heterozygous DMRΔ and M. spretus mice (M) as well as their wild-type littermates (WT). (MAT) DMRΔ × M. spretus; (PAT) M. spretus × DMRΔ; (B) C57BL/6; (S) M. spretus. The arrows on the right refer to the allele-specific products.
Figure 3.
Figure 3.
Deletion of the Kcnq1ot1 promoter. (A) The endogenous locus is depicted as in Figure 1. The promoter Δ targeting vector contains a 244-bp deletion of the Kcnq1ot1 minimum promoter region and two DNase I hypersensitive sites, as well as the neomycin resistance gene (neo) and the Cre recombinase gene driven by a testes-specific promoter (testes-cre), flanked by loxP sites (open triangles). The loxP site at the 5′ end of the KvDMR (closed triangle) contains point mutations that reduce its recombination frequency with the downstream sites. The positions of the probes used in B (numbered colored boxes) and the fragments that are detected are indicated. (H) HindIII; (R1) EcoRI; (K) KpnI; (F) FspI; (P) PstI; (S) SspI; (RV) EcoRV; (B) BamHI; (N) NheI. (B) Southern blot analysis of DNAs prepared from wild-type (WT) and heterozygous PromΔ +neo mice after digestion with restriction enzymes and hybridization with the external probes as indicated in A. The asterisks denote the presence of additional bands that are the result of methylation of FspI sites. In wild-type cells, a 15.1-kb band derives from methylation of the maternal allele and the 6.7-kb band from the unmethylated paternal allele. In targeted ES cells, the targeted paternal allele produces both a new 9.4-kb band created by a FspI site in the neo gene and an additional 18.8-kb band when that site is methylated. (C) DNA from PromΔ (−neo) and wild-type (WT) mice was amplified by PCR using primers that span the neo and testes cre sequences that yield a 250-bp product derived from amplification of the nontargeted allele and a 400-bp product from the PromΔ (−neo) allele (Table 1). (D) RNA protection of placental RNA from wild-type (WT) and M. spretus × PromΔ mice (PromΔ) was performed using probe C located at the 3′ end of the DNA repeats in KvDMR and an rpL32 ribosomal protein RNA probe as a control. Radiolabeled probes were incubated in the presence (+) and absence (−) of RNase and yeast RNA.
Figure 4.
Figure 4.
Effect of Kcnq1ot1 promoter deletion on imprinting. Allele-specific RT–PCR assays were used to analyze the expression of the genes listed on the left using primers specific for each transcript (Table 2). Analysis was performed on E11.5 placental RNAs and in embryo RNAs (for Cdkn1c), isolated from offspring of reciprocal crosses between heterozygous PromΔ and M. spretus mice (M) as well as their wild-type littermates (WT). (MAT) PromΔ × M. spretus; (PAT) M. spretus × PromΔ; (B) C57BL/6; (S) M. spretus. The arrows on the right refer to the allele-specific products.
Figure 5.
Figure 5.
Termination of the Kcnq1ot1 transcript. (A) The endogenous locus is depicted as in Figure 1. The Term targeting vector contains a poly(A)-based transcriptional stop element (stop sign) that is inserted 1.5 kb downstream of the Kcnq1ot1 transcription start site. The rest of the vector is described in the legend for Figure 3. The positions of the probes used in B and the positions of the restriction fragments that are detected are indicated. (N) NheI; (H) HindIII; (R1) EcoRI; (K) KpnI; (P) PstI; (S) SspI; (RV) EcoRV; (B) BamHI; (Ah) AhdI. (B) Southern blot analysis of DNAs prepared from wild-type (WT) and Term +neo mice after digestion with restriction enzymes and hybridization with the external probes as indicated in A. (C) DNA from Term (−neo) and wild-type (WT) cells was amplified by PCR using primers as described in the legend for Figure 3C. (D,E) RNase protection of E13.5 placental RNAs of wild-type (WT) and heterozygous Term embryos. Probe D spans the integration site of the poly(A) terminator and probe E is derived from downstream of the poly(A) terminator (see diagram). An rpL32 ribosomal protein RNA probe was included as a control. Radiolabeled probes were incubated in the presence (+) and absence (−) of RNase and yeast RNA.
Figure 6.
Figure 6.
Effect of premature termination of the Kcnq1ot1 transcript on imprinting. (A) Allele-specific RT–PCR assays were used to analyze the expression of the genes listed on the left using primers specific for each transcript (Table 2). Analysis was performed on E11.5 placental RNAs and in embryo RNAs (for Cdkn1c), isolated from offspring of reciprocal crosses between heterozygous TermΔ and M. spretus mice (M) as well as their wild-type littermates (WT). (MAT) TermΔ × M. spretus; (PAT) M. spretus × TermΔ; (B) C57BL/6; (S) M. spretus. The arrows on the right refer to the allele-specific products. (B) Allele-specific RNase protection analysis was used to determine the levels of Tssc4 RNA in E11.5 and E13.5 placentae isolated from offspring of heterozygous Term × M. spretus mice (M) as well as their wild-type littermates (WT). An rpL32 ribosomal protein RNA probe was included to control for RNA amount. Radiolabeled probes were incubated in the presence (+) and absence (−) of RNase and yeast RNA. (MAT) TermΔ × M. spretus; (PAT) M. Spretus × TermΔ.
Figure 7.
Figure 7.
Deletion of DNA repeats in the Kcnq1ot1 transcript. (A) The endogenous locus is depicted as in Figure 1. The RepΔ targeting vector contains a 657-bp deletion of five repeats within the Kcnq1ot1 transcript. The rest of the vector is described in the legend for Figure 2. The positions of the probes used in B and the sizes and positions of the restriction fragments that are detected are indicated. (Ap) ApaLI; (H) HindIII; (R1) EcoRI; (K) KpnI; (P) PstI; (Af) AfeI; (S) SspI; (RV) EcoRV; (B) BamHI. (B) Southern blot analysis of DNAs prepared from wild-type (WT) and RepΔ +neo mice after digestion with restriction enzymes and hybridization with the external probes as indicated in A. The asterisk denotes the presence of a 10.4-kb band that results from the methylation of an AfeI site on the untargeted maternal chromosome. (C) DNA from RepΔ (−neo) and wild-type (WT) cells was amplified by PCR using primers as described in the legend for Figure 3C. (D) RNase protection of placental (P) and embryo (E) RNAs of wild-type (WT) and RepΔ embryos in which the RepΔ is inherited either maternally (MAT) or paternally (PAT). Probe C spans the 3′ end of the repeat region that is deleted. An rpL32 ribosomal protein RNA probe was included to control for RNA quantity. Radiolabeled probes were incubated in the presence (+) and absence (−) of RNase and yeast RNA.
Figure 8.
Figure 8.
Effect of deletion of DNA repeats in the Kcnq1ot1 transcript on imprinting. Allele-specific RT–PCR assays were used to analyze the expression of the genes listed on the left using primers specific for each transcript (Table 2). Analysis was performed on E11.5 placental RNA and in embryo RNAs (for Cdkn1c), isolated from offspring of reciprocal crosses between heterozygous RepΔ and M. spretus mice (M) as well as their wild-type littermates (WT). (MAT) RepΔ × M. spretus; (PAT) M. spretus × RepΔ; (B) C57BL/6; (S) M. spretus. The arrows on the right refer to the allele-specific products.
Figure 9.
Figure 9.
CpG methylation analysis at KvDMR. A 537-bp region spanning the Kcnqt1ot1 promoter, which includes 23 CpG dinucleotides, was amplified from E11.5 and E13.5 embryos and yolk sac following treatment with sodium bisulfite. The asterisk refers to a polymorphism between M. spretus and C57BL/6 that was used to distinguish the parental alleles. DNA from M. spretus × Term embryos (A) and M. spretus × RepΔ embryos (B). Black ovals indicate methylated CpGs, open ovals indicate unmethylated CpGs, and gray ovals represent unreadable sequence.
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