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The chicken ß-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain

Piroska E. Szabó, Shih-Huey E. Tang, Michael R. Reed^*, Francisco J. Silva, Walter M. K. Tsark and Jeffrey R. Mann

Division of Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010-3011, USA
^* Present address: Life Sciences Division, Motorola, 757 South Raymond Avenue, Pasadena, California 91105-3250, USA

View larger version (33K): [in a new window]   Fig. 1. (A) Gene regulation at the Igf2 and H19 loci. (M), Maternal allele; (P), Paternal allele. (B) Nucleotide structure of the 2.4 kb ICR and the 2.4 kb (ChßGI)2. Vertical lines are CpGs or GpCs. CTCF-binding sites are indicated (gray rectangles) and are in the 5'-CCGCnnGGnGGCAG-3' orientation. Sequence of the ICR is as published elsewhere (Ishihara et al., 1998) (GenBank Accession Number, AF049091) and is defined here as a BglII fragment at –2.0 to –4.4 kb RTSS of H19. To obtain the ChßGI sequence, we sequenced the outer 1.2 kb XbaI fragment of plasmid pJC13-1 (Chung et al., 1993). This sequence has been deposited with GenBank (Accession Number, AY040835). (C) Comparison of the present ICR substitution (gray bar) with three independent ICR deletions (open bars): deletion a (Thorvaldsen et al., 1998), deletion b (Drewell et al., 2000) and deletion c (Srivastava et al., 2000). H19 transcription start site (arrow). X, XbaI; B, BamHI; Bg, BglII; H, HindIII; Bs, BspEI; K, KpnI.

View larger version (31K): [in a new window]   Fig. 2. Targeting at the Igf2/H19 ICR. The ICR was replaced by the (ChßGI)2, keeping the CTCF sites in the same orientation. The sequences involved are described in Fig. 1B. BamHI and probe a (Pr.a) blot, one clone shown. All seven clones obtained [EcoRI and probe b (Pr.b) blot] underwent conservative recombination. The neo selection cassette was flanked by loxP sites allowing for excision with Cre.

View larger version (39K): [in a new window]   Fig. 3. Phenotype of 18.5 dpc fetuses inheriting the (ChßGI)2. (A) RT-PCR SNuPE assays. +/–(P) and +/+ lanes; top row is presumptive inactive allele. –(M)/+ lanes (in boxes) (these fetuses were obtained from the reciprocal mating, hence bottom row is presumptive inactive allele). Parental origin of alleles is on the right. Value above each band is the amount of RNA contributed by the presumptive inactive allele as a percent of the total. (B) (a) +/+ fetus; (b) +/–(P) fetus. (C) Northern blots. Values under bands are the mean relative amounts of RNA standardized according to Gapd mRNA. All four Igf2 transcripts were reduced to the same extent in +/–(P) mice (only the largest transcript is shown). Number of embryos used: liver, one in all lanes; kidney, two in +/+ and –(M)/+ lanes, and four in +/–(P) lanes. (D) Northern blots, 11.5 dpc midgestation embryos. One embryo was used for each lane. Other details as in C.

View larger version (39K): [in a new window]   Fig. 4. (ChßGI)2 methylation. (A) Southern blots of tissues of 18.5 dpc fetuses. L, liver; M, carcass (mostly muscle); K, kidney. This assay tests for methylation at the central methylation-sensitive Hpa (HpaII) (CCGG) and Hha (HhaI) (GCGC) sites indicated below the blots (if methylated, the 2.6 kb Eco (EcoRI) and Bam (BamHI) band should remain intact). Msp (MspI) cuts at CCGG, regardless of CpG methylation. The CTCF sites are indicated (vertical arrows). The 2.6 kb Eco, Bam fragment consists of the 2.4 kb (ChßGI)2 plus an additional 0.2 kb of downstream sequence (see Fig. 2). Major bands expected on complete cutting are shown below blots. The complete ChßGI sequence was the probe used. Number of embryos used: +/–(P), one embryo per lane for L and M, and three embryos per lane for K; –(M)/+, one embryo per lane for L and M, and two embryos per lane for K. (B) Purification and methylation state of germ cells of 18.5 dpc +/–(P) fetuses. In the flow cytometry graph, EGFP+ cells above the line were sorted for analysis. Each row of squares represents the methylation state of each of 22 sequential CpGs, 5'3' orientation as in Fig. 1B (white square, unmethylated; black square, methylated). Each row represents a separate sequencing reaction. The CpG of the CTCF site is indicated (arrow).

View larger version (23K): [in a new window]   Fig. 5. Summary of results. In –(M)/+ fetuses (middle), the maternal (ChßGI)2 acts as a chromatin insulator, substituting for this property of the maternal ICR in +/+ fetuses. Allele-specific expression of Igf2 and H19 was unaffected. In +/–(P) fetuses (bottom) the paternal (ChßGI)2 acted as a chromatin insulator as did the maternal (ChßGI)2. Consequently both Igf2 alleles were strongly repressed and total Igf2 RNA was very low. Both H19 promoters had access to the enhancers in cis, and H19 was expressed biallelically. Other details are as described in the legend to Fig. 1.

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