An upstream repressor element plays a role in Igf2 imprinting

Abstract

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.

Fig. 1. Igf2 allelic methylation and histone acetylation patterns. (A) Map of the mouse Igf2 gene indicating the promoters (P0–P3), the differentially methylated (DMR1) region including HpaII sites 2–5, restriction sites for E (EcoRI), X (XbaI), N (NgoMI; NgoMIV), D (DraI; only present on the M.spretus allele), the EcoRI–XbaI probe and the position of the PCR fragment used in the immunoprecipitation experiments. (B) Igf2 differential methylation at site 4 was measured by Southern blot analysis of kidney genomic DNA from SD7 hetero zygous mice using the restriction enzymes shown in each lane. After digestion with NgoMI, both the domesticus allele (3 kb) and the spretus allele (1.3 kb) yield a 1.25 kb band. Similar results were obtained using DNA from liver but, in this case, the paternal allele was only 70% methylated. (C) The acetylation state of nucleosomes in DMR1 was determined by immunoprecipitation of mononucleosomes from SD7 heterozygous mice using anti-acetylated H4 histone. DNA from input and bound fractions was extracted and 1 or 3 µl subjected to quantitative radiolabeled PCR with primers specific for sequences from the κ-light chain, β-actin and Igf2 upstream regions (left panel). The Igf2 PCR product was digested with HpaII to identify a polymorphic site on this fragment, present only on the spretus (paternal) allele, analyzed by gel electrophoresis (right panel) and the results quantitated using a phosphoimager. The ratio between the maternal and paternal alleles in the bound fraction was found to be 5.5-fold more than that measured for the input DNA. In a separate experiment, we also determined that the PCR product obtained from total DNA is produced equally from the two alleles (data not shown).

Fig. 2. DMR1 contains a methylation-sensitive repressor element. Transient transfection into 293 human kidney cells was used to measure repressor activity on a pTK-CAT reporter construct. (A) Map showing the different constructs used in the transient transfection experiments. (B) CAT assays of reporter constructs. The results are shown in graphic form relative to the basic TK-CAT vector. These values were determined by scanning autoradiograms. For each lane, the particular construct used is indicated by a modular diagram made up of fragments from DMR1, the TK promoter and the CAT reporter gene. The last construct (disruption) is the same as the 340 bp fragment but contains a loxP insertion (see text). Constructs were either unmethylated (–) or in vitro methylated by MHpaII (+). The HpaII sites (2–5) located within each fragment are indicated in the diagram. Most of these experiments were repeated 4–5 times and the results shown represent average values (± SD).

Fig. 3. Repression is mediated by protein interactions with DMR1. (A) Co-transfection competition experiments were carried out using a reporter construct containing a 680 bp fragment from DMR1 (see Figure 2A) and competitor plasmid DNA containing only the 680 bp fragment (10- or 20-fold excess in methylated or unmethylated form), and the results (average ± SD) are shown as in Figure 2. The pTk-CAT vector was unaffected by the addition of either unmethylated or methylated competitor DNA (data not shown). (B) Gel shift analysis of a ³²P-end-labeled Igf2 upstream oligonucleotide using 293 cell nuclear extract. Competition (left) was carried out using a 45-fold excess of non-radioactive wild-type (wt), methylated or disrupted oligonucleotides (see Materials and methods). Gel shift analysis was also carried out (middle) with the same extract treated with two different concentrations of GCF2 antibody to bring about immunodepletion. In a separate experiment, we removed the antibody complex from the Sepharose beads and confirmed by western analysis that it contained GCF2. A mock experiment in which the extract was passed over protein A–Sepharose beads without antibody is also shown. Extract or pure recombinant GCF2 protein was reacted (right) with either the labeled Igf2 upstream oligonucleotide (I) or an oligonucleotide containing the EGFR recognition sequence (E). A small amount of immunodepleted extract was added to the pure protein in order to stabilize it and enhance binding.

Fig. 4. Genomic targeting of a loxP element into DMR1 HpaII site 4. (A) The targeting construct. A 5 kb BamHI fragment containing DMR1 was subcloned from a genomic library and a loxP-Neo^r-loxP cassette (bottom line) was then inserted into HpaII site 4, which resides within a GCF2 consensus sequence (see Kageyama and Pastan, 1989; Reed et al., 1998). (B) Southern blot analysis of genomic DNA from wild-type (WT), targeted and cre-deleted ES cells restricted with BglII and probed with the fragments indicated in the map. Only data from the left hand probe are shown in the blot. The targeted allele appears under-represented in this figure because of the presence of DNA from accompanying feeder cells. The nature of the disruption was verified by amplifying and sequencing this region from the cre-deleted mice.

Fig. 5. Analysis of Igf2 maternal allele expression in targeted mice. (A) RT–PCR on total RNA extracted from 8–10 whole 12.5 d.p.c. embryos, which were first genotyped by PCR in order to identify and group the R⁺/Igf2^– and R^–/Igf2^– individuals. PCR was carried out on 1, 3 or 9 µl of cDNA in the presence of [α-³²P]dCTP, and the products were run on a 5% acrylamide gel. PCR for β-actin cDNA was carried out in the same tube and served as a control. Igf2 transcription in R^– embryos was 2.9-fold higher than in R⁺ embryos as determined by phosphoimager analysis, after normalization for β-actin. Similar results were also obtained using pooled embryos from an additional inde pendent targeted mouse line. We also carried out expression analysis on individual embryos. In this case, the level of Igf2 mRNA was normalized against β-actin, and the average relative amount of Igf2 in the R⁺ embryos set at 1 (R⁺, 1.0 ± 0.2, n = 5; R^–, 2.8 ± 0.6, n = 6, P <0.001). (B) RT–PCR was carried out on total RNA from 8–10 whole 12.5 d.p.c. embryos obtained from crosses between M.musculus mice carrying one targeted Igf2 allele (R^–) and homozygous SD7 mice (R⁺). The PCR product was then cut with BsaAI, which recognizes a polymorphic site present only in the M.spretus allele, and was run on a 7% acrylamide gel. In the left panel, the R^– allele is derived from the mother while, in the right panel, this allele is paternally derived. The R^– maternal allele is 2.8-fold more active than the R⁺ allele when compared with the spretus paternal allele, as determined by phosphoimager analysis.

Fig. 6. Model of the Igf2 imprinting mechanism. The diagram shows the maternal and paternal Igf2/H19 alleles with the DMR1 methylated and packaged into a closed chromatin structure containing deacetylated histones on the paternal copy, while the DMR1 on the maternal copy is unmethylated and packaged into a more open structure containing acetylated histones. Enhancer sequences (E) are located 3′ to the H19 gene. On the maternal allele, these sequences activate H19, and Igf2 activity is repressed by elements in DMR1 that bind the GCF2 protein. On the paternal allele, DMR1-mediated repression is abrogated either because enhancer sequences overcome this inhibitory element or because of a closed methylated chromatin structure.