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. 2008 Jun;22(6):1476-88.
doi: 10.1210/me.2007-0474. Epub 2008 Mar 20.

A complex deoxyribonucleic acid looping configuration associated with the silencing of the maternal Igf2 allele

Xinwen Qiu  1 , Thanh H Vu, Qiucheng Lu, Jian Qun Ling, Tao Li, Aiju Hou, Shu Kui Wang, Hui Ling Chen, Ji Fan Hu, Andrew R Hoffman
Affiliations

A complex deoxyribonucleic acid looping configuration associated with the silencing of the maternal Igf2 allele

Xinwen Qiu et al. Mol Endocrinol. 2008 Jun.

Abstract

Alternate interactions between the H19 imprinting control region (ICR) and one of the two Igf2 differentially methylated regions has been proposed as a model regulating the reciprocal imprinting of Igf2 and H19. To study the conformation of this imprint switch, we performed a systematic structural analysis across the 140 kb of the mouse Igf2-H19 region, which includes enhancers located both between the two genes as well as downstream of H19, by using a scanning chromosome conformation capture (3C) technique. Our results suggest that on the active paternal Igf2 allele, the various enhancers have direct access to the Igf2 promoters, whereas the imprinted silent maternal Igf2 allele assumes a complex three-dimensional knotted loop that keeps the enhancers away from the Igf2 promoters and allows them to interact with the H19 promoter. This complex DNA looping of the maternal allele is formed by interactions involving differentially methylated region 1, the ICR, and enhancers. Binding of CTC-binding factor to the maternal, unmethylated ICR in conjunction with the presence of multicomplex components including interchromosomal interactions, create a barrier blocking the access of all enhancers to Igf2, thereby silencing the maternal Igf2. This silencing configuration exists in newborn liver, mouse embryonic fibroblast, and embryonic stem cells and persists during mitosis, conferring a mechanism for epigenetic memory.

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Figures

Figure 1
Figure 1
Expression of Igf2 and H19 A, Allelic expression in newborn liver (Liver NB), liver 21 d after birth (Liver 21d), SF1-G, and MEF. Igf2 is expressed from the paternal allele (Pat, 84 bp), whereas H19 is expressed from the maternal allele (Mat, 199bp). Controls (Maternal, Paternal) were from genomic DNAs. B, Igf2 expression by standard RT-PCR. M, 100- and 10-bp markers. C, Relative mRNA levels by real-time Q-PCR. The steady-state mRNA levels were calculated with reference to β-actin control, relative to ES cells (SF1-G), which were set at 1.0. Error bars represent sem.
Figure 2
Figure 2
3C Analysis of Intrachromosomal Interactions across 140-kb Igf2/H19 Region in Mitotic MEF Cells A, Map of Igf2/H19 region showing location of 24 target RI sites (a, b, 1–16, 6x, 7x, 9x, 10, and 11x) and the relative position of DMRs, MAR3, ICR, and intergenic (Ig), endoderm (Endo), and mesoderm (Meso) enhancers (Enh). One-directional, forward primers (arrows) that were identical in both M. musculus and M. spretus mouse strains were used in the 3C analysis. Each target primer (E #1 through E #16) was analyzed against one of the 24 forward primer sets. B, 3C interactions in mitotic MEF. Cells were treated with 1 mm nocodazole for 14 h. Each panel from top (E #1) to bottom (E #14) depicts 3C products across the 24 RI locations (columns a, b, and 1–16). In each column, two primers consisting of a target primer (vertical arrow) and a specified primer (marked by the column number) were used to amplify 3C cross-linked products. 3C products were sized on a polyacrylamide-urea gel. The horizontal line across the panel signifies 200 bp. The vertical line indicates alignment at DMR1, intergenic enhancer, and ICR. M at the top indicates size marker. M, Maternal; P, paternal.
Figure 3
Figure 3
Local and Long-Range Interaction in Interphase MEF Cells A, 3C interaction across the140-kb Igf2/H19 region in MEF cells. Each panel depicts 3C products across the 24 RI locations. In each column, a target primer (vertical arrow) and a specified primer (column number) were used to amplify 3C products. Further details are described in Fig. 1B. B, Parental allele-specific pattern of 3C interaction in MEF between DMR1 RI #3 (panel E #3, StuI digestion) and the 24 RI sites (top panel) and the interaction between ICR Eco #12A (panel E #12A, HpaII digestion) and the 24 sites (bottom panel). PCR products were labeled at the last PCR cycle (hot-stop protocol). A control L7 template added before digestion indicates completion of the restriction digest (arrow). M at the top indicates size marker. M, Maternal; P, paternal.
Figure 4
Figure 4
Local and Long-Range Interaction in Newborn Liver A, 3C interaction across the140-kb Igf2/H19 region in newborn liver. Details are in Fig. 2A. B, Parental allele-specific pattern of 3C interaction in newborn liver between DMR1 RI #3 (panel E #3, StuI digestion) and the 24 RI sites (top panel) and the interaction between ICR Eco #12A (panel E #12A, HpaII digestion) and the 24 sites (bottom panel). PCR products were labeled at the last PCR cycle (hot-stop protocol). A control L7 template indicates complete digestion (arrow). M at the top indicates size marker. M, Maternal; P, paternal.
Figure 5
Figure 5
Local and Long-Range Interaction in ES Cells A, 3C analysis across 140-kb Igf2/H19 region in ES cells (SF1-G). Details are in Fig. 2A. B, Parental allele-specific pattern of 3C interaction in normal SF1-G between DMR1 RI #3 (E #3, StuI digestion) and the 24 RI sites (top panel) and the interaction between ICR Eco #12A (HpaII digestion) across the 140-kb region (bottom panel). Hot-stop PCR was employed. A control L7 template added before digestion indicates completion of the restriction digest. Note that the intensity of the 3C product in Fig. 5B, E #3 (column 3, lower panel) was weaker than expected. This may be attributable to variation in the handling/loading of this sample. See explanation in supplemental Note N7. C, Parental allele-specific pattern of 3C interaction in mitotic SF1-G. The ES cells were treated with 1 mm nocodazole for 14 h as shown in Fig. 2B. Analysis of allele-specific 3C interaction is detailed in Fig. 4B. A control L7 template indicates complete digestion. M at the top indicates size marker. M, Maternal; P, paternal.
Figure 6
Figure 6
Interaction of Enhancers and Igf2 Promoter in Newborn Liver A, 3C analysis was performed using a reverse primer (Eco 14R) and one of each of 32 forward primers across the Igf2/H19 region. Note that the 32 sites include the 24 sites of the Igf2/H19 region in Figs. 1–4 and as well as four upstream and four downstream sites. Cross-linked liver DNA (50 ng) was amplified for 36 cycles. Major 3C products of correct size were observed in the 140-kb region (columns a through 30). A correct PCR product from Eco 14 forward/reverse primers was observed at column 14. B, Digestion with KpnI was used to identify the two parental alleles. Paternal allele (M. spretus) has the KpnI restriction site. All 3C products derived from the paternal chromosome contain a common KpnI fragment of 105 and 101 bases (two strands of DNA, seen as a doublet). M at the top indicates size marker. M, Maternal; P, paternal.
Figure 7
Figure 7
Knot Model of the Inactive Igf2 Allele A, A complex looping configuration organized by a CTCF complex binding at the unmethylated ICR. Two internal loops formed by three-point interaction (ICR-intergenic enhancer-H19 enhancer) are superimposed on a larger loop that brings the ICR-CTCF-enhancers complex close to the Igf2 DMR1 region. Relative location of Insulin and Igf2 promoters (P0, P1–P3), and location of the RI sites (a, b, and 1–16) are shown. The intergenic enhancer and H19 downstream enhancers (endoderm and mesoderm) are marked. Active H19 transcription is shown as an arrow. B, Side view of the knot model. The triple loops bring all enhancers (intergenic, endoderm, and mesoderm) to one side and the Igf2 promoter region to the other side of the ICR-CTCF complex. Further modification, interacting with other protein complexes (such as CHD8, EZH2, and SUZ12; see text) and other interchromosomal regions, may create a huge multicomplex (shown as a blocking wall) that covers the entire Igf2 12-kb promoter region, which sequesters all the enhancers preventing direct Igf2 activation. Endo, Endoderm; Ig, intergenic; Meso, mesoderm.
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