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. 2008 Oct;28(20):6473-82.
doi: 10.1128/MCB.00204-08. Epub 2008 Jul 28.

CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop

Tao Li  1 , Ji-Fan Hu, Xinwen Qiu, Jianqun Ling, Huiling Chen, Shukui Wang, Aiju Hou, Thanh H Vu, Andrew R Hoffman
Affiliations

CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop

Tao Li et al. Mol Cell Biol. 2008 Oct.

Abstract

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.

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Figures

FIG. 1.
FIG. 1.
Intrachromosomal interaction between the ICR and Igf2 promoters. (A) Schematic presentation of Igf2, H19, DMRs, and EcoRI sites used for 3C assay. The orientation and location of the 3C primers are shown by arrows under each EcoRI restriction site. (B and C) Ligated 3C products between the ICR (EcoRI sites 5 and 6) and EcoRI sites 1 to 4 located up- or downstream of the Igf2 promoters. Chromatin was fixed with formaldehyde, digested with the restriction enzyme EcoRI, and ligated with T4 DNA ligase. The ligated DNA was amplified by PCR with primers covering two EcoRI sites (5 and 6) in the ICR and four EcoRI sites (1 to 4) near the Igf2 promoters. Allele-specific intrachromosomal looping was distinguished by the use of two restriction enzyme polymorphisms (HpaII and DpnII) in the ICR. The ligated intrachromosomal DNA was amplified with the primers in the same orientation to reduce the background. b, bases.
FIG. 2.
FIG. 2.
Allele-specific ChIP assay across DMR0, DMR1, and the promoter region of Igf2. (A, top) Scheme of the Igf2/H19 imprinting domain. The exons are depicted as solid boxes. DMRs are shown as underlines, and the polymorphic restriction enzyme sites are shown as vertical arrows. The allelic interaction of CTCF with the Igf2 promoters (P0 to P3) and DMRs was identified by using four polymorphic restriction enzymes (Dde1, Hpa2, Ava1, and Msp1) that distinguish M. spretus from C57BL/c. (A, bottom) ChIP of CTCF, Suz12, and dimethylated H3-K27 (mK27) in F1 mouse fibroblasts derived from breeding M. spretus males with C57BL/c females. Cross-linked DNA-protein complexes were immunoprecipitated with antisera against CTCF, Suz12, and dimethyl-H3-K27 (mK27), followed by PCR amplification with specific primers for the DMR0, DMR1, and Igf2 promoters (P1 to P3). Allelic ChIP products were distinguished by polymorphic restriction enzymes (RE). N-Ct lane, negative control (no antibody); input lane, genomic DNA collected before antibody precipitation (positive control). The M/P ratio is the ratio of the maternal to the paternal alleles after normalization with the input DNA. (B) ChIP of CTCF, Suz12, and dimethylated H3-K27 in human fibroblasts. Alleles are labeled A and B because the parental alleles are not known. (C) DNA methylation of CpG dinucleotides at the Igf2 promoters. After sodium bisulfite treatment, genomic DNA fragments were amplified with primers 4875 and 4876 for promoter P2 and primers 4878 and 4879 for promoter P3. Each line represents a single sequenced PCR molecule. Black circles represent methylated CpG dinucleotides, and open circles represent unmethylated CpG dinucleotides. Note the DNA hypomethylation in both promoters. (D) Oligonucleotide pull-down assay for CTCF and Suz12 with Igf2 promoter P2 and P3 DNA fragments labeled with biotin-streptavidin. Wild-type (−) and methylated (+) DNA fragments were end labeled with biotin and incubated with nuclear extracts. After incubation, DNA fragments were pulled down with streptavidin beads. Proteins bound to the DNA fragments were eluted and detected by Western blotting with antibodies directed against CTCF and Suz12. (Input) Aliquots of nuclear proteins, collected before oligonucleotide pull down, were analyzed in parallel with the samples in lanes 1 to 4 and detected by Western blotting. b, bases.
FIG. 3.
FIG. 3.
(A) Interaction between CTCF and Suz12 as measured by co-IP and Western immunoblot assays. Nuclear proteins were immunoprecipitated, respectively, with anti-CTCF and anti-Suz12 antibodies and washed with detergent buffers. After separation on a 7.5% SDS-polyacrylamide gel, immunoprecipitates were immunodetected with appropriate antibodies to CTCF and Suz12. LD, low-detergent buffer; MD, medium-detergent buffer; HD, high-detergent buffer; Ct, negative IP control with no antibody. (Input) Aliquots of nuclear proteins, collected before CTCF and Suz12 IP, were analyzed in parallel with the samples in lanes 1 to 8 and detected by Western blotting. (B) Oligonucleotide pull down of CTCF and Suz12. At the top are the sequences of the wild-type (Wt) and mutated (Mut) CTCF-binding sites in synthesized oligonucleotide fragments. Consensus CTCF-binding sites are in bold and are partially replaced by ATATAT in mutated oligonucleotides. (Input) Aliquots of nuclear proteins, collected before CTCF oligonucleotide pull down, were analyzed in parallel with the samples in lanes 1 to 6 and detected by Western blotting. At the bottom is Western blotting of CTCF and Suz12 in nuclear proteins pulled down by wild-type (−), methylated (+), and mutated (+) CTCF oligonucleotide fragments. DNA fragments were end labeled with biotin and incubated with nuclear extracts. After incubation, DNA fragments were pulled down with streptavidin beads. Proteins bound to the DNA fragments were eluted and detected with CTCF and Suz12 antibodies. (C) Two-hybrid interactions between CTCF and Suz12. Lane 1 shows background expression of firefly luciferase from the pG5luc vector as determined by cotransfection with the pACT and pBIND vectors, which did not contain CTCF or Suz12, into HBF1 human fibroblast cells. Lanes 2 and 3 show two controls used to determine the background activity of individual CTCF or Suz12 (no interaction). In lanes 4 and 5, The reporter vector was cotransfected with CTCF and SUZ12 in fusion with the VP16 transcription activation domain (pACT constructs) or the GAL4 DNA-binding domain (pBIND constructs). In lane 6, pBIND-Id and pACT-MyoD were used as the positive control encoding two proteins known to interact in vivo. Luciferase activity was measured as relative luminescence units (RLU). *, P < 0.01 compared with the three control groups. The values shown are averages ± standard deviations (n = 6). (D) In vitro binding assay with recombinant proteins. Lanes 1 to 3 show the CTCF-and-Suz12 interaction; lanes 4 to 6 show the CTCF-and-CBX2 interaction. Input, reaction mixture aliquot collected before particle pull down and analyzed in parallel with the samples (CTCF-GST and BSA) by Western blotting. NC, negative control with an equal amount of BSA.
FIG. 4.
FIG. 4.
ChIP of CTCF, Suz12, and methylated H3-K27 in transgenic mouse skin tissues. (A) ICR deletion model. Mouse fibroblasts, kindly provided by M. S. Bartolomei, were cultured from neonates carrying a 3.8-kb deletion of the ICR (35). These mice were generated by reciprocal crosses of C57BL/6(CAST) with F1 ICR heterozygotes maintained in a C57BL/6 background. Heterozygous fetuses inherit either a maternal [−(M)/+] or a paternal [+/−(P)] ICR deletion. Allelic ChIP products were distinguished by polymorphic restriction enzymes Cac81 and Mwo1. N-Ct, negative control (no antibody). b, bases. (B) ICR mutation model. Fetal liver tissues, kindly provided by P. E. Szabo, were derived by breeding male FVB/NJ.CAST/Ei(N7) and female 129SI/ImJ mice to produce F1 mice that are heterozygous for a mutation in the ICR (34). Wild-type (+/+) mice carry both alleles from strain CAST/Ei. −(M)/+ mice carry the maternally inherited mutated ICR from strain 129SI/ImJ (129) and the paternally inherited ICR from strain CAST/Ei. Since the wild-type mice are homozygous, the parental alleles cannot be distinguished. N-cont., negative control (no antibody). b, bases.
FIG. 5.
FIG. 5.
Loss of Igf2 imprinting induced by RNAi knockdown of Suz12. (A) Quantitation of Suz12 mRNA by RT-PCR in Suz12 knockdown cells. The T/C ratio is the ratio of Suz12 mRNA in RNAi-treated cells to that in control cells (N-Ct). (B) Igf2 imprinting in Suz12 knockdown cells with polymorphic restriction enzyme DpnII. The M/P ratio is the ratio of Igf2 mRNA from the maternal allele to that from the paternal allele. (C) Measurement of β-actin mRNA serves as the PCR control. Lanes 1 and 2, stealth RNAi control; lanes 3 to 8, RNAi-treated cells; lane 9, 100-bp DNA molecular size marker. Three Suz12 RNAi duplex oligonucleotides that target distinct locations of Suz12 were separately transfected into MBW2 cells that maintain normal Igf2 imprinting (6). As the Suz12 RNAi oligonucleotides are rapidly degraded and the preexisting Suz12 protein has a relatively long half-life, we transfected each group of MBW2 cells three times with Suz12 RNAi oligonucleotides. RNAi-1 to -3 are three individual stable clones with duplicated RT-PCR measurements. (D) Suz12 Western blotting in RNAi knockdown fibroblasts. Suz12 was knocked down by three different RNAi oligonucleotides, and equal amounts of proteins were used for detection by Western blotting with β-actin as the internal control. (E) H3-K27 ChIP assay of Igf2 promoters P2 and P3 in Suz12 knockdown fibroblasts. Experimental conditions are the same as in Fig. 2A. N-Ct., control without Suz12 RNAi. Spr., M. spretus; b, bases.
FIG. 6.
FIG. 6.
Simplified model of regulation of Igf2 imprinting by the CTCF-mediated PRC2-Igf2 promoter suppressive complex. CTCF binds to the unmethylated ICR and the Igf2 promoters on the maternal allele and forms a long-distance intrachromosomal loop through CTCF dimerization. CTCF recruits PRC2 via Suz12, resulting in methylated H3-K27 and inactive chromatin around the Igf2 promoters. On the paternal allele, the methylated ICR does not bind CTCF and the CTCF-PRC2 suppression complex cannot be formed, resulting in unmethylated H3-K27 in the active Igf2 promoters. Similarly, CTCF-mediated ICR-promoter looping is abolished when the CTCF binding site in the ICR is deleted or mutated. Loss of H3-K9 methylation at the maternal promoters leads to loss of Igf2 imprinting.
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