1. CTCF-Dependent Chromatin Insulator Is Linked to Epigenetic Remodeling
Ko Ishihara, Mitsuo Oshimura, Mitsuyoshi Nakao 
Molecular Cell 
Volume 23, Issue 5, Pages (September 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Interaction of CTCF with CHD8
(A) Structures of CTCF and CHD8. CTCF consists of amino-terminal (N), zinc finger, and carboxyl-terminal (C) domains. Yeast two-hybrid screening using the zinc finger domain (amino acids 268–577) as a bait identified CHD8 (amino acids 2240–2582). CHD8 is characterized by two chromodomains, helicase/ATPase, and two BRK domains.
(B) Direct binding between CTCF and CHD8 in vitro. Bacterially expressed, GST-fused N domain (CN), zinc finger domain (CZ), and C domain (CC) of CTCF were immobilized on glutathione-agarose beads and incubated with His-tagged carboxyl-terminal region of CHD8. The input represents 10% of the protein in the reaction mixture.
(C) Specific interaction between the zinc finger domain of CTCF and the carboxyl-terminal region of CHD8 in yeast two-hybrid assay. Yeast strain AH109 was transformed by GAL4 binding domain-fused CN, CZ, or CC of CTCF, together with GAL4 activation domain-fused carboxyl terminus of CHD8 that had been identified by our original screening. Yeast colonies were grown on a plate lacking tryptophan and leucine (SD, −Trp/−Leu) as transformation controls and on a selection plate further lacking histidine and adenine (SD, −Trp/−Leu/−His/−Ade) as a result of the two-hybrid interaction.
(D) Association of CHD8 with cancer-specific missense mutations of CTCF. The zinc finger mutations R339W and R448Q in Wilms' tumor, K344E in breast cancer, and H345R in prostate cancer are previously reported (Filippova et al., 2002). GST pull-down analysis shows that these mutations do not affect the interaction between CTCF and CHD8.
(E) Complex formation of endogenous CTCF and CHD8 in HeLa cells. CTCF was present in the immunoprecipitates with CHD8 but not in the species-matched control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 Existence of CHD8 at Known CTCF Binding Sites in Chromatin
(A) Chromatin immunoprecipitation (IP). Crosslinked DNA-protein complexes were immunoprecipitated with anti-CHD8, anti-CTCF, and control antibodies, followed by PCR amplification with specific primers for H19, BRCA1, c-myc, and β-globin gene regions (as shown by red bars in [B]). Genomic DNA in the input cell lysates was used as a positive control. The use of no antibody control was indicated with a minus (−).
(B) Schematic representation of known CTCF binding sites. The DMR insulator of the H19 gene on the maternal allele prevents the downstream enhancer from acting on genes upstream of DMR. The upstream regions of BRCA1 and c-myc genes have CTCF binding sites that locate at the boundaries between active and inactive chromatin. The 5′HS5 site in the β-globin locus control region blocks an interaction between the enhancers and neighboring genes. As a negative control, the region for exon 4/5 of the H19 gene has no CTCF binding sites. CTCF (or CTCF binding sites), enhancer elements, and PCR-amplified regions are shown by green and orange circles and red bars, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Involvement of CHD8 in CTCF-Dependent Enhancer-Blocking Activity of the H19 DMR Insulator
(A) Enhancer-blocking assays. The indicated reporter constructs were introduced into HeLa cells for testing luciferase activities led by enhancer-promoter interaction. Luciferase activities from pIHLE were normalized to 100. Luciferase, luciferase gene; P, mouse H19 promoter; Enh, SV40 enhancer; Ins, H19 DMR insulator; and Mut, H19 DMR insulator with mutated CTCF binding sites.
(B) RNAi-mediated CTCF knockdown (K.D.). Western blot analysis was carried out using nuclear extracts from HeLa cells carrying CTCF-targeted shRNA expression and control vectors. Lamin A and CHD8 are shown as a control. The relative amounts of proteins are indicated.
(C) The effect of CTCF knockdown on enhancer-blocking activity. CTCF-knockdown and control cells were transfected with pIHLIE and pIHLME (shown in [A]). Luciferase activities from pIHLME were separately normalized to 100 in each cell type.
(D) Tetracycline-inducible CHD8 knockdown. CHD8-targeted shRNA expression vector containing the tetracycline operator sequence was stably introduced into HeLa cells, together with the tetracycline repressor expression vector. Western blot analysis was performed using stable transfectants that were cultured for 4 days with or without tetracycline (K.D. or control cells, respectively).
(E) Effect of CHD8 knockdown on enhancer-blocking activity of CTCF-dependent insulator. Tetracycline-inducible CHD8 knockdown cells were stably transfected with the indicated reporter constructs and cultured for 10 days with or without tetracycline. Luciferase activities in control cells were normalized to 100 within each use of the reporter constructs. Values are given as means and standard deviations of results from more than three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 Binding of CTCF and CHD8 to H19 DMR Insulator
(A) The pIHLIE reporter construct. PCR-amplified regions are shown.
(B) Binding of CHD8 to the insulator in CTCF knockdown cells.
(C) Binding of CTCF to the insulator in CHD8 knockdown cells. HeLa cells were transfected with the shRNA expression vector and selected with puromycin for 48 hr. The pIHLIE was then introduced into the control and knockdown cells for 48 hr. The cells were subjected to chromatin immunoprecipitation with indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5
CHD8 Is Required for CTCF-Dependent Insulator and Imprinted Expression
(A) Model of IGF2/H19 imprinting dependent on the DMR insulator. Unmethylated DMR on the maternal allele is bound by CTCF-CHD8 complex (green and blue) to prevent the shared enhancer from interacting with IGF2. Methylated DMR on the paternal allele abrogates CTCF binding, allowing interaction between the enhancer and IGF2 promoter. Due to the enhancer-blocking activity of the DMR insulator, IGF2 is expressed exclusively from the paternal allele.
(B) RNAi-mediated CHD8 knockdown in mouse hybrid cells carrying a human chromosome 11 of maternal origin (A911M). ShRNA expression vectors targeting CHD8 and mock control were stably transfected into A911M cells. Western blot analysis was carried out using stable transformants carrying the CHD8 knockdown (C1–C4) and mock (V1–V4) vectors.
(C) Expression analysis of IGF2/H19. Total RNA from the stable transformants (V1, V3, C1, and C3) was isolated for RT-PCR of human IGF2/H19 transcripts. IGF2 as well as H19 was activated in CHD8 knockdown A911M cells. Mouse hybrid cells carrying human chromosome 11 of paternal origin (A911P) were used as a control.
(D) Chromatin immunoprecipitation analysis in control (V3) and CHD8 knockdown (C3) cells. Formaldehyde crosslinked chromatin was immunoprecipitated with anti-CTCF antibodies. PCR amplification was done as shown in Figure 2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6
CHD8 Participates in Epigenetic Modifications at CTCF Binding Sites Adjacent to Heterochromatin
(A) DNA methylation assay in c-myc insulator region. Tetracycline-inducible CHD8 knockdown HeLa cells were cultured for 10 days with or without tetracycline (+ or −, respectively). Genomic DNA was isolated, digested with methylation-sensitive enzyme (AciI), and PCR amplified using specific primers for c-myc locus (CM1–CM4). The appearance of amplified fragments indicated the gain of DNA methylation in the region tested in the CHD8 knockdown cells.
(B) A restriction map of the upstream region of the c-myc gene. The open box shows the CTCF binding site. EcoRI digestion outside the map was used for a control.
(C) Altered DNA methylation in the CTCF sites (BR1 and BR2) of BRCA1 promoter region under CHD8 knockdown. Genomic DNA was digested with methylation-sensitive enzymes (HpaII or AciI) and was used for PCR amplification.
(D) Restriction map of the upstream region of the BRCA1 gene.
(E) Chromatin immunoprecipitation using anti-acetyl histone H3 antibodies. Acetylated H3 was present in the CTCF site (CM2) of the c-myc insulator in control cells (Tet−), but not in CHD8 knockdown cells (Tet+). The region for exon 4/5 of the H19 gene did not change the acetylation status.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions