N-CoR Mediates DNA Methylation-Dependent Repression through a Methyl CpG Binding Protein Kaiso  Ho-Geun Yoon, Doug W. Chan, Albert B. Reynolds, Jun Qin, Jiemin Wong  Molecular Cell  Volume 12, Issue 3, Pages 723-734 (September 2003) DOI: 10.1016/j.molcel.2003.08.008

Figure 1 The Identification of Kaiso as a Component of the Purified N-CoR Complex (A) The scheme for purification of N-CoR complex from HeLa nuclear extracts. (B) Immunopurified N-CoR and its associated proteins were resolved by 8% SDS-PAGE and stained with Coomassie blue. Also shown are three peptides identified by mass spectrometry, which match with Kaiso. Three other newly identified proteins are TIF1γ (gi4325109), KIAA0677 (gi7662245), and HsEg5/TRIP5 (gi4758656). (C) IP-Western experiments showing that in HeLa nuclear extracts Kaiso is associated with N-CoR but not SMRT. Purification of the SMRT complex was as described (Li et al., 2000). (D) N-CoR and Kaiso can be mutually depleted from HeLa nuclear extracts using their specific antibodies. For depletion, 100 μl of extracts and 1, 2, and 4 μl of N-CoR or Kaiso antibody were used. (E) Silver staining of immunopurified Kaiso and N-CoR complexes. The proteins verified by Western are indicated, whereas the common bands are marked by asterisks. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 2 Kaiso Interacts with N-CoR through Its POZ Domain (A) In vitro GST pull-down experiments showing that Kaiso binds N-CoR but not SMRT, HDAC3, TBL1, and TBLR1. (B) The binding of N-CoR is mapped to the POZ domain of Kaiso. (C) The Kaiso-interaction domain is mapped to the RD1 region of N-CoR. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 3 The Kaiso-Containing N-CoR Complex Exhibits Methylation-Dependent DNA Binding Activity (A) Both in vitro translated Kaiso and recombinant MeCP2 can bind to the Sm probe in a methylation-dependent manner. (B) Kaiso but not MeCP2 can bind to the methylated CpG1 probe. Note that addition of a Kaiso-specific monoclonal antibody led to a supershift of the DNA-Kaiso complex. (C) HeLa nuclear extracts were used for gel shift assay, and a methyl CpG1 specific complex could be supershifted with antibodies against Kaiso and N-CoR but not SMRT. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 4 Immunopurified N-CoR Complex Possesses Specific Binding Activity to the Methylated CpG1 (A) The panel on the left shows the experimental scheme. The N-CoR complex was precipitated first with a N-CoR-specific antibody. The resulting beads were incubated with the radiolabeled probe, and after washes, the retained probe was gel resolved and detected by autoradiography. (B) Kaiso in HeLa nuclear extracts cofractionates with N-CoR and HDAC3 in a gel filtration analysis using a Superose 6 column. Again, the methyl CpG1 binding activity was examined as in (A). Note that the methyl CpG1 probe binding activity correlates with the presence of the N-CoR complex. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 5 Repression by Kaiso Correlates with Its Interaction with the N-CoR Complex (A) HeLa cells were transfected with indicated expression constructs and a luciferase reporter bearing a TK promoter and four GAL4 binding sites. Note that the repression domain resided in the POZ domain. The structures of different constructs used are shown in the middle panel. Also shown in the lower panel are the levels of protein expression from samples used for luciferase assay. (B) The repression by Kaiso is sensitive to TSA. The concentrations of TSA used were 50, 150, and 300 nM, respectively. (C) ChIP assay revealed recruitment of N-CoR complex by Kaiso. HeLa cells were transfected with Gal-Kaiso or Gal-DBD control, and the luciferase reporter and ChIP assays were as described in Experimetal Procedures. Note that the presence of the N-CoR complex was detected only for Gal-Kaiso in the promoter region (P) but not in the downstream coding region (C). Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 6 The Kaiso/N-CoR Complex Binds to the MTA2 Gene Promoter In Vivo in a Methylation-Dependent Manner (A) A diagram showing the position of the CpG1 sequence in the MTA2 gene. The distance between P1 and P2 region is 3.9 kb. (B) In HeLa cells, the MTA2 gene is repressed in a HDAC- and DNA methylation-dependent manner. (C) Genomic PCR detecting unmethylated (U) and methylated (M) CpG1 sites. (D) ChIP assays revealed the effect of TSA and 5-aza-dC treatment on the binding of the Kaiso/N-CoR complex to the MTA2 promoter. Note that the association of Kaiso and N-CoR was detected only in the promoter region but not in the coding region. (E) The effect of TSA and 5-aza-dC treatment on histone modifications at the MTA2 promoter region. Note that both treatments led to increased histone acetylation and decreased H3 Lys methylation. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)

Figure 7 Knockdown Experiments Using siRNAs Reveal a Causal Role for Kaiso in Targeting the N-CoR Complex for Repression of the MTA2 Gene (A) Western blotting analysis showing the specific effect of individual siRNA in a knockdown of its target protein. Coactivator SRC3 serves as a control. (B) IP-Western revealed the association of TBL1 and HDAC3 with N-CoR in the absence of Kaiso. (C) RT-PCR revlealed elevated expression of MTA2 in HeLa cells treated with siRNA against N-CoR, HDAC3, and Kaiso. (D) ChIP assays were used to analyze the effect of different siRNAs on the binding of the Kaiso/N-CoR complex to the MTA2 promoter. (E) ChIP assays were used to analyze the effect of different siRNAs on histone modifications over the MTA2 promoter region. Note that both HDAC3 and Kaiso were required for maintaining histone hypoacetylation and hypermethylation on H3-K9 over the MTA2 promoter. Molecular Cell 2003 12, 723-734DOI: (10.1016/j.molcel.2003.08.008)