N-CoR Mediates DNA Methylation-Dependent Repression through a Methyl CpG Binding Protein Kaiso

Abstract

The identification and characterization of molecular mechanisms utilized by cells to mediate transcriptional repression at methylated loci are fundamental to understanding the biological consequences of DNA methylation. Here we demonstrate that Kaiso, a methyl CpG binding protein belonging to the BTB/POZ family of transcription factors, is a component of the human N-CoR complex. In vitro, the Kaiso/N-CoR complex binds specific CpG-rich sequences in a methylation-dependent manner. In vivo, Kaiso targets the N-CoR complex to the MTA2 gene promoter in a methylation-dependent manner. Importantly, we demonstrate that Kaiso is required for transcriptional repression of the methylated MTA2 locus. Furthermore, this repression also requires a functional N-CoR deacetylase complex, which brings about histone hypoacetylation and methylation of H3 lysine 9 to the MTA2 locus. Thus, our data demonstrate a critical role for a methyl CpG binding protein in mediating DNA methylation-dependent repression and reveal the mechanism by which it represses transcription.

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

“…DNA methylation is only one component in the downregulation of gene transcription. Methyl CpG binding proteins can bind methylated DNA and repress transcription (Bird and Wolffe, 1999;Yoon et al 2003;Kuzmichev et al 2004). In addition, specific modification of histones and binding of proteins that recognize these modifications can repress gene expression (Reviewed by Ruthenburg et al 2007 andTaverna et al 2007).…”

Decreased intranuclear mobility of acute myeloid leukemia 1-containing fusion proteins is accompanied by reduced mobility and compartmentalization of core binding factor β

“…Both AML1-ETO and AML1-MTG16 (in pSP64(polyA), Promega) vectors for in vitro transcription experiment were linearized with AflII restriction enzyme and the SP6 mMessage mMachine kit (Ambion) was applied for the in vitro mRNA synthesis according to the manufacturer's instruction. The injected Xenopus oocytes or the mammalian 293T cells following transient expression of HA-AML1-ETO and HA-AML1-MTG16 proteins were processed for ChIP analysis as described (Yoon et al, 2003;Stewart et al, 2005) using primers located in the proximal region of AML1 consensus sequence of human RUNX3 gene. The extract from oocytes or 293T cells was sonicated in a cold room to break chromatin into fragments averaging 500 bp in length (Yoon et al, 2003;Stewart et al, 2005) and subsequently used for ChIP experiment with a specific mouse monoclonal HA-tagged (6E2) antibody (Cell Signaling) and/or rabbit polyclonal AML1(Ab-1) antibody (Oncogene).…”

“…The injected Xenopus oocytes or the mammalian 293T cells following transient expression of HA-AML1-ETO and HA-AML1-MTG16 proteins were processed for ChIP analysis as described (Yoon et al, 2003;Stewart et al, 2005) using primers located in the proximal region of AML1 consensus sequence of human RUNX3 gene. The extract from oocytes or 293T cells was sonicated in a cold room to break chromatin into fragments averaging 500 bp in length (Yoon et al, 2003;Stewart et al, 2005) and subsequently used for ChIP experiment with a specific mouse monoclonal HA-tagged (6E2) antibody (Cell Signaling) and/or rabbit polyclonal AML1(Ab-1) antibody (Oncogene). The final PCR reactions were performed with inclusion of [a-32 P]dATP; the PCR products were visualized by autoradiography following fractionation on a 6% native polyacrylamide gel.…”

“…A RUNX3-Luc reporter construct containing one copy of the AML1 consensus DNA-binding site was integrated into chromatin via replication-coupled chromatin assembly following injection of its double-stranded DNA into the nuclei of oocytes . Molecular interactions in vivo between AML1-ETO or AML1-MTG16 and the RUNX3 promoter AML1-binding site in oocytes or 293T cells was investigated by chromatin immunoprecipitation (ChIP) assay (Yoon et al, 2003). PCR amplification of input genomic DNA and of anti-HA or anti-AML1 immunoprecipitants from both oocytes and 293T cells gave PCR products of the expected size (150 bp; Figure 1c and d).…”

Dynamic and reversibility of heterochromatic gene silencing in human disease

“…The knock-down of Kaiso leads to premature zygotic gene activation and subsequent developmental arrest and apoptosis, mimicking the phenotype of hypomethylated embryos deficient in DNMT1 [60]. In HeLa cells, Kaiso is coupled with the NCoR corepressor to mediate DNA methylation-dependent gene silencing [61].…”