Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso

Abstract

Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys 2 His 2 zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence.protein-DNA interaction | NMR spectroscopy | X-ray crystallography | folding upon binding | intrinsic disorder I n eukaryotes, DNA methylation is a common epigenetic modification that is central to the maintenance of genome stability, gene silencing, genomic imprinting, development, and disease (1, 2). Methyl-CpG-binding proteins (MBPs) mediate these processes by binding to methylated DNA signals and recruiting chromatin remodeling corepressor complexes, resulting in compaction of chromatin into its transcriptionally inactive state (3). To date, three classes of MBPs that recognize 5-methyl cytosine (5mC) have been identified. The SRA domain family has specificity for hemimethylated sites and is required for maintenance methylation during DNA replication (4). In contrast, the methyl CpG-binding domain (MBD) and Kaiso families of MBPs function as essential mediators of epigenetically controlled gene silencing by recognizing symmetrically methylated CpG sites (5). Kaiso is a MBP belonging to the BTB/POZ (broad complex, tramtrak, bric à brac/pox virus and zinc finger) subfamily of transcription factors that recognize cognate DNA sequences through a C-terminal zinc finger domain; the N-terminal BTB/POZ domain mediates protein-protein interactions (6). Kaiso is a POZ protein that participates in both methyl-dependent and sequence-specific transcriptional repression, using its three Cys 2 His 2 zinc fingers to recognize either two consecutive symmetrically methylated CpG dinucleotides (mCpG) (7, 8) or a TCCTGCNA consensus (termed "KBS") (9). Although originally it was reported that Kaiso only requires zinc fingers 2 and 3 for DNA recog...

The physics of epigenetics

“…Some crystal structures of proteins that specifically recognize methylated DNA have recently become available (Buck-Koehntop et al, 2012;Liu et al, 2014). The detailed analysis of the binding modes revealed that although similarities between them exist (Liu et al, 2013), it is yet unclear why three distinct families of methylated DNA-binding proteins were needed in the course of evolution.…”

A Structural Perspective on Readout of Epigenetic Histone and DNA Methylation Marks

“…It contains an amino-terminal BTB/POZ (BR-C, ttk and bab/Pox virus and Zn finger) domain involved in protein -protein interactions and three mCG-DNA-binding carboxy-terminal zinc fingers (Cys 2 His 2 coordination) that repress transcription by recruiting chromatin-remodeling compression machinery to target genes (reviewed in Clouaire and Stancheva 2008). Recently, structural studies of the zinc-finger domains of Kaiso bound to a pair of sequential symmetrically methylated 5mCpG/5mCpG DNA sites from the E-cadherin promoter region have elucidated the details underlying the recognition process, in which 1 Kaiso molecule is bound per DNA duplex (Buck-Koehntop et al 2012). The side chains of the first two zinc fingers target the major groove through base-specific recognition mediated by classical and C-H † †O hydrogen bonds, as well as phosphate backbone contacts.…”

Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence

“…The ability of ZnF proteins to respond to methylation in this way is significant because ''sequences longer than CpG would be necessary for the regulation of gene expression by methylation'' (Holliday 1996). The structures of three ZnF domains bound to 5mC-containing DNA were solved recently from the transcription factors Kaiso, Zfp57, and Klf4 (Buck-Koehntop et al 2012;Liu et al 2012Liu et al , 2014. Here we investigate the binding of ZnF domains to oxidized modifications of 5mC.…”