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Molecular Basis for Recognition of Methylated and Specific DNA

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Molecular Basis for Recognition of Methylated and Specific DNA Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso Bethany A. Buck-Koehntopa, Robyn L. Stanfielda, Damian C. Ekierta, Maria A. Martinez-Yamouta, H. Jane Dysona, Ian A. Wilsona,b, and Peter E. Wrighta,b,1 aDepartment of Molecular Biology and bSkaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 Contributed by Peter E. Wright, August 7, 2012 (sent for review July 19, 2012) Methylation of CpG dinucleotides in DNA is a common epigenetic in cancer cells (14). Specific Kaiso-binding sites are found in the modification in eukaryotes that plays a central role in maintenance promoters of several target genes (matrilysin, cyclin-D1, siamois, of genome stability, gene silencing, genomic imprinting, develop- c-myc,andWnt11) regulated by the Wnt signaling pathway, ment, and disease. Kaiso, a bifunctional Cys2His2 zinc finger protein which plays a critical role in early development and tumor implicated in tumor-cell proliferation, binds to both methylated CpG progression (9, 15, 16). The cytoplasmic armadillo repeat pro- (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) tein p120 catenin (p120ctn) binds Kaiso and relieves Kaiso- and represses transcription by recruiting chromatin remodeling co- mediated transcriptional repression (6, 17, 18). The interaction repression machinery to target genes. Here we report structures of of Kaiso with p120ctn has been implicated in regulation of ca- the Kaiso zinc finger DNA-binding domain in complex with its non- nonical and noncanonical Wnt signaling pathways through methylated, sequence-specific DNA target (KBS) and with a symmet- a mechanism that involves KBS recognition (15, 16). Addi- rically methylated DNA sequence derived from the promoter region tionally, there is increasing evidence that Kaiso expression lev- of E-cadherin. Recognition of specific bases in the major groove of els are elevated in tumorigenesis, indicating that Kaiso may play the core KBS and mCpG sites is accomplished through both classical a direct role in modulating cancer through transcriptional and methyl CH···O hydrogen-bonding interactions with residues in control of aberrantly methylated and KBS-containing genes (14, the first two zinc fingers, whereas residues in the C-terminal exten- 19, 20). It still is unclear whether the transcriptional regulation sion following the third zinc finger bind in the opposing minor of the two distinctly different DNA sequences is synergistic in groove and are required for high-affinity binding. The C-terminal nature or mutually exclusive. region is disordered in the free protein and adopts an ordered struc- Although structures of the SRA (UHRF1) (21–23) and MBD ture upon binding to DNA. The structures of these Kaiso complexes (MBD1, MeCP2, and MBD2) (24–26) domains in complex with provide insights into the mechanism by which a zinc finger protein methylated DNA have been determined, the structural basis for can recognize mCpG sites as well as a specific, nonmethylated reg- recognition of both mCpG and KBS sites by the Kaiso family is ulatory DNA sequence. unknown. Here we report crystal and solution structures of the Kaiso zinc finger domain in complex with its nonmethylated, se- protein–DNA interaction | NMR spectroscopy | X-ray crystallography | quence-specific DNA site (KBS) (Fig. 1A) and the crystal struc- folding upon binding | intrinsic disorder ture of the complex with an oligonucleotide derived from the promoter region of E-cadherin, a known Kaiso-binding site (7, 27) n eukaryotes, DNA methylation is a common epigenetic mod- that contains two sequential, symmetrically methylated CpG sites Iification that is central to the maintenance of genome stability, (MeECad) (Fig. 1B). In normal cells, E-cadherin is an important gene silencing, genomic imprinting, development, and disease mediator of cell–cell adhesion, but hypermethylation of the pro- (1, 2). Methyl-CpG–binding proteins (MBPs) mediate these pro- moter in several cancers results in down-regulation of E-cadherin cesses by binding to methylated DNA signals and recruiting expression and has been correlated with increasing invasion and chromatin remodeling corepressor complexes, resulting in com- metastasis (28). The current studies provide insights into the paction of chromatin into its transcriptionally inactive state (3). mechanism by which Kaiso recognizes both methylated DNA and To date, three classes of MBPs that recognize 5-methyl cytosine the unmethylated KBS DNA sequence. Remarkably, the molec- (5mC) have been identified. The SRA domain family has speci- ular interactions that mediate binding to these distinct DNA ficity for hemimethylated sites and is required for maintenance target sites are very similar. methylation during DNA replication (4). In contrast, the methyl CpG-binding domain (MBD) and Kaiso families of MBPs function Results and Discussion as essential mediators of epigenetically controlled gene silencing Overall Structure of the Kaiso:DNA Complexes. The crystal structure by recognizing symmetrically methylated CpG sites (5). Kaiso is a of the Kaiso:KBS complex was determined at 2.4-Å resolution MBP belonging to the BTB/POZ (broad complex, tramtrak, bric à using single-wavelength anomalous dispersion from the zinc, and brac/pox virus and zinc finger) subfamily of transcription factors that the Kaiso:MeECad complex then was solved by molecular re- recognize cognate DNA sequences through a C-terminal zinc finger placement at 2.8-Å resolution as detailed in SI Methods. The domain; the N-terminal BTB/POZ domain mediates protein–pro- tein interactions (6). Kaiso is a POZ protein that participates in both methyl-dependent and sequence-specific transcriptional re- Author contributions: B.A.B.-K., M.A.M.-Y. and P.E.W. designed research; B.A.B.-K., R.L.S., fi D.C.E., and M.A.M.-Y. performed research; B.A.B.-K., R.L.S., D.C.E., H.J.D., I.A.W., and P.E.W. pression, using its three Cys2His2 zinc ngers to recognize either two BIOCHEMISTRY consecutive symmetrically methylated CpG dinucleotides (mCpG) analyzed data; and B.A.B.-K., H.J.D., I.A.W., and P.E.W. wrote the paper. (7,8)oraTCCTGCNAconsensus(termed“KBS”) (9). Although The authors declare no conflict of interest. originally it was reported that Kaiso only requires zinc fingers 2 and Data deposition: Atomic coordinates and structure factors have been deposited in Protein 3 for DNA recognition (9), we determined that Kaiso in fact Data Bank, www.pdb.org [PDB ID codes 4F6M (crystal structure of the KBS complex), 4F6N fi (crystal structure of the MeECad complex), and 2LT7 (NMR structures)] and resonance requires all three zinc ngers plus adjacent regions for structural assignments in the Biological Magnetic Resonance Bank, http://www.bmrb.wisc.edu stability and high-affinity binding to both sequence classes (10). [accession no. 18462 (NMR assignments)]. Transcriptional repression by Kaiso has implications in both 1To whom correspondence should be addressed. E-mail: [email protected]. – development and cancer (11 13). Kaiso binds to and silences This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aberrantly methylated tumor-suppressor and DNA-repair genes 1073/pnas.1213726109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213726109 PNAS | September 18, 2012 | vol. 109 | no. 38 | 15229–15234 Downloaded by guest on September 27, 2021 heteronuclear single-quantum coherence (HSQC) spectrum of free Kaiso but undergo large shifts in the spectrum of the KBS complex, indicating formation of folded structure in the presence of DNA (Fig. 2A). Backbone {1H}-15N heteronuclear nuclear Overhauser effect (NOE) measurements (Fig. 2B)confirm that this region is highly flexible in the free protein but becomes or- dered, with NOE values similar to those of the rest of the protein, in the presence of KBS. The structure induced in the C-terminal region is stabilized by incorporation into the ZF3 β-sheet, by hydrophobic packing and hydrogen-bonding interactions with ZF2, and by interactions with the phosphate backbone (Figs. S1 and S3A). Molecular Determinants of KBS and MeECad DNA Recognition. In both the KBS and MeECad complexes, the zinc fingers are an- chored to the DNA by an extensive network of direct and water- mediated hydrogen bonds to the phosphate backbone as well as by van der Waals interactions with the sugar rings (Fig. 3A). Subtle differences are observed in the phosphate backbone interactions for the KBS and MeECad complexes (Fig. S2). ZF3 is anchored firmly to the DNA backbone by a network of hydrogen bonds from a cluster of tyrosine residues (Fig. 3B). Residues 590–594 form a structured loop, which is anchored to the side chain of Glu-547 in the ZF2–ZF3 linker by hydrogen bonds involving Arg-590 and Ser-591 backbone amides, and packs, by way of hydrophobic interactions, against the helix of ZF2, thereby positioning the Fig. 1. (A) Sequence of oligonucleotide containing the KBS motif (TCCT- GCNA). The box shows the core sequence equivalent to the methylated DNA site shown in B.(B) Sequence of the MeECad oligonucleotide containing two symmetrically methylated CpG sites (boxed). (C) Overlay of the Kaiso:KBS (dark colors, yellow DNA) and Kaiso:MeECad (light colors, gray DNA) X-ray crystal structures, superimposed on the protein backbone. The three Cys2His2 zinc finger DNA-binding domains are colored red (ZF1), green (ZF2), and blue (ZF3), and the zinc atoms are shown as gray spheres. The C-terminal extension is colored purple. In the crystal structure of the KBS complex, the protein backbone is ordered between residues 482–597, whereas the MeECad com- plex is ordered between residues 481–600. (D) Superposition of the crystal structure (red protein and DNA) and the 20 lowest-energy structures in the NMR ensemble of the Kaiso:KBS complex. The structures are superimposed on their protein backbones. The zinc finger domains in the NMR structures are colored pink (N-terminal extension and ZF1), pale green (ZF2), pale blue (ZF3), and magenta (C-terminal extension).
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