Solution structure of human MBD1 CXXC1

Methylation of a CpG dinucleotide at cytosine C5 (mCpG) is a major modification in vertebrate genomes associated with epigenetic gene silencing. CpG methylation recruits proteins which specifically recognise this motif and these methylated DNA binding proteins then recruit enzymes which chemically and physically alter chromatin, inducing transcriptional repression. Although most CpG motifs are methylated, short (500–2000 bp) CG-rich regions, known as CpG islands (CGIs), found within *60 % of human gene promoters remain non-methylated (Bird 2002). How these CGIs contribute to epigentic regulation is an area of continuing research. To investigate the possibility that proteins are targeted to these CGIs, Voo et al. (2000) used a ligand screen to identify proteins that bind non-methylated CpGs. They identified a protein, CFP1 (formerly CGBP) that contains a cysteine rich zinc finger–CXXC (ZF-CXXC) domain. Since this discovery, several more CXXC containing proteins have been identified as reviewed recently by Long et al. (2013). CXXC domains can be differentiated into three groups, type 1 which bind CpG and contain the KFGG motif, type 2 which do not bind CpG and lack the KFGG motif, and type 3 which lack the extended loop of types 1 and 2 but do bind to CpG (Fig. 3a). The conserved CXXCXXCX4/5CXXCXXC and CXXRC motifs that provide the amino acid residues that coordinate the two zinc ions means that the main domain architecture, despite sequence variation between the three types, is similar in all CXXC domains. CXXC domains are found in various chromatin binding proteins: CFP1 is a member of the SETDB1 complex responsible for H3K4 methylation; MLL protein is also a H3K4 methyltrasferase; KDM2A and KDM2B are H3K36 demethylase enzymes. DNMT1 and MBD1 are unique amongst CXXC domain containing proteins in that they can also bind hemi-methylated and methylated CpG respectively as well as non-methylated CpG. As well as the methylated DNA binding domain, MBD1 also contains a transcriptional repressor domain (TRD) and depending on splice variant, 2 or 3 CXXC domains. The TRD is capable of transcriptional repression independently of the MBD domain, and MBD1 induces transcriptional repression by recruiting the histone H3-K9 methyl transferase enzyme SETDB1. After DNA replication, this histone methylation is heritably maintained due to MBD1 remaining at the replication fork through its interaction with chromatin assembly factor 1. Once replication has occurred, DNMT1 activity fully methylates hemimethylated CpG motifs allowing MBD1 to rebind to the methyl-CpG (Sarraf and Stancheva 2004). There are several published structures of type 1 (Allen et al. 2006; Cierpicki et al. 2010; Song et al. 2011; Xu et al. 2011) and type 3 (Xu et al. 2012) (unpublished data, PDB depositions 4BBQ, 4O64) CXXC domains with and without DNA bound that have revealed how the CXXC domain interacts with CpG DNA. The CXXC domains bind perpendicular to the DNA helical axis, with their DNA binding loops packed into the major groove. The type 1 DNA binding motif consists of three residues, in the case of hMLL, KKQ, which hydrogen bond to the CpG sequence. The main chain carbonyls of K1185 and K1186 & Brian O. Smith [email protected]