Allen, M. D. et al. Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase. EMBO J. 25, 4503-4512

University of Cambridge, Cambridge, England, United Kingdom
The EMBO Journal (Impact Factor: 10.43). 11/2006; 25(19):4503-12. DOI: 10.1038/sj.emboj.7601340
Source: PubMed


Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.

Download full-text


Available from: Mark Bycroft
  • Source
    • "ing promoters and gene regulatory units in up to 70% of genes (Blackledge and Klose, 2011; Deaton and Bird, 2011; Gardiner-Garden and Frommer, 1987). Recent studies suggested that an unmethylated CpG dinucleotide was bound by a CxxC-zinc finger (CxxC-ZF) domain (Allen et al., 2006; Blackledge et al., 2010; Cierpicki et al., 2010; Lee et al., 2001; Song et al., 2011; Thomson et al., 2010; Voo et al., 2000) that was characterized by two CGXCXXC repeats and conserved cysteine residues that bound to two zinc ions (Long et al., 2013; Xu et al., 2011). It was reported that the CxxC-ZF domain was involved in targeting chromatin-modifying proteins to CpG islands, which may affect gene expression (Blackledge et al., 2010; Long et al., 2013; Smith and Shilatifard, 2010; Thomson et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The transcription of ribosomal RNA genes (rDNA) is a rate-limiting step in ribosome biogenesis and changes profoundly in response to environmental conditions. Recently we reported that JmjC demethylase KDM2A reduces rDNA transcription on starvation, with accompanying demethylation of dimethylated Lys 36 of histone H3 (H3K36me2) in rDNA promoter. Here, we characterized the functions of two domains of KDM2A, JmjC and CxxC-ZF domains. After knockdown of endogenous KDM2A, KDM2A was exogenously expressed. The exogenous wild-type KDM2A demethylated H3K36me2 in the rDNA promoter on starvation and reduced rDNA transcription as endogenous KDM2A. The exogenous KDM2A with a mutation in the JmjC domain lost the demethylase activity and did not reduce rDNA transcription on starvation, showing that the demethylase activity of KDM2A itself is required for the control of rDNA transcription. The exogenous KDM2A with a mutation in the CxxC-ZF domain retained the demethylase activity but did not reduce rDNA transcription on starvation. It was found that the CxxC-ZF domain of KDM2A bound to the rDNA promoter with unmethylated CpG dinucleotides in vitro and in vivo. The exogenous KDM2A with the mutation in the CxxC-ZF domain failed to reduce H3K36me2 in the rDNA promoter on starvation. Further, it was suggested that KDM2A that bound to the rDNA promoter was activated on starvation. Our results demonstrate that KDM2A binds to the rDNA promoter with unmethylated CpG sequences via the CxxC-ZF domain, demethylates H3K36me2 in the rDNA promoter in response to starvation in a JmjC domain-dependent manner, and reduces rDNA transcription.
    Preview · Article · Feb 2014 · Cell Structure and Function
  • Source
    • "Point mutations that have been proved to abolish the binding ability to non-methylated CpGs (C1155A and K1186A) (31) resulted in a loss of transforming activity (Figure 4A and B), while the Q1162A substitution, which has no effect on non-methylated CpG-specific binding, did not compromise transformation. Mutations in the KFGG motif (K1178A and K1179A) that severely attenuate the binding ability to non-methylated CpGs (31) also abolished transforming activity. The artificial protein (P′C′) composed of the minimum structures of the PWWP domain and the CXXC domain localized in the nucleosome fraction (Figure 4C and D) and associated with nucleosomes (Figure 4E). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mixed-lineage leukemia (MLL) maintains the expression of cellular memory genes during development, while leukemic MLL fusion proteins aberrantly maintain expression of hematopoietic stem cell program genes such as HOXA9 to cause leukemia. However, the molecular mechanism of gene activation is unclear. Here we show that only two functional modules are necessary and sufficient for target recognition: those that bind to non-methylated CpGs and di-/tri-methylated histone H3 lysine 36 (H3K36me2/3). An artificial protein composed of the two targeting modules and an interaction domain for AF4-family coactivators can functionally substitute for MLL fusion proteins. Because H3K36me2/3 markers are indicative of active transcription, MLL fusion proteins target previously active CpG-rich genes and activate transcription by recruiting coactivators thereto. Our results indicate that such chromatin context-dependent gene activation is the fundamental mechanism by which MLL fusion proteins maintain the expression of the cellular memory/hematopoietic stem cell program genes.
    Full-text · Article · Jan 2014 · Nucleic Acids Research
  • Source
    • "TET proteins have a catalytic domain (CD) in the C-terminus that hydroxylates methylcytosine. Moreover, TET1 and TET3 have CXXC domains that bind to nonmethylated CpG sequences (Allen et al. 2006; Xu et al. 2012). The CD consists of a cysteine-rich domain (CRD) and a double-stranded betahelix domain (DSBH). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene expression is controlled by alterations in the epigenome, including DNA methylation and histone modification. Recently, it was reported that 5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by proteins in the ten-eleven translocation (TET) family. This conversion is believed to be part of the mechanism by which methylated DNA is demethylated. Moreover, histones undergo modifications such as phosphorylation and acetylation. In addition, modification with O-linked-N-acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) was recently identified as a novel histone modification. Herein, we focused on TET3, the regulation of which is still unclear. We attempted to elucidate the mechanism of its regulation by biochemical approaches. First, we conducted mass spectrometric analysis in combination with affinity purification of FLAG-TET3, which identified OGT as an important partner of TET3. Co-immunoprecipitation assays using a series of deletion mutants showed that the C-terminal H domain of TET3 was required for its interaction with OGT. Furthermore, we showed that TET3 is GlcNAcylated by OGT, although the GlcNAcylation did not affect the global hydroxylation of methylcytosine by TET3. Moreover, we showed that TET3 enhanced its localization to chromatin through the stabilization of OGT protein. Taken together, we showed a novel function of TET3 that likely supports the function of OGT.
    Preview · Article · Dec 2013 · Genes to Cells
Show more