Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase

Centre for Protein Engineering, Cambridge, UK.
The EMBO Journal (Impact Factor: 10.43). 11/2006; 25(19):4503-12. DOI: 10.1038/sj.emboj.7601340
Source: PubMed

ABSTRACT 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.

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Available from: Mark Bycroft, Sep 26, 2015
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    • "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). "
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    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.
    Nucleic Acids Research 01/2014; 42(7). DOI:10.1093/nar/gkt1394 · 9.11 Impact Factor
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    • "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). "
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    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.
    Genes to Cells 12/2013; 19(1). DOI:10.1111/gtc.12107 · 2.81 Impact Factor
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    • "In addition to targeting the function of MLL1-fusion proteins, inhibitors that target the association of MLL1-fusion proteins to target DNA might also have potential therapeutic implications. In this regard, inhibitors that disrupt the interaction between MLL1's CXXC motif and unmethylated CpG containing target DNA are being investigated as molecular drug targets in MLL1-related leukemias (Allen et al., 2006; Cierpicki et al., 2010). In addition to these molecular targets, recent studies have also identified other cooperating pathways such as positive transcription elongation factor-b (pTEFb) recruitment (Marschalek, 2010; Mueller et al., 2007; Mueller et al., 2009; Shapiro, 2006), activation of FMS-like tyrosine kinase 3 (FLT3) (Armstrong et al., 2003; Brown et al., 2005; Stubbs et al., 2008; Yao et al., 2005), glycogen synthase kinase 3 (GSK3) (Wang et al., 2008), heat shock protein-90 (HSP-90) (Yao et al., 2005; Yocum et al., 2006), myeloid cell leukemia sequence-1 (MCL-1) expression (Chen et al., 2009), and RAS pathways (Liang et al., 2006) that are implicated in MLL1-induced leukemogenesis. "
    Acute Leukemia - The Scientist's Perspective and Challenge, 12/2011; , ISBN: 978-953-307-553-2
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