Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia - PubMed (original) (raw)

Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia

Tomasz Cierpicki et al. Nat Struct Mol Biol. 2010 Jan.

Abstract

The gene MLL (encoding the protein mixed-lineage leukemia) is the target of chromosomal translocations that cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain, which binds to nonmethylated CpG DNA sites. We present the solution structure of the MLL CXXC domain in complex with DNA, showing how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. On the basis of the structure, we generated point mutations that disrupt DNA binding. Introduction of these mutations into the MLL-AF9 fusion protein resulted in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9-locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also provide support for viewing this interaction as a potential target for therapeutic intervention.

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Figures

Figure 1

Figure 1

Structure of the CXXC domain – DNA complex and details of CpG recognition. a) representative conformer of the complex is shown in green (CpG binding loop 1182-1188 in magenta) and central 6 base pair region of DNA shown in blue and cyan; zinc atoms are shown as black spheres. b) Structure of the complex representing the view perpendicular to DNA axis; N- and C-terminal residues are labeled. Sidechains of Arg1150, Ser1152 and Leu1197 are shown in red. Cysteine resides coordinating zinc atoms are yellow. c) Schematic of protein-DNA contacts. Hydrogen bond, electrostatic, and van der Waals interactions of the protein backbone and sidechains with DNA are shown as red and blue arrows, respectively. The interaction of Arg1150 with minor groove is shown with green arrow. Ovals represent protein residues involved in base-specific (cyan) and electrostatic/van der Waals (green) contacts with DNA. d) Hydrogen bonds involving the carbonyls of Lys1185 and Lys1186 and N4-amine groups of Cyt106 and Cyt118. e) Hydrogen bonds formed between sidechains of Gln1187, Lys1186 and Gua107, Gua119, respectively. f) Close contacts between the protein backbone and cytosines in the CpG motif. The positions of H5 protons that are substituted by CH3 groups in methylated DNA are shown as gray spheres with the van der Waals radii of a methyl group. Backbone H and O atoms in intimate contact with the DNA are shown as magenta spheres with appropriate van der Waals radii.

Figure 2

Figure 2

Design of mutations impairing interaction of the CXXC domain with DNA. a) Surface representation of the CXXC domain - DNA complex with indicated mutation sites. Residues involved in electrostatic interactions with DNA (Lys1185; Arg1154, Lys1193) are shown in green, Gln1187 forming hydrogen bond with guanine base in magenta, and Cys1188 located in close proximity to the DNA backbone in yellow. b) Relative dissociation constants (Kd) for binding of wildtype and mutant CXXC domains to DNApal determined using NMR titration. The colors of bars are the same as in panel a. Error bars indicate s.d. The Kd value for C1188D could not be determined due to very weak binding. The additional set of resides (shown in red) comprises mutation of N-terminal (Arg1150) and C-terminal (L1197 and M1200) residues to alanines; position of these residues on the structure of the complex (panel a) is omitted for clarity. c) Example of 15N-1H HSQC spectra showing titration of the Q1187A mutant (red) with increasing concentrations of DNApal (cyan - 1:1 protein-DNA ratio; blue - 1:4 ratio). d) Comparison of spectra of C1188D mutant without (red) and with DNApal in a 1:4 ratio (blue).

Figure 3

Figure 3

DNA binding activity of the MLL CXXC domain is required for MLL-AF9 to protect Hoxa9 from DNA methylation and induce Hoxa9 and mir-196b transcript expression, but not for binding to the locus. a) Relative methylation levels of CpGs in the upstream Hoxa9 locus in Mll null MEFs transfected with either MCSVneo, MSCVneo-MLL-AF9(C1188A), or MSCVneo-MLL-AF9(C1188D) and pSuper, after one week of puromycin selection. Bisulfite treatment, PCR and sequencing on genomic DNA samples were performed three times, and the results of one representative experiment are shown. Expression levels of Hoxa9 (b) and mir196b (c) in bone marrow progenitor cells transduced with MSCVneo vector, MLL-AF9(C1188A), or MLL-AF9(C1188D). Cells were harvested after one week culture in methylcellulose and expression levels of Hoxa9 and mir196b were quantified with real-time RT-PCR. Shown are average relative expression levels (s.d.). (d) ChIP assay performed on Phoenix cells transfected with FLAG tagged MSCV-MLL-AF9(C1188A) or MSCV-MLL-AF9(C1188D). Chromatin was immunoprecipitated with the indicated antibodies and real time PCR was performed with primers that localize near mir-196b in the upstream region of the HOXA9 locus. Samples were run in triplicate and were normalized to GAPDH and input chromatin, with error bars showing s.d.

Figure 4

Figure 4

DNA binding activity of the MLL CXXC domain in MLL-AF9 is required for increased proliferative capacity and immortalization in a bone marrow progenitor serial replating assay. a) Average numbers of colonies for each of the four weeks after plating or re-plating in methylcellulose are shown for bone marrow progenitor cells expressing MLL-AF9 or MLL-AF9 with various CXXC domain point mutations, with error bars showing standard error. Also shown are the relative Kd values for binding of the wild type and mutated MLL CXXC domains to DNA. b) Digital photographs showing colony (above) and cell (below) morphologies of transduced bone marrow cells at the end of week 4 of the colony assay.

Figure 5

Figure 5

DNA binding activity of the MLL CXXC domain is required for MLL-AF9 to cause leukemia in vivo. a) Survival curve of mice transplanted with bone marrow progenitor cells infected with either MSCVneo-MLL-AF9(C1188A) or MSCVneo-MLL-AF9(C1188D). No mice transplanted with empty vector MSCVneo died (data not shown). b) Peripheral blood from mice at time of sacrifice (MLL-AF9(C1188A)) or at two months after bone marrow transplants (MSCVneo and MLL-AF9(C1188D)).

Figure 6

Figure 6

Model of the regulation of Hoxa9 locus transcription by the CXXC domain of MLL-AF9. a) The CXXC domain of MLL-AF9 protects specific CpG sequences within the Hoxa9 locus from methylation and maintains transcription within the locus; b) disruption of DNA binding function of CXXC domain by C1188D mutation results in increased methylation of the same CpGs, increased H3K9 trimethylation, and silencing of Hoxa9 and mir196b.

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