The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation.
Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.
Birke
Marco
M
Department of Genetics, University of Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany.
Schreiner
Silke
S
García-Cuéllar
María-Paz
MP
Mahr
Kerstin
K
Titgemeyer
Fritz
F
Slany
Robert K
RK
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D005982:Glutathione Transferase / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D008969:Molecular Sequence Data; D051788:Myeloid-Lymphoid Leukemia Protein; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011519:Proto-Oncogenes; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014157:Transcription Factors; D015533:Transcriptional Activation
C408982:CPG-oligonucleotide; D004268:DNA-Binding Proteins; C497422:KMT2A protein, human; C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051788:Myeloid-Lymphoid Leukemia Protein; D015254:DNA Modification Methylases; D011495:Histone-Lysine N-Methyltransferase; D005982:Glutathione Transferase; D003850:Deoxyribonuclease I