Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.
The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex.
From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
Anamika
A
Patel
Valarie E
VE
Vought
Stephen
S
Swatkoski
Susan
S
Viggiano
Benny
B
Howard
Venkatasubramanian
V
Dharmarajan
Kelsey E
KE
Monteith
Gillian
G
Kupakuwana
Kevin E
KE
Namitz
Stephen A
SA
Shinsky
Robert J
RJ
Cotter
Michael S
MS
Cosgrove
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D051788:Myeloid-Lymphoid Leukemia Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C485622:ASH2L protein, human; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C497422:KMT2A protein, human; D009687:Nuclear Proteins; C470552:RBBP5 protein, human; D014157:Transcription Factors; C502547:WDR5 protein, human; D051788:Myeloid-Lymphoid Leukemia Protein; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine; D003545:Cysteine
Automethylation; Chromatin Histone Modification; Enzyme Mechanisms; Epigenetics; Histone Methylation; Leukemia; Self-methylation