title: Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint.
abstract: Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
authors.forename: Han
authors.initials: H
authors.lastname: Liu
authors.forename: Shugaku
authors.initials: S
authors.lastname: Takeda
authors.forename: Rakesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Todd D
authors.initials: TD
authors.lastname: Westergard
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Brown
authors.forename: Tej K
authors.initials: TK
authors.lastname: Pandita
authors.forename: Emily H-Y
authors.initials: EH
authors.lastname: Cheng
authors.forename: James J-D
authors.initials: JJ
authors.lastname: Hsieh
mesh_terms: D000483:Alleles; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000502:physiology; D005799:Genes, Dominant / Q000235:genetics; D042822:Genomic Instability / Q000502:physiology; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000235:genetics; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D023461:Myeloid Progenitor Cells / Q000378:metabolism; D051788:Myeloid-Lymphoid Leukemia Protein / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase / Q000502:physiology*; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D015398:Signal Transduction; D014178:Translocation, Genetic / Q000235:genetics
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D006657:Histones; C497422:KMT2A protein, human; D044786:S-Phase Kinase-Associated Proteins; D051788:Myeloid-Lymphoid Leukemia Protein; D010768:Phosphoserine; D011495:Histone-Lysine N-Methyltransferase; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D008239:Lysine
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