A Novel Function of MLL in the S Phase Checkpoint: MLL Is Phosphorylated by ATR Upon DNA Damage, Which Blocks Its Degradation by the SCF^skp2 Proteasome, Leading to Its Accumulation That Directly Inhibits DNA Replication.

Abstract 450

Cell cycle checkpoints are implemented to safeguard our genome. Accordingly, checkpoint deregulation can result in human cancers. Although the S phase checkpoint plays an essential role in preventing genetic aberrations, the detailed molecular makeup of this signaling cascade, especially how it is executed in higher eukaryotes remains largely unknown. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a histone H3 lysine 4 methyl transferase that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathways must be involved, which remains to be discovered. Here, we demonstrate a novel function of MLL in executing the S phase DNA damage checkpoint response.

We found that MLL was accumulated in the S phase upon DNA damage triggered by various agents including UV, ionizing radiation, etoposide, hydroxyurea and aphidocholin, which was observed in all of the cell lines examined including HeLa, 293T, NIH 3T3, and BJ-1 cells. During a normal cell cycle progression, MLL was recognized and degraded by the SCF^skp2 proteasome in the S phase. Upon DNA damage, MLL was phosphorylated and thereby no longer recognized by SCF^skp2, leading to its ultimate accumulation in the S phase. To determine the importance of DNA-damage induced MLL accumulation, we investigated whether MLL deficiency compromises S phase checkpoint in response to DNA damage. MLL knockout or knockdown cells displayed radioresistant DNA synthesis (RDS) and chromatid type genomic abnormalities (two hallmarks of S phase checkpoint defect). Using genetically well-defined mouse embryonic fibroblasts (MEFs), we identified ATR, but not ATM or DNA-PK, as the kinase required for the MLL accumulation. Furthermore, MLL with mutation of the ATR phosphorylation site failed to accumulate upon DNA damage and thus was unable to rescue the RDS and genomic instability phenotypes of MLL deficient cells. In summary, MLL is phosphorylated by ATR upon DNA damage, which disrupts its interaction with SCF^skp2, leading to its accumulation in the S phase that is essential for the proper DNA damage checkpoint execution.

We further dissected the mechanism by which MLL participates in the S phase checkpoint execution. We demonstrated that ATR -mediated phosphorylation of Chk1 remained intact in the absence of MLL, which positions MLL downstream to the DNA damage signaling cascade. CDC45 loading onto the replication origin constitutes the critical step of origin firing and thus ushers DNA replication - a step that is normally inhibited upon DNA damage signaling. Using co-immunoprecipitation and chromatin-immunoprecipitation assays, we demonstrated that S phase-accumulated MLL interacts with the MCM complex at the late replication origin, prevents the loading of CDC45, and thereby inhibits DNA replication. In other words, CDC45 was aberrantly loaded in the absence of MLL, which explains the observed RDS defects associated with the loss of MLL. To determine whether MLL leukemogenic fusions incur S phase checkpoint defects, we employed a MLL-CBP knockin mouse model. The RDS phenotype was observed in murine myeloid progenitor cells (MPCs) with haploinsufficiency of MLL. More importantly, MPCs expressing one knockin allele of MLL-CBP exhibited even greater S phase checkpoint defects, suggesting that MLL fusion further compromised DNA damage checkpoint. Taken together, our study establishes a previously unrecognized activity of MLL in direct inhibition of late origin firing upon DNA damage signaling, the deregulation of which may contribute to the pathogenesis of MLL leukemias.