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 SCFskp2 proteasome in the S phase. Upon DNA damage, MLL was phosphorylated and thereby no longer recognized by SCFskp2, 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 SCFskp2, 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.
No relevant conflicts of interest to declare.
Mammalian embryogenesis is achieved through precisely organized temporal and spatial control of gene activities, including epigenetic regulation. MLL is a conserved epigenetic regulator that belongs ...to the Trithorax protein family. The SET domain of MLL exerts a histone methyltransferase (HMT) activity that specifically catalyzes methylation of histone H3 lysine 4 (H3K4), an epigenetic mark for active transcription. MLL is site-specifically proteolyzed by a threonine endopeptidase, Taspase1, to generate a MLL N320/MLLC180 heterodimer. To investigate the in vivo functions of Taspase1, we generated Taspase1 -/- animals. Disruption of Taspase1 resulted in homeotic transformation and impaired cell proliferation. Importantly, uncleaved MLL displayed diminished HMT activity. Taspase1 deficiency in MEFs showed down-regulation of Cyclin Es, As and Bs, accompanied by reduced H3K4 tri-methylation at the respective promoters. We discovered that MLL interacts with E2Fs transcription factors to target the cyclin loci for H3K4 tri-methylation. The biological roles of Taspase1-mediated cleavage of MLL were specifically investigated by the generation and analyses of transgenic MLL nc/nc mice that express a non-cleavable form of MLL. MLLnc/nc mice exhibited multiple homeotic transformation phenotypes, indicating the prerequisite of MLL proteolysis for proper Hox expression. Unexpectedly, MLLnc/nc animals also showed defects in the outgrowth of the hypoglossal nerve, a phenotype associated with the deficiency of HGF-MET signaling. Importantly, this defect was also observed in Taspase1-/- and MLL-/- embryos, indicating a novel involvement of MLL in the HGF-MET signaling pathway. HGF-MET signaling modulates diverse physiological and pathological processes, including proliferation, migration and invasion. The molecular mechanisms by which MLL engages in the HGF-MET signaling pathway were characterized using HepG2 hepatocellular carcinoma cells. Cell migration and invasion assays demonstrated the requirement of MLL for HGF-induced migration/invasion. Consistently, silencing of MLL abolished up-regulation of critical HGF-downstream effecter genes, matrix metalloprotease (MMP) 1 and 3. The induction of MMP1/3 is dependent on Ets2, a transcription factor activated by HGF-MET pathway. Importantly, we revealed a novel complex of MLL and Ets2 by co-immunoprecipitation. Lastly, the chromatin immunoprecipitation assay demonstrated that HGF directs the recruitment of the MLL-Ets2 complex to the promoter of MMP1, where MLL confers H3K4 tri-methylation to activate MMP1. In summary, my thesis reveals a critical post-translational regulation of MLL through Taspase1-mediated proteolysis and discovers a novel participation of MLL in the HGF-MET signaling pathway that is commonly dysregulated in human cancers.
The MLL (mixed lineage leukemia) gene encodes a highly conserved 3,969 aa histone H3 K4 methyl transferase, of which chromosome translocations result in poor prognostic infant and therapy-related ...leukemias. Mysteriously, the pathognomonic MLL leukemia fusions consist of a common amino(N)-terminal ∼1400 aa of MLL fused in frame with more than 60 different partners with no shared characteristics. The most well known genetic function of MLL is to antagonize polycomb group of proteins for proper Hox gene expression. Consequently deregulated Hox genes caused by MLL translocations contribute to the MLL leukemogenesis. Besides Hox genes, it remains largely undetermined whether MLL participates in other biological processes. The 500kD MLL precursor undergoes evolutionarily conserved proteolytic maturation mediated by Taspase1 (Hsieh et al. 2003, MCB, 23, 186–194; Hsieh et al. 2003, Cell, 115, 293–303). Our recent studies on Taspase1 knockout cells established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins and possibly Cdk inhibitors (Takeda et al. 2006, Genes & Development , 20, 2397–2409). As MLL actively participates in the cell cycle regulation, we investigated the regulation of MLL through cell cycle transition. We uncovered a unique biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M phase progression. Conversely, over-expression of MLL blocks S phase progression. Remarkably, MLL degradation initiates at its N-terminal ∼1400 aa that is retained in all MLL leukemia fusions. We examined prevalent MLL-fusions, including MLL-AF4, MLL-AF9, MLL-ELL and MLL-ELL, and observed their increased resistance to degradation. Furthermore, the same resistance was observed with the leukemogenic MLL-lacZ but not the non-leukemogenic MLL-Myc tag fusion. Thus, non-oscillating expression of MLL-fusions through the cell cycle, resulted from impaired degradation, likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) to assure the temporal necessity of MLL in coordinating cell cycle progression. Future studies aim at providing a comprehensive analysis on the cell cycle consequences associated with MLL-fusions using genetically modified cells derived from mice carrying various MLL-fusion knockin alleles, including MLL-AF4, MLL-AF9, and MLL-CBP.
Taspase1 was identified as the threonine endopeptidase that cleaves MLL for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1−/− mice. Taspase1 ...deficiency results in non−cleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in cell cycle. Taspase1−/− animals are smaller in size. Taspase1−/− MEFs exhibit impaired proliferation and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down−regulation of Cyclin Es, As, and Bs and up−regulation of p16Ink4a. We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The un−cleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, CHIP assays demonstrate a markedly decreased histone H3 K4 tri−methylation at Cyclin E1 and E2 genes in Taspase1−/− cells. Furthermore, MLLnc/nc;2nc/nc MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1−/− cells are resistant to oncogenic transformation and Taspase1 is over−expressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.
Sexual development in
Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for ...nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes
tmcB and
tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of
tmcB was much less sensitive. The mRNA level of phosphodiesterase (
pde) was higher and that of its inhibitor (
pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus,
tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of
tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.
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