Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive.

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

The mammalian cell cycle is precisely controlled by cyclin-dependent kinases (CDKs) and related pathways such as the RB and p53 pathways. Recent research on long non-coding RNAs (lncRNAs) indicates that many lncRNAs are involved in the regulation of critical cell cycle regulators such as the cyclins, CDKs, CDK inhibitors, pRB, and p53. These lncRNAs act as epigenetic regulators, transcription factor regulators, post-transcription regulators, and protein scaffolds. These cell cycle-regulated lncRNAs mainly control cellular levels of cell cycle regulators via various mechanisms, and may provide diversity and reliability to the general cell cycle. Interestingly, several lncRNAs are induced by DNA damage and participate in cell cycle arrest or induction of apoptosis as DNA damage responses. Therefore, deregulations of these cell cycle regulatory lncRNAs may be involved in tumorigenesis, and they are novel candidate molecular targets for cancer therapy and diagnosis.

Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR–Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2–Cdk1 in the regulation of late origin firing. Activity of cyclin A2–Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2–Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2–Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2–Cdk1 is a key regulator of S-phase programs.

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Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (␥H2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by ␥H2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.Initiation of mitosis in mammals is triggered by the abrupt activation of cyclin B-Cdc2. Activation of Cdc2 is regulated by a complex process that includes the binding to its regulatory subunit, cyclin B, whose level rises at late S phase, and peaks in mitosis (1-4). Just before mitosis, most cyclin B-Cdc2 complexes exist in an inactive state because of the inhibitory phosphorylation of Cdc2 on Thr-14 and Tyr-15, whose phosphorylation is catalyzed by Wee1 and Myt1, and whose dephosphorylation is catalyzed by Cdc25 phosphatases.Mitotic catastrophe was first identified in certain mutant fission yeast strains as a lethal phenotype characterized by gross abnormalities in chromosome segregation during mitosis (5, 6). A similar lethal phenotype was also observed in mammalian cells and found to result from premature mitosis (7, 8) or a failure to undergo complete mitosis (9, 10). However, there is still no broadly accepted definition of the term "mitotic catastrophe," presumably because the major processes involved have not been described in molecular and genetic terms. For example, some researchers have argued that mitotic catastrophe is fundamentally different from apoptosis (11) because overexpression of anti-apoptotic genes including Bcl-2 and MDR1 can actually enhance the frequency of catastrophic mitosis (12, 13). In contrast, a recent study demonstrated that mitotic catastrophe induced by DNA damage is dependent on caspase activation, suggesting that it constitutes a special case of apoptosis (14,15).In response to blocks to DNA replication, eukaryotes activate checkpoint pathways that prevent genomic instability. In yeast models, spontaneous chromosomal breaks and gross chromosomal rearrangements has been detected in mutant strains that cannot respond to stalled or incomplete DNA replicat...