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.

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono-and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.[Keywords: Histone; methylation; G9a; GLP] Supplemental material is available at http://www.genesdev.org.

Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.cancer cell microenvironment ͉ multidrug resistant-associated protein ͉ oxidative stress ͉ somatic mutation ͉ ubiquitin-proteasome system

Oxidative and electrophilic stresses are sensed by Keap1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-Keap1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low Keap1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer-derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in Keap1-null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2. [Cancer Res 2008;68(5):1303-9]

No abstract

Meiotic recombination of S. cerevisiae contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs). Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core, and participates in DSB processing. Although these proteins are also involved in DSB formation, Mre11 is not necessarily holding them. The C-terminal region of Mre11 is required only for DSB formation and binds to some meiotic proteins. The N-terminal half specifies nuclease activities that are collectively required for DSB processing. Mre11 has a DNA-binding site for DSB formation and another site for DSB processing. It has two regions to bind to Rad50. Mre11 repairs methyl methanesulfonate-induced DSBs by reactions that require the nuclease activities and those that do not.

Background: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein.