The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC Cdh1 ) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC Cdh1 ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. INTRODUCTIONDuplication of large mammalian genomes requires that DNA replication initiate at thousands of chromosomal origins. In order for an origin to be competent for replication, it must first be bound by a multiprotein complex, the prereplication complex (preRC). PreRCs are constructed in a stepwise process through the chromatin binding of the origin recognition complex (ORC), which then recruits both the Cdc6 ATPase and Cdt1, two proteins that are required for the stable loading of the minichromosome maintenance complex (MCM). The Cdc6 and Cdt1-dependent loading of MCM complexes at origins licenses them for replication during the G1 phase of the cell cycle. Sufficient preRCs must be assembled during G1 to promote complete replication, but new preRCs must not assemble after S phase begins because relicensing of previously fired origins leads to rereplication and genome instability (Vaziri et al., 2003;Melixetian et al., 2004;Zhu et al., 2004;Archambault et al., 2005). For these reasons, preRC assembly is one of the most highly regulated events in the control of DNA replication. Cells restrict preRC assembly to the G1 period through a combination of overlapping mechanisms that regulate individual preRC components (reviewed in Bell and Dutta, 2002;Blow and Hodgson, 2002;Nishitani and Lygerou, 2002;Diffley, 2004;Machida et al., 2005;Fujita, 2006).Cdc6 is not only an essential factor for preRC construction, but it has also been implicated in the activation of the cell cycle checkpoint that prevents entry into mitosis while DNA replication is incomplete (Clay-Farrace et al., 2003;Oehlmann et al., 2004;Lau et al., 2006). These observations suggest that Cdc6 functions not only during G1, but also in later cell cycle stages. Moreover, Cdc6 plays a role in setting the threshold for...

Degradation of damaged proteins by members of the protein quality control system is of fundamental importance in maintaining cellular homeostasis. In mitochondria, organelles which both generate and are targets of reactive oxygen species (ROS), a number of membrane bound and soluble proteases are essential components of this system. Here we describe the regulation of Podospora anserina LON (PaLON) levels, an AAA(+) family serine protease localized in the matrix fraction of mitochondria. Constitutive overexpression of PaLon results in transgenic strains of the fungal ageing model P. anserina showing increased ATP-dependent serine protease activity. These strains display lower levels of carbonylated (aconitase) and carboxymethylated proteins, reduced secretion of hydrogen peroxide and a higher resistance against exogenous oxidative stress. Moreover, they are characterized by an extended lifespan without impairment of vital functions such as respiration, growth and fertility. The reported genetic manipulation proved to be a successful intervention in organismal ageing and it led to an increase in the healthy lifespan, the healthspan, of P. anserina.

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Rasdependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the ''pointed'' domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.T he function of posttranslational modification by ligation of a small ubiquitin-like modifier (SUMO) to a target protein appears to be diverse and substrate specific. RanGAP1 was the first protein shown to be modified by addition of SUMO (1-3). Unmodified RanGAP1 is diffusely cytoplasmic, whereas sumoylation targets RanGAP1 to nuclear pore complexes. Sumoylation also directs targeted proteins such as promyelocytic leukemia and the homeodomain-interacting protein kinase 2 to distinct subnuclear domains (nuclear bodies or speckles; refs. 4 and 5). SUMO modification appears to activate the heat shock transcription factors 1 and 2 (6, 7), whereas sumoylation may negatively regulate c-JUN and c-MYB activity (8, 9). Sumoylation also affects protein stability if the modified lysine is also used for ubiquitination. Both inhibitor of NF-B␣ and murine double minute 2 are targeted for degradation by ubiquitination, but competition for the target lysine by SUMO stabilized these factors (10-12). Like ubiquitin, SUMO conjugation and deconjugation is a very dynamic process. Sumoylation studies are limited because of the rapid removal of the modification by SUMO-specific isopeptidases in cellular extracts. Thus, for each of these examples, the functional consequences of sumoylation were derived from conservative substitution of the modified lysine of the target protein.Like ubiquitination, sumoylation is a three-step process involving an E1-activating enzyme heterodimer Aos͞Uba2, the E2-conjugating enzyme Ubc9 and substrate-specific E3 ligases (for recent reviews, see refs. 13 ...

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.Chromosomal translocations disrupt master regulatory genes that control cellular proliferation, apoptosis, and the lineage decisions that affect stem cell self-renewal and differentiation of progenitor cells (15,29). The myeloid translocation gene on chromosome 8 (MTG8, also known as eighttwenty-one or ETO) is disrupted by t(8;21) in up to 15% of acute myeloid leukemia cases (7,26,27). MTG8 is the founding member of a gene family that includes the myeloid translocation gene on chromosome 16 (MTG16 or ETO2), which is disrupted by t(16;21), and myeloid translocation gene-related 1 (MTGR1) (5, 6, 12, 18). t(8;21) and t(16;21) fuse MTG8 and MTG16, respectively, to the DNA binding domain of Runtrelated 1 (RUNX1, also known as acute myeloid leukemia 1 or AML1) (7,12,26,27). The resulting fusion proteins repress RUNX1-regulated genes (11,20,25). For RUNX1-MTG8, this repression requires the MTG8 sequences, leading to the hypothesis that MTG8 is a transcriptional corepressor (20). Consistent with this hypothesis, MTG8 associates with multiple corepressors, including N-CoR/SMRT, mSin3, and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3 (1,13,14,23,34).MTG family members display approximately 85% sequence similarity (3) and contain four conserved subdomains with up to 95% identity (5, 8). Based on homology to MTG8, it was anticipated that MTG16 and MTGR1 also act as transcriptional corepressors. MTG16 is 92% homologous to MTG8, and the murine form of MTG16, Eto2, interacts with multiple HDACs and N-CoR (1). In contrast to MTG8, Eto2 failed to interact with mSin3A (1). The MTG family members also heterodimerize, and this property allowed the identification of MTGR1 as a RUNX1-MTG8-associated protein (18). Although it associates with MTG8 and the t(8;21) fusion protein, the molecular function of MTGR1 is unknown.While two of the three MTG family members are disrupted by chromosomal translocations, the MTG family members are widely expressed, suggesting that this gene f...

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Canonical Wnt signaling is mediated by a molecular "switch" that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of ␤-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of ␤-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of ␤-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.Canonical Wnt signaling plays a critical regulatory role in development and in stem cell functions and cellular differentiation (41, 43). Wnts initiate a signaling cascade that leads to the nuclear accumulation of ␤-catenin, which associates with T-cell factor 4 (TCF4), releasing the TCF4-associated transcriptional corepressors and recruiting coactivators to stimulate TCF4-dependent transcription. The intestinal epithelium has been a rich source of information about this pathway. For example, Tcf4 is required for small intestinal stem cell selfrenewal; mice lacking this transcription factor exhaust the capacity for continued replenishment of the epithelium in utero (24). Conversely, hyperactive Wnt signaling is closely associated with colorectal carcinoma, most commonly via inactivation of the APC tumor suppressor, which regulates the levels of ␤-catenin (41, 43). At the end point of the Wnt signaling cascade, ␤-catenin opposes the action of transcriptional corepressors for binding to TCFs to regulate genes that affect cell fate decisions.Two members of the myeloid translocation gene (MTG) family of transcriptional corepressors, MTG on chromosome 8 (MTG8; also known as ETO or RUNX1T1) and MTG on chromosome 16 (MTG16; also known as ETO2 or CBFA2T3), are disrupted by chromosomal translocations in acute myeloid leukemia (10, 29), which suggests that these factors are key regulators of cellular proliferation or differentiation. As would be expected of master regulatory factors, these targets of chromosomal translocations in acute leukemia are commonly mutated in other tumor types as well. A genomewide screen to detect mutations of genes in colorectal and breast can...

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