A Role for Proapoptotic BID in the DNA-Damage Response

SummaryThe BCL-2 family of apoptotic proteins encompasses key regulators proximal to irreversible cell damage. The BH3-only members of this family act as sentinels, interconnecting specific death signals to the core apoptotic pathway. Our previous data demonstrated a role for BH3-only BID in maintaining myeloid homeostasis and suppressing leukemogenesis. In the absence of Bid, mice accumulate chromosomal aberrations and develop a fatal myeloproliferative disorder resembling chronic myelomonocytic leukemia. Here, we describe a role for BID in preserving genomic integrity that places BID at an early point in the path to determine the fate of a cell. We show that BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage distinct from its proapoptotic function. We further demonstrate that this role is mediated through BID phosphorylation by the DNA-damage kinase ATM. These results establish a link between proapoptotic Bid and the DNA-damage response

Regulation of Glucose Homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2-/-ksr1-/- (DKO) mice were compared to wild type, mark2-/-, and ksr1-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage

The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease

Genetic and Physical Interactions between Tel2 and the Med15 Mediator Subunit in Saccharomyces cerevisiae

International audienceBACKGROUND: In budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases. PRINCIPAL FINDINGS: To uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15 mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants. SIGNIFICANCE: Altogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit

Mutability and mutational spectrum of chromosome transmission fidelity genes

It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetochore function. Extensive screening of systematic mutant collections has shown that ~350 genes with functions as diverse as RNA processing and proteasomal activity mutate to cause a Ctf phenotype and at least 692 genes are required for faithful chromosome segregation. The enrichment of random Ctf alleles in only 13 of ~350 possible Ctf genes suggests that these genes are more easily mutable to cause genome instability than the others. These observations inform our understanding of recurring CIN mutations in human cancers where presumably random mutations are responsible for initiating the frequently observed CIN phenotype of tumors

Identification of Novel Human Damage Response Proteins Targeted through Yeast Orthology

Studies in Saccharomyces cerevisiae show that many proteins influence cellular survival upon exposure to DNA damaging agents. We hypothesized that human orthologs of these S. cerevisiae proteins would also be required for cellular survival after treatment with DNA damaging agents. For this purpose, human homologs of S. cerevisiae proteins were identified and mapped onto the human protein-protein interaction network. The resulting human network was highly modular and a series of selection rules were implemented to identify 45 candidates for human toxicity-modulating proteins. The corresponding transcripts were targeted by RNA interference in human cells. The cell lines with depleted target expression were challenged with three DNA damaging agents: the alkylating agents MMS and 4-NQO, and the oxidizing agent t-BuOOH. A comparison of the survival revealed that the majority (74%) of proteins conferred either sensitivity or resistance. The identified human toxicity-modulating proteins represent a variety of biological functions: autophagy, chromatin modifications, RNA and protein metabolism, and telomere maintenance. Further studies revealed that MMS-induced autophagy increase the survival of cells treated with DNA damaging agents. In summary, we show that damage recovery proteins in humans can be identified through homology to S. cerevisiae and that many of the same pathways are represented among the toxicity modulators

Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3−/− mice. Lipid metabolism disorders in Sik3−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance