Mechanistic insights into aging, cell-cycle progression, and stress response.

The longevity of an organism depends on the health of its cells. Throughout life cells are exposed to numerous intrinsic and extrinsic stresses, such as free radicals, generated through mitochondrial electron transport, and ultraviolet irradiation. The cell has evolved numerous mechanisms to scavenge free radicals and repair damage induced by these insults. One mechanism employed by the yeast Saccharomycescerevisiae to combat stress utilizes the Anaphase Promoting Complex (APC), an essential multi-subunit ubiquitin-protein ligase structurally and functionally conserved from yeast to humans that controls progression through mitosis and G1. We have observed that yeast cells expressing compromised APC subunits are sensitive to multiple stresses and have shorter replicative and chronological lifespans. In a pathway that runs parallel to that regulated by the APC, members of the Forkhead box (Fox) transcription factor family also regulate stress responses. The yeast Fox orthologs Fkh1 and Fkh2 appear to drive the transcription of stress response factors and slow early G1 progression, while the APC seems to regulate chromatin structure, chromosome segregation, and resetting of the transcriptome in early G1. In contrast, under non-stress conditions, the Fkhs play a complex role in cell-cycle progression, partially through activation of the APC. Direct and indirect interactions between the APC and the yeast Fkhs appear to be pivotal for lifespan determination. Here we explore the potential for these interactions to be evolutionarily conserved as a mechanism to balance cell-cycle regulation with stress responses.

Troglitazone inhibits histone deacetylase activity in breast cancer cells.

We previously demonstrated that the PPARgamma agonist Troglitazone (TRG), a potent antiproliferative agent, in combination with the anthracycline antibiotic Doxorubicin (DOX), is an effective killer of multiple drug resistant (MDR) human cancer cells. Cell killing was accompanied by increased global histone H3 acetylation. Presently, we investigated the epigenetic and cell killing effects of TRG in estrogen receptor (ER) positive MCF7 breast cancer cells. MCF7 cells were treated with the Thiazolidinediones (TZDs) TRG and Ciglitazone (CIG), the non-TZD PPARgamma agonist 15PGJ2, and the histone deacetylase inhibitors (HDACi's) Trichostatin A (TSA), sodium butyrate and PXD101. Using MTT cell viability assays, Western analyzes and mass spectrometry, we showed a dose-dependent increase in cell killing in TRG and HDACi treated cells, that was associated with increased H3 lysine 9 (H3K9) and H3K23 acetylation, H2AX and H3S10 phosphorylation, and H3K79 mono- and di-methylation. These effects were mediated through an ER independent pathway. Using HDAC activity assays, TRG inhibited HDAC activity in cells and in cell lysates, similar to that observed with TSA. Furthermore, TRG and TSA induced a slower migrating HDAC1 species that was refractory to HDAC2 associations. Lastly, TRG and the HDACi's decreased total and phosphorylated AKT levels. These findings suggest that TRG's mode of killing may involve downregulation of PI3K signaling through HDAC inhibition, leading to increased global histone post-translational modifications.

Involvement of homologous recombination repair after proton-induced DNA damage.

Protection from chronic exposure to cosmic radiation, which is primarily composed of protons, in future manned missions to Mars and beyond is considered to be a key unresolved issue. To model the effects of cosmic radiation on a living cell, we used Saccharomyces cerevisiae cells harboring various deletions of DNA repair genes to investigate the response of cells to DNA strand breaks caused by exposure to 250 MeV proton irradiation (linear energy transfer of 0.41 keV/microm). In our study, DNA strand breaks induced by exposure to protons were predominantly repaired via the homologous recombination and postreplication repair pathways. We simulated chronic exposure to proton irradiation by treating cells from colonies that survived proton treatment, after several rounds of subculturing, to a second proton dose, as well as additional cell stressors. In general, cells cultured from proton surviving colonies were not more sensitive to secondary cell stressors. However, cells from rad52delta colonies that survived proton treatment showed increased resistance to secondary stressors, such as gamma-rays (1.17 and 1.33 MeV; 0.267 keV/microm), ultraviolet (UV) and proton irradiation and elevated temperatures. Resistance to secondary stressors was also observed in rad52delta cells that survived exposure to gamma-rays, rather than protons, but this was not observed to occur in rad52delta cells after UV irradiation. rad52delta cells that survived exposure to protons, followed by gamma-rays (proton surviving colonies were cultured prior to gamma-ray exposure), exhibited an additive effect, whereby these cells had a further increase in stress resistance. A genetic analysis indicated that increased stress resistance is most likely due to a second-site mutation that suppresses the rad52delta phenotype. We will discuss possible origins of these second-site mutations.

Chromatin assembly in a yeast whole-cell extract.

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH: ubiquinone reductase of Neurospora mitochondria.

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH: ubiquinone reductase (Complex I) from Neurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain of N. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functional nuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH: ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.

Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position-independent functions in assembly but not in gene regulation.

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.

Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I in Neurospora crassa by repeat induced point mutation (RIP).

We have used the procedure of sheltered RIP to generate mutants of the 78-kDa protein of the peripheral arm of Neurospora crassa complex I. The nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. This demonstrates that the gene does not serve an essential function. Sequence analysis of one allele shows that 61 transition mutations were created resulting in 39 amino-acid changes including the introduction of four stop codons. Mutant strains grow at a slower rate than wild-type and exhibit a decrease in the production of conidia. Electron paramagnetic spectroscopy of mutant mitochondria suggest that they are deficient in Fe-S clusters N-1, N-3, and N-4.

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria.

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.