Jörg Fuchs

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.

Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3.

Both DNA methylation and post‐translational histone modifications contribute to gene silencing, but the mechanistic relationship between these epigenetic marks is unclear. Mutations in two Arabidopsis genes, the KRYPTONITE (KYP) histone H3 lysine 9 (H3K9) methyltransferase and the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase, cause a reduction of CNG DNA methylation, suggesting that H3K9 methylation controls CNG DNA methylation. Here we show that the chromodomain of CMT3 can directly interact with the N‐terminal tail of histone H3, but only when it is simultaneously methylated at both the H3K9 and H3K27 positions. Furthermore, using chromatin immunoprecipitation analysis and immunohistolocalization experiments, we found that H3K27 methylation colocalizes with H3K9 methylation at CMT3‐controlled loci. The H3K27 methylation present at heterochromatin was not affected by mutations in KYP or in several Arabidopsis PcG related genes including the Enhancer of Zeste homologs, suggesting that a novel pathway controls heterochromatic H3K27 methylation. Our results suggest a model in which H3K9 methylation by KYP, and H3K27 methylation by an unknown enzyme provide a combinatorial histone code for the recruitment of CMT3 to silent loci.

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.

Telomere sequence localization and karyotype evolution in higher plants

Data for chromosomal localization of theArabidopsis-type of telomeric sequence repeats (TTTAGGG)n are compiled for 44 species belonging to 14 families of angiosperms, gymnosperms and bryophytes. For 23 species and seven families this is the first report. Species of all families, except theAlliaceae, revealed these sequences at their chromosome termini. This indicates thatArabidopsis-type telomeric repeats are highly conserved. It is inferred that they represent the basic telomere sequence of higher plant phyla. In theAlliaceae, a deviating sequence (and mechanism?) for the stabilization of chromosome termini has possibly evolved secondarily. Nine species revealed interstitial telomeric sequences in addition to the terminal ones, in three species (Vicia faba, Pinus elliottii, P. sylvestris) also at centromeric positions. Interstitial telomeric sequences may indicate karyotype reconstructions, in particular alterations of chromosome numbers by chromosome fusion — or inversions with one breakpoint within the terminal array of repeats. They may contribute to stabilization of chromosome breaks, especially centric fissions, and increase the frequency of meiotic and illegitimate recombination.

Chromosome territory arrangement and homologous pairing in nuclei of Arabidopsis thaliana are predominantly random except for NOR-bearing chromosomes

Differential painting of all five chromosome pairs of Arabidopsis thaliana revealed for the first time the interphase chromosome arrangement in a euploid plant. Side-by-side arrangement of heterologous chromosome territories and homologous association of chromosomes 1, 3 and 5 (on average in 35–50% of nuclei) are in accordance with the random frequency predicted by computer simulations. Only the nucleolus organizing region (NOR)-bearing chromosome 2 and 4 homologs associate more often than randomly, since NORs mostly attach to a single nucleolus. Somatic pairing of homologous ∼100 kb segments occurs less frequently than homolog association, not significantly more often than expected at random and not simultaneously along the homologs. Thus, chromosome arrangement in Arabidopsis differs from that in Drosophila (characterized by somatic pairing of homologs), in spite of similar genome size, sequence organization and chromosome number. Nevertheless, in up to 31.5% of investigated Arabidopsis nuclei allelic sequences may share positions close enough for homologous recombination.

Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell-derived neural progenitor cells

Mouse embryonic stem (ES) cells were used as an experimental model to study the effects of electromagnetic fields (EMF). ES‐derived nestin‐positive neural progenitor cells were exposed to extremely low frequency EMF simulating power line magnetic fields at 50 Hz (ELF‐EMF) and to radiofrequency EMF simulating the Global System for Mobile Communication (GSM) signals at 1.71 GHz (RF‐EMF). Following EMF exposure, cells were analyzed for transcript levels of cell cycle regulatory, apoptosis‐related, and neural‐specific genes and proteins; changes in proliferation; apoptosis; and cytogenetic effects. Quantitative RT‐PCR analysis revealed that ELF‐EMF exposure to ES‐derived neural cells significantly affected transcript levels of the apoptosis‐related bcl‐2, bax, and cell cycle regulatory “growth arrest DNA damage inducible” GADD45 genes, whereas mRNA levels of neural‐specific genes were not affected. RF‐EMF exposure of neural progenitor cells resulted in down‐regulation of neural‐specific Nurr1 and in up‐regulation of bax and GADD45 mRNA levels. Short‐term RF‐EMF exposure for 6 h, but not for 48 h, resulted in a low and transient increase of DNA double‐strand breaks. No effects of ELF‐ and RF‐EMF on mitochondrial function, nuclear apoptosis, cell proliferation, and chromosomal alterations were observed. We may conclude that EMF exposure of ES‐derived neural progenitor cells transiently affects the transcript level of genes related to apoptosis and cell cycle control. However, these responses are not associated with detectable changes of cell physiology, suggesting compensatory mechanisms at the translational and posttranslational level.

How do Alliaceae stabilize their chromosome ends in the absence of TTTAGGG sequences

TheArabidopsis-type telomeric repeats (5′-TTTAGGG-3′) are highly conserved. In most families of different plant phyla they represent the basic sequence of telomeres that stabilize and protect the chromosome termini. The results presented here show that Alliaceae and some related liliaceous species have no tandemly repeated TTTAGGG sequences. Instead, their chromosomes reveal highly repetitive satellite and/or rDNA sequences at the very ends. These apparently substitute the original plant telomeric sequences in Alliaceae. Both sequence types are very active in homologous recombination and may contribute to the stabilization of chromosome termini via compensation of replication-mediated shortening.

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1–1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Division of the Nucleolus and Its Release of CDC14 during Anaphase of Meiosis I Depends on Separase, SPO12, and SLK19

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesins Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12delta and slk19delta mutants.

The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana

Post-translational modifications of proteins by addition of ubiquitin can regulate protein degradation and localization, protein-protein interactions and transcriptional activation. In the ubiquitylation system, substrate specificity is primarily determined by the E2 ubiquitin-conjugating enzyme (UBC) and the E3 ubiquitin ligase. The Arabidopsis thaliana genome contains 37 genes encoding UBC homologs. However, the biological functions of these genes remain largely uncharacterized. Here, we report reverse genetic characterization of AtUBC1 and AtUBC2. While the loss-of-function single mutants Atubc1-1 and Atubc2-1 only show weak phenotypes, the double mutant Atubc1-1 Atubc2-1 shows a dramatically reduced number of rosette leaves and an early-flowering phenotype. Consistent with these results, the transcript levels of the floral repressor genes FLOWERING LOCUS C (FLC), MADS ASSOCIATED FLOWERING 4 (MAF4) and MAF5 are reduced in the double mutant. Loss-of-function mutants of HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, which were previously reported to encode an E3 involved in histone H2B ubiquitylation, also show an early-flowering phenotype and reduced levels of FLC, MAF4 and MAF5 transcripts. In both Atubc1-1 Atubc2-1 and hub2-2 mutants, H2B mono-ubiquitylation is drastically reduced. Taken together, our results indicate that E2s AtUBC1/AtUBC2 and E3s HUB1/HUB2 together mediate H2B ubiquitylation, which is involved in the activation of floral repressor genes as well as in other processes as indicated by the pleiotropic phenotypes of the mutants.