More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.
Human herpesviruses (HHVs) can reside in a lifelong non-infectious state displaying limited activity in their host and protected from immune responses. One possible way by which HHV-6 achieves this state is by integrating into the telomeric ends of human chromosomes, which are highly repetitive sequences that protect the ends of chromosomes from damage. Various stress conditions can reactivate latent HHV-6 thus increasing the severity of multiple human disorders. Recently, we have identified Chlamydia infection as a natural cause of latent HHV-6 reactivation. Here, we have sought to elucidate the molecular mechanism of HHV-6 reactivation. HHV-6 efficiently utilizes the well-organized telomere maintenance machinery of the host cell to exit from its inactive state and initiate replication to form new viral DNA. We provide experimental evidence that the shortening of telomeres, as a consequence of interference with telomere maintenance, triggers the release of the integrated virus from the chromosome. Our data provide a mechanistic basis to understand HHV-6 reactivation scenarios, which in light of the high prevalence of HHV-6 infection and the possibility of chromosomal integration of other common viruses like HHV-7 have important medical consequences for several million people worldwide.
Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.
Chromosome rearrangements are frequently associated with human cancers. Such rearrangement can result from a DNA break followed by an erroneous repair. Mammalian common fragile sites are one of the most extensively studied naturally occurring breakage prone regions of the genome. It has been proposed that fragile sites are recombination hotspots and that increased recombination activity at these loci contribute to cancer. We examined this hypothesis using a model organism, budding yeast Saccharomyces cerevisiae, where a homolog of the mammalian common fragile sites has been identified. Unexpectedly, our results showed that the rate of recombination at the fragile sites was not any higher than non fragile sites, even under the conditions that promoted chromosome breakage at the fragile sites. However, we found that the frequency of loss of heterozygosity (LOH) and translocation, the type of recombination outcomes known to contribute to cancer, to be significantly elevated at fragile sites under certain conditions. These findings suggest that the fragile sites might indeed contribute to cancer more frequently than non-fragile loci, but the reason for this is likely to be due the nature of the recombination outcome(s) rather than higher rates of recombination.
Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.
Pseudomonas putida strains are among the microorganisms that have acquired the capability to use toxic and xenobiotic compounds, such as nicotine, for growth. Although nicotine degradation by Pseudomonas was first discovered more than 50 years ago, the underlying molecular mechanisms remain unclear. In the last few years, we have made significant efforts to identify the key genes for the hydroxylation of 3-succinoylpyridine (SP) through genomic library screening and purification of wild-type enzymes. However, these efforts did not result in identifying any genes related to SP hydroxylation. In this study, by using comparative genetic analysis, we report the identification of 3 key genes, spmA, spmB and spmC from P. putida S16. The heterotrimeric enzyme encoded by these genes requires molybdopterin-cytosine dinucleotide as a cofactor. The proteomes of strain S16 grown on nicotine or glycerol contain 1,292 observed proteins, and provide a detailed view of enzymes involved in nicotine degradation. Our comparative analysis of the proteomic profiles of nicotine grown versus glycerol grown bacterial cells reveals a wide range of cellular processes and functions related to nicotine catabolism.
How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments.
Heat stress, caused by fluctuations in ambient temperature, occurs frequently in nature. How organisms adapt and maintain regular patterns of growth over a range of environmental conditions remain poorly understood. Our work in the simple unicellular yeast Schizosaccharomyces pombe suggests that the heat stress-associated transcription must be repressed by the evolutionary conserved Hsp70-Hsp40 chaperone complex to allow cells to adapt the polarized growth machinery to elevated temperature.
Nematodes of the genus Caenorhabditis enter a developmental diapause state after hatching in the absence of food. To better understand the relative contributions of distinct regulatory modalities to gene expression changes associated with this developmental transition, we characterized genome-wide changes in mRNA abundance and translational efficiency associated with L1 diapause exit in four species using ribosome profiling and mRNA-seq. We found a strong tendency for translational regulation and mRNA abundance processes to act synergistically, together effecting a dramatic remodeling of the gene expression program. While gene-specific differences were observed between species, overall translational dynamics were broadly and functionally conserved. A striking, conserved feature of the response was strong translational suppression of ribosomal protein production during L1 diapause, followed by activation upon resumed development. On a global scale, ribosome footprint abundance changes showed greater similarity between species than changes in mRNA abundance, illustrating a substantial and genome-wide contribution of translational regulation to evolutionary maintenance of stable gene expression.
Working with a set of four related animal species, we have studied a conserved developmental and metabolic transition at the level of protein production and regulation of RNA levels. Strikingly, regulatory effects at the level of RNA accumulation and protein synthesis act together to achieve the observed metabolic shift. In addition to a general conservation of the underlying basis for the regulation of individual genes, alterations of these two processes—mRNA production and protein synthesis—can compensate for one another during evolution to maintain stable amounts of functional gene products. A salient feature of the observed regulation was the storage of idle mRNAs encoding key members of the protein synthesis machinery during metabolic arrest (diapause). Maintenance of this pool facilitates re-activation upon feeding, with the rapid regeneration of protein synthesis capacity an early and critical function during adaptation to a major metabolic shift.
Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts.
Mounting evidence in several model organisms collectively demonstrates a role for the DNA-protein complexes known as chromatin insulators in orchestrating the functional domain organization of the eukaryotic genome. Several DNA elements displaying features of insulators, viz barrier and/or directional enhancer-blocking activity, have been identified in yeast, Drosophila, sea urchin, vertebrates and plants; however, proteins that bind these DNA sequences eliciting insulator activities are far less known. Here we identify a novel protein, COMPASS-like (CMPl), which is expressed exclusively by the ambulacrarian group of metazoans and interacts directly with the sea urchin sns5 insulator. Sns5 lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter, where it acts buffering the H1 promoter from the H2A enhancer influence. Intriguingly, we find that CMPl role is absolutely required for the sns5 activity, therefore imposing the different level of accumulation of the linker and nucleosomal transcripts. Overall, our findings add an interesting and novel facet to the chromatin insulator field, highlighting the surprisingly low evolutionary conservation of trans-acting factors binding to chromatin insulators. This opens the possibility that multiple lineage-specific factors modulate chromatin organization in different metazoans.
Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm5s2U34 is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm5s2U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non-canonical pathway. Thus, loss of mcm5s2U causes global effects on gene expression due to perturbation of cellular signaling.
Ribosomes translate the messages of the genetic code into functional proteins with the help of transfer RNAs (tRNAs), which carry a specific amino acid at one end and recognize three letters of the genetic code (a codon) with the other. tRNAs are subject to extensive chemical modifications, which are thought to enhance the efficiency, fidelity and specificity of translation. Many of these modifications are conserved across all domains of life, underscoring their biological importance. Despite intensive biochemical characterization, the physiological roles of most tRNA modifications are unknown. The tRNA modification mcm5s2U34 is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. In order to test this model, we determined ribosome distributions at each codon in yeast strains lacking mcm5s2U. The changes we found appeared too small to affect protein output. Instead, we observed a non-canonical activation of a yeast stress response pathway. Thus, loss of mcm5s2U causes widespread perturbation of cellular signaling, independent of any codon-specific translation defects.
The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex.
In multicellular organisms, differentiated cells have a cell-type specific profile of gene expression. Sperm production is particularly specialised, and as a result over 5% of all genes are expressed exclusively in the sperm precursor cells, termed primary spermatocytes. Expression of these genes depends on a particular transcription regulation complex (tMAC) only active in spermatocytes. In this paper we show that a factor, Nxt1, whose previously characterised function is in transport of RNA from the cell nucleus to the cytoplasm, is also required for expression of many testis-specific transcripts. Spermatocytes deficient for Nxt1 fail to express many tMAC-dependent genes, and we show that this effect is due in part to reduced transcription. We further show that processing of the RNA, via splicing, can partially offset the need for Nxt1 in expression of tMAC-dependent genes. Our data reveal an unexpected link between the core RNA processing pathway and a tissue-specific transcription factor.
Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.
Before anaphase onset, the presence of securin Pds1 prevents the cleavage of cohesin, a protein complex that holds sister chromatids together, but the degradation of Pds1 leads to robust cohesion dissolution, resulting in synchronous separation of all chromosomes. Since sister chromatids are attached to spindle microtubules before anaphase onset, spindle elongation segregates sister chromatids to each daughter cell after cohesion dissolution. A fundamental question is how spindle elongation is temporally regulated in order to coordinate with sister chromatid separation. Cyclin-dependent kinases (CDKs) play a key role in the cell cycle, and it is well established that S-phase CDK promotes DNA synthesis. We found that hyperactive S-phase CDK delays spindle elongation in budding yeast, while hyperactive mitotic CDK leads to premature spindle elongation. We further provide evidence indicating that mitotic CDK promotes spindle elongation by activating a protein phosphatase that antagonizes S-phase CDK, thus the balance of S-phase and mitotic CDK controls the timing of spindle elongation. Interestingly, hyperactive mitotic CDK induces uncoupled spindle elongation and sister chromatid separation in the absence of securin, suggesting that the temporal control of spindle elongation and cohesin cleavage is essential for faithful chromosome segregation.
Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants.
Transposable elements (TEs) are selfish DNA sequences. Together with their immobilized derivatives, they account for a large fraction of eukaryotic genomes. TEs can affect nearby gene activity, either directly by disrupting regulatory sequences or indirectly through the host mechanisms used to prevent TE proliferation. A comparison of Arabidopsis thaliana genomes reveals rapid TE degeneration. We asked what drives TE degeneration and how often TE variation affects nearby gene expression. To answer these questions, we studied the interplay between TEs, DNA sequence variation, and short interfering RNAs (siRNAs) in three A. thaliana strains. We find sequence variation in genes and adjacent TEs to be correlated, from which we conclude either that TEs insert more often near polymorphic genes or that TEs next to polymorphic genes are less efficiently purged from the genome. We also noticed that processes that cause deletions within TEs and ones that silence TEs appear to be linked, because siRNA targeting is a predictor of sequence loss in accessions. Our work provides insight into the contribution of TEs to gene expression plasticity, and it links TE silencing mechanisms to the evolution of TE variation between genomes, thereby linking TE silencing mechanisms to expression plasticity.
In many human fungal pathogens, genes required for disease remain largely unannotated, limiting the impact of virulence gene discovery efforts. We tested the utility of a cross-species genetic interaction profiling approach to obtain clues to the molecular function of unannotated pathogenicity factors in the human pathogen Cryptococcus neoformans. This approach involves expression of C. neoformans genes of interest in each member of the Saccharomyces cerevisiae gene deletion library, quantification of their impact on growth, and calculation of the cross-species genetic interaction profiles. To develop functional predictions, we computed and analyzed the correlations of these profiles with existing genetic interaction profiles of S. cerevisiae deletion mutants. For C. neoformans LIV7, which has no S. cerevisiae ortholog, this profiling approach predicted an unanticipated role in the Golgi apparatus. Validation studies in C. neoformans demonstrated that Liv7 is a functional Golgi factor where it promotes the suppression of the exposure of a specific immunostimulatory molecule, mannose, on the cell surface, thereby inhibiting phagocytosis. The genetic interaction profile of another pathogenicity gene that lacks an S. cerevisiae ortholog, LIV6, strongly predicted a role in endosome function. This prediction was also supported by studies of the corresponding C. neoformans null mutant. Our results demonstrate the utility of quantitative cross-species genetic interaction profiling for the functional annotation of fungal pathogenicity proteins of unknown function including, surprisingly, those that are not conserved in sequence across fungi.
HIV/AIDS patients, cancer chemotherapy patients, and organ transplant recipients are highly susceptible to infection by opportunistic fungal pathogens, organisms common in the environment that are harmless to normal individuals. Understanding how these pathogens cause disease requires the identification of genes required for virulence and the determination of their molecular function. Our work addresses the latter problem using the yeast Cryptococcus neoformans, which is estimated to cause 600,000 deaths annually worldwide in the HIV/AIDS population. We describe a method for determining gene function in which C. neoformans genes are expressed in deletion mutants of all nonessential genes of the well-studied model yeast S. cerevisiae. By examining the impact on growth (enhancement or suppression) we generated “cross-species” genetic interaction profiles. We compared these profiles to the published genetic interaction profiles of S. cerevisiae deletion mutants to identify those with correlated patterns of genetic interactions. We hypothesized that the known functions of S. cerevisiae genes with correlated profiles could predict the function of the pathogen gene. Indeed, experimental tests in C. neoformans for two pathogenicity genes of previously unknown function found the functional predictions obtained from genetic interaction profiles to be accurate, demonstrating the utility of the cross-species approach.
Mutations in the BREVIPEDICELLUS (BP) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP, generated by EMS, fast neutrons, x-rays, and aberrant T–DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution.
Chromosome evolution involves both small-scale (e.g. single nucleotide) changes, as well as large-scale rearrangements such as inversions, translocations, and fusion events. We investigated mutations of the BREVIPEDICELLUS gene of Arabidopsis, which is a master regulator of inflorescence architecture. These mutations are not due to single nucleotide changes, but rather to large deletions, some spanning nearly one million base pairs. Molecular and biochemical analyses reveal that the chromosome breakpoints cluster in an area that is rich in repetitive elements and harbor multiple binding sites for nuclear matrix proteins. Data mining revealed intriguing correlations between the breakpoint cluster and hotspots of genetic recombination, regions of the chromosome that have undergone several rearrangement events during evolution, and changes in histone protein modifications. We propose that these unstable regions are chromosome fragile sites that assist in marking a boundary between gene-poor, transcriptionally repressed centromeric chromatin and a more relaxed chromatin domain that is gene-rich.
During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.
Meiotic recombination is important for pairing and sustained association of homologous chromosomes (homologs), thereby ensuring proper homolog segregation and normal fertility. DNA synthesis is thought to be required for meiotic recombination, but few genes coding for DNA synthesis factors have been studied for possible meiotic functions because their essential roles in the mitotic cell cycle make it difficult to study their meiotic functions due to the lethality of corresponding null mutations. Current models for meiotic recombination only include leading strand DNA synthesis. We found that the Arabidopsis gene encoding the DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis promotes meiotic recombination via a specific pathway for crossovers (COs) that involves the formation of double Holliday Junction (dHJ) intermediates. Therefore, lagging strand DNA synthesis is likely important for meiotic recombination. Because DNA synthesis is a highly conserved process and meiotic recombination is highly similar among budding yeast, mammals, and flowering plants, the proposed function of lagging strand synthesis for meiotic recombination might be a general feature of meiosis.
Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation.
Throughout human evolution, one question has remained constant: can we live forever? We are continuously bombarded with products, diets, and exercise regimens that supposedly add years to our life. Is there an alterable program, whether genetic or environmental, that can be tweaked to increase longevity? Medical advances have led to a dramatic increase in average lifespan over the last century. However, the maximum human lifespan has curiously remained constant. Recent research indicates that in many organisms a genetic program exists to control lifespan. The conservation of this genetic lifespan program extends into yeast where numerous longevity genes have been isolated and characterized. Interestingly, mutations that reduce genomic instability, glucose utilization, or oxidative damage extend lifespan in multiple organisms. Here we characterize one such set of genes, the FOXOs. In animals, these genes increase lifespan and suppress tumors, but have yet to be associated with longevity in yeast. By confirming that these genes play a similar role in yeast, we provide a tool to identify downstream factors triggered by the FOXOs, a feat which has not yet been accomplished in other systems. Considering the conservation of these factors, it is likely that our discoveries in yeast will be directly applicable to research into human cancer and aging.
In eukaryotes, histone H3 lysine 9 methylation (H3K9me) mediates silencing of invasive sequences to prevent deleterious consequences including the expression of aberrant gene products and mobilization of transposons. In Arabidopsis thaliana, H3K9me maintained by SUVH histone methyltransferases (MTases) is associated with cytosine methylation (5meC) maintained by the CMT3 cytosine MTase. The SUVHs contain a 5meC binding domain and CMT3 contains an H3K9me binding domain, suggesting that the SUVH/CMT3 pathway involves an amplification loop between H3K9me and 5meC. However, at loci subject to read-through transcription, the stability of the H3K9me/5meC loop requires a mechanism to counteract transcription-coupled loss of H3K9me. Here we use the duplicated PAI genes, which stably maintain SUVH-dependent H3K9me and CMT3-dependent 5meC despite read-through transcription, to show that when PAI sRNAs are depleted by dicer ribonuclease mutations, PAI H3K9me and 5meC levels are reduced and remaining PAI 5meC is destabilized upon inbreeding. The dicer mutations confer weaker reductions in PAI 5meC levels but similar or stronger reductions in PAI H3K9me levels compared to a cmt3 mutation. This comparison indicates a connection between sRNAs and maintenance of H3K9me independent of CMT3 function. The dicer mutations reduce PAI H3K9me and 5meC levels through a distinct mechanism from the known role of dicer-dependent sRNAs in guiding the DRM2 cytosine MTase because the PAI genes maintain H3K9me and 5meC at levels similar to wild type in a drm2 mutant. Our results support a new role for sRNAs in plants to prevent transcription-coupled loss of H3K9me.
Methylation of histone H3 at the lysine 9 position (H3K9me) is a fundamental chromatin modification that suppresses expression from invasive and repetitive sequences such as transposons. In plant genomes, regions modified by H3K9me are maintained with precise boundaries. However, at junctions where H3K9me target regions are subject to read-through transcription from outside promoters, the stability of H3K9me patterns is jeopardized by transcription-coupled processes that remove this modification. We show that maintenance of H3K9me patterns at such vulnerable sites requires small RNAs corresponding to the H3K9me target region. We use a sensitive reporter system to show that, in the absence of small RNAs, target regions subject to read-through transcription undergo an immediate reduction in H3K9me levels, followed by further losses in progeny plants upon inbreeding. Our results support a new function for small RNAs in maintaining accurate H3K9me patterns in the plant genome.
Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10−3 inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.
Organisms strike a balance between genetic continuity and change. Most cells are well adapted to their niches and therefore invest heavily in mechanisms that maintain accurate DNA replication. When cell populations are confronted with changing environmental conditions, “mutator” clones with high mutation rates emerge and readily adapt to the new conditions by rapidly acquiring beneficial mutations. However, deleterious mutations also accumulate, raising the question: what level of mutational burden can cell populations sustain before collapsing? Here we experimentally determine the maximal mutation rate in haploid yeast. We observe that yeast can withstand a 1,000-fold increase in mutation rate without losing colony forming capacity. Yet no strains survive a 10,000-fold increase in mutation rate. Escape mutants with an “anti-mutator” phenotype frequently emerge from cell populations undergoing this error-induced extinction. The diversity of antimutator changes suggests that strong mutator phenotypes in nature may be inherently transient, ensuring that rapid adaptation is followed by genetic attenuation which preserves the beneficial, adaptive mutations. These observations are relevant to microbial populations during infection as well as the somatic evolution of cancer cells.
The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5′-to-3′ resection of DNA ends, generating 3′ single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.
A double-strand break (DSB) is a devastating form of DNA damage. Fortunately, cells are equipped with two DSB repair pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). The Mre11-Rad50-Nbs1 (MRN) protein complex recognizes DSBs and initiates HR repair. The Mre11 subunit harbors a nuclease domain that is essential for repair in fission yeast and mammals, although the function is unknown. Here we show that Mre11 nuclease activity is required to release the Ku complex from DNA ends in fission yeast. While the initiation of repair, i.e. the generation of single-stranded DNA (ssDNA) overhangs in Mre11-nuclease dead mutants, is unaffected, we find that an essential downstream step involving the localization of Replication Protein A (RPA) to ssDNA is substantially decreased due to the inability to release Ku and MRN from the DNA end. In contrast, a DNA processing factor called Ctp1, which binds to Nbs1, is essential for the initiation of repair as well as the release of Ku and MRN from DNA ends. Importantly, we find that efficient localization of RPA, which is essential for efficient DSB repair by HR, requires the release of Ku and MRN from the DNA by the combined action of Mre11 nuclease and Ctp1.