A generic strategy for CRISPR-Cas9-mediated gene tagging.

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.

A genomics approach identifies senescence-specific gene expression regulation.

Replicative senescence is a fundamental tumor-suppressive mechanism triggered by telomere erosion that results in a permanent cell cycle arrest. To understand the impact of telomere shortening on gene expression, we analyzed the transcriptome of diploid human fibroblasts as they progressed toward and entered into senescence. We distinguished novel transcription regulation due to replicative senescence by comparing senescence-specific expression profiles to profiles from cells arrested by DNA damage or serum starvation. Only a small specific subset of genes was identified that was truly senescence-regulated and changes in gene expression were exacerbated from presenescent to senescent cells. The majority of gene expression regulation in replicative senescence was shown to occur due to telomere shortening, as exogenous telomerase activity reverted most of these changes.

Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.

The mechanism of activation of the alternative lengthening of telomeres (ALT) pathway of mammalian chromosome-end maintenance has been unclear. We have now discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT-associated PML (promyelocytic leukemia) bodies (APBs), the presence of extrachromosomal telomeric DNA species, an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and intertelomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We determined that ALT induction is positively regulated by the proteins RAD17 and BLM and negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly supports the hypothesis that ALT is a consequence of histone management dysfunction.

C. elegans survivors without telomerase.

In most eukaryotic organisms with a linear genome, the telomerase complex is essential for telomere maintenance and, thus, for genomic integrity. Proper telomerase function in stem and germ cell populations counteracts replication-dependent telomere shortening. On the other hand, repression of telomerase expression in most somatic tissues limits the proliferative potential of these cells through the induction of a permanent cell cycle arrest termed senescence upon critical telomere erosion. Thus, senescence, induced by telomere shortening and subsequent DNA damage signaling, is an essential tumor suppressive mechanism, emphasized by the fact that repression of telomerase is lost in about 90% of cancers, endowing them with unlimited proliferative potential. In 10% of cancers telomeres are maintained using the recombination-based alternative mechanism of telomere lengthening (ALT). To date, ALT and ALT-like mechanisms have only been described in the context of individual cells such as cancer cells and yeast. Now, several "survivor" strains of the nematode Caenorhabditis elegans have been generated that can propagate despite mutations of the telomerase gene. These nematode strains represent the first multi-cellular organism with canonical telomerase that can survive in the absence of a functional telomerase pathway.

Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast.

BACKGROUND: Gene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed global regulation during different stress conditions in fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA, translation, and protein profiles. RESULTS: We observed a strong overall concordance between changes in mRNAs and co-directional changes in translation, for both induced and repressed genes, in response to three conditions: oxidative stress, heat shock, and DNA damage. However, approximately 200 genes each under oxidative and heat stress conditions showed discordant regulation with respect to mRNA and translation profiles, with genes and patterns of regulation being stress-specific. For oxidative stress, we also measured dynamic profiles for 2,147 proteins, comprising 43% of the proteome. The mRNAs induced during oxidative stress strongly correlated with increased protein expression, while repressed mRNAs did not relate to the corresponding protein profiles. Overall changes in relative protein expression correlated better with changes in mRNA expression than with changes in translational efficiency. CONCLUSIONS: These data highlight a global coordination and fine-tuning of gene regulation during stress that mostly acts in the same direction at the levels of transcription and translation. In the oxidative stress condition analyzed, transcription dominates translation to control protein abundance. The concordant regulation of transcription and translation leads to the expected adjustment in protein expression only for up-regulated mRNAs. These patterns of control might reflect the need to balance protein production for stress survival given a limited translational capacity.

Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans.

To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.

A change in nuclear pore complex composition regulates cell differentiation.

Nuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.

A siRNA-based screen for genes involved in chromosome end protection.

Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG) and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci). We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1), a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach.

Negative regulation of meiotic gene expression by the nuclear poly(a)-binding protein in fission yeast.

Meiosis is a cellular differentiation process in which hundreds of genes are temporally induced. Because the expression of meiotic genes during mitosis is detrimental to proliferation, meiotic genes must be negatively regulated in the mitotic cell cycle. Yet, little is known about mechanisms used by mitotic cells to repress meiosis-specific genes. Here we show that the poly(A)-binding protein Pab2, the fission yeast homolog of mammalian PABPN1, controls the expression of several meiotic transcripts during mitotic division. Our results from chromatin immunoprecipitation and promoter-swapping experiments indicate that Pab2 controls meiotic genes post-transcriptionally. Consistently, we show that the nuclear exosome complex cooperates with Pab2 in the negative regulation of meiotic genes. We also found that Pab2 plays a role in the RNA decay pathway orchestrated by Mmi1, a previously described factor that functions in the post-transcriptional elimination of meiotic transcripts. Our results support a model in which Mmi1 selectively targets meiotic transcripts for degradation via Pab2 and the exosome. Our findings have therefore uncovered a mode of gene regulation whereby a poly(A)-binding protein promotes RNA degradation in the nucleus to prevent untimely expression.

The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs.

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

The fission yeast homeodomain protein Yox1p binds to MBF and confines MBF-dependent cell-cycle transcription to G1-S via negative feedback.

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle-regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident.

Translational control of gene expression from transcripts to transcriptomes.

The regulation of gene expression is fundamental to diverse biological processes, including cell growth and division, adaptation to environmental stress, as well as differentiation and development. Gene expression is controlled at multiple levels from transcription to protein degradation. The regulation at the level of translation, from specific transcripts to entire transcriptomes, adds considerable richness and sophistication to gene regulation. The past decade has provided much insight into the diversity of mechanisms and strategies to regulate translation in response to external or internal factors. Moreover, the increased application of different global approaches now provides a wealth of information on gene expression control from a genome-wide perspective. Here, we will (1) describe aspects of mRNA processing and translation that are most relevant to translational regulation, (2) review both well-known and emerging concepts of translational regulation, and (3) survey recent approaches to analyze translational and related posttranscriptional regulation at genome-wide levels.

The BioGRID Interaction Database: 2008 update.

The Biological General Repository for Interaction Datasets (BioGRID) database (http://www.thebiogrid.org) was developed to house and distribute collections of protein and genetic interactions from major model organism species. BioGRID currently contains over 198 000 interactions from six different species, as derived from both high-throughput studies and conventional focused studies. Through comprehensive curation efforts, BioGRID now includes a virtually complete set of interactions reported to date in the primary literature for both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. A number of new features have been added to the BioGRID including an improved user interface to display interactions based on different attributes, a mirror site and a dedicated interaction management system to coordinate curation across different locations. The BioGRID provides interaction data with monthly updates to Saccharomyces Genome Database, Flybase and Entrez Gene. Source code for the BioGRID and the linked Osprey network visualization system is now freely available without restriction.

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly.

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Delta strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.

Global transcriptional responses of fission and budding yeast to changes in copper and iron levels: a comparative study.

BACKGROUND: Recent studies in comparative genomics demonstrate that interspecies comparison represents a powerful tool for identifying both conserved and specialized biologic processes across large evolutionary distances. All cells must adjust to environmental fluctuations in metal levels, because levels that are too low or too high can be detrimental. Here we explore the conservation of metal homoeostasis in two distantly related yeasts. RESULTS: We examined genome-wide gene expression responses to changing copper and iron levels in budding and fission yeast using DNA microarrays. The comparison reveals conservation of only a small core set of genes, defining the copper and iron regulons, with a larger number of additional genes being specific for each species. Novel regulatory targets were identified in Schizosaccharomyces pombe for Cuf1p (pex7 and SPAC3G6.05) and Fep1p (srx1, sib1, sib2, rds1, isu1, SPBC27B12.03c, SPAC1F8.02c, and SPBC947.05c). We also present evidence refuting a direct role of Cuf1p in the repression of genes involved in iron uptake. Remarkable differences were detected in responses of the two yeasts to excess copper, probably reflecting evolutionary adaptation to different environments. CONCLUSION: The considerable evolutionary distance between budding and fission yeast resulted in substantial diversion in the regulation of copper and iron homeostasis. Despite these differences, the conserved regulation of a core set of genes involved in the uptake of these metals provides valuable clues to key features of metal metabolism.

A network of multiple regulatory layers shapes gene expression in fission yeast.

Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and unexpected relationships between distinct aspects of gene expression. Translation and polyadenylation are aligned on a global scale with both the lengths and levels of mRNAs: efficiently translated mRNAs have longer poly(A) tails and are shorter, more stable, and more efficiently transcribed on average. Transcription and translation may be independently but congruently optimized to streamline protein production. These rich data sets, all acquired under a standardized condition, reveal a substantial coordination between regulatory layers and provide a basis for a systems-level understanding of multilayered gene-expression programs.

Autoregulation of ribosome biosynthesis by a translational response in fission yeast.

Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3Delta mutants. Importantly, nearly all of the 40S ribosomal protein-encoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.

A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo.

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man.