A R-loop sensing pathway mediates the relocation of transcribed genes to nuclear pore complexes.

Nuclear pore complexes (NPCs) have increasingly recognized interactions with the genome, as exemplified in yeast, where they bind transcribed or damaged chromatin. By combining genome-wide approaches with live imaging of model loci, we uncover a correlation between NPC association and the accumulation of R-loops, which are genotoxic structures formed through hybridization of nascent RNAs with their DNA templates. Manipulating hybrid formation demonstrates that R-loop accumulation per se, rather than transcription or R-loop-dependent damages, is the primary trigger for relocation to NPCs. Mechanistically, R-loop-dependent repositioning involves their recognition by the ssDNA-binding protein RPA, and SUMO-dependent interactions with NPC-associated factors. Preventing R-loop-dependent relocation leads to lethality in hybrid-accumulating conditions, while NPC tethering of a model hybrid-prone locus attenuates R-loop-dependent genetic instability. Remarkably, this relocation pathway involves molecular factors similar to those required for the association of stalled replication forks with NPCs, supporting the existence of convergent mechanisms for sensing transcriptional and genotoxic stresses.

NPCs and APBs: two HUBs of non-canonical homology-based recombination at telomeres?

Apart from a few rare exceptions, the maintenance of functional telomeres by recombination-based mechanisms is restricted to accidental and/or pathological situations. Originally described in the yeast S. cerevisiae, this mode of telomere repair has gained interest with the discovery of telomerase negative cancers that use alternative lengthening of telomeres (ALT cancer) dependent on homologous recombination. In both yeast and humans, it has been shown that recombination at telomeres is spatially regulated and occurs preferentially at the nuclear pore complexes (NPCs) in yeast and at ALT-associated promyelocytic leukemia nuclear bodies (APBs) in human cells. Here, we discuss the potential relationships between these two membrane-less structures and their role in enabling unconventional recombination pathways.

Telomeric C-circles localize at nuclear pore complexes in Saccharomyces cerevisiae.

As in human cells, yeast telomeres can be maintained in cells lacking telomerase activity by recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). A hallmark of ALT human cancer cells are extrachromosomal telomeric DNA elements called C-circles, whose origin and function have remained unclear. Here, we show that extrachromosomal telomeric C-circles in yeast can be detected shortly after senescence crisis and concomitantly with the production of survivors arising from "type II" recombination events. We uncover that C-circles bind to the nuclear pore complex (NPC) and to the SAGA-TREX2 complex, similar to other non-centromeric episomal DNA. Disrupting the integrity of the SAGA/TREX2 complex affects both C-circle binding to NPCs and type II telomere recombination, suggesting that NPC tethering of C-circles facilitates formation and/or propagation of the long telomere repeats characteristic of type II survivors. Furthermore, we find that disruption of the nuclear diffusion barrier impairs type II recombination. These results support a model in which concentration of C-circles at NPCs benefits type II telomere recombination, highlighting the importance of spatial coordination in ALT-type mechanisms of telomere maintenance.

The Set1 N-terminal domain and Swd2 interact with RNA polymerase II CTD to recruit COMPASS.

Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is important for gene regulation. Set1/COMPASS associates with the RNA polymerase II C-terminal domain (CTD) to establish proper levels and distribution of H3K4 methylations. However, details of CTD association remain unclear. Here we report that the Set1 N-terminal region and the COMPASS subunit Swd2, which interact with each other, are both needed for efficient CTD binding in Saccharomyces cerevisiae. Moreover, a single point mutation in Swd2 that affects its interaction with Set1 also impairs COMPASS recruitment to chromatin and H3K4 methylation. A CTD interaction domain (CID) from the protein Nrd1 can partially substitute for the Set1 N-terminal region to restore CTD interactions and histone methylation. However, even when Set1/COMPASS is recruited via the Nrd1 CID, histone H2B ubiquitylation is still required for efficient H3K4 methylation, indicating that H2Bub acts after the initial recruitment of COMPASS to chromatin.

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules.

Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies and tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing internal runs of T's. This bias is marked for runs of at least 15 T's, but is already detectable for runs of at least 9 T's and therefore concerns more than 20% of expressed transcripts in mouse brain and liver. Finally, we outline that bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of repeat-associated genes such as processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.

Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps.

Plant genomes are often characterized by a high level of repetitiveness and polyploid nature. Consequently, creating genome assemblies for plant genomes is challenging. The introduction of short-read technologies 10 years ago substantially increased the number of available plant genomes. Generally, these assemblies are incomplete and fragmented, and only a few are at the chromosome scale. Recently, Pacific Biosciences and Oxford Nanopore sequencing technologies were commercialized that can sequence long DNA fragments (kilobases to megabase) and, using efficient algorithms, provide high-quality assemblies in terms of contiguity and completeness of repetitive regions1-4. However, even though genome assemblies based on long reads exhibit high contig N50s (>1 Mb), these methods are still insufficient to decipher genome organization at the chromosome level. Here, we describe a strategy based on long reads (MinION or PromethION sequencers) and optical maps (Saphyr system) that can produce chromosome-level assemblies and demonstrate applicability by generating high-quality genome sequences for two new dicotyledon morphotypes, Brassica rapa Z1 (yellow sarson) and Brassica oleracea HDEM (broccoli), and one new monocotyledon, Musa schizocarpa (banana). All three assemblies show contig N50s of >5 Mb and contain scaffolds that represent entire chromosomes or chromosome arms.

Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae.

Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer.

Quantitative Fitness Analysis Identifies exo1∆ and Other Suppressors or Enhancers of Telomere Defects in Schizosaccharomyces pombe.

Synthetic genetic array (SGA) has been successfully used to identify genetic interactions in S. cerevisiae and S. pombe. In S. pombe, SGA methods use either cycloheximide (C) or heat shock (HS) to select double mutants before measuring colony size as a surrogate for fitness. Quantitative Fitness Analysis (QFA) is a different method for determining fitness of microbial strains. In QFA, liquid cultures are spotted onto solid agar and growth curves determined for each spot by photography and model fitting. Here, we compared the two S. pombe SGA methods and found that the HS method was more reproducible for us. We also developed a QFA procedure for S. pombe. We used QFA to identify genetic interactions affecting two temperature sensitive, telomere associated query mutations (taz1Δ and pot1-1). We identify exo1∆ and other gene deletions as suppressors or enhancers of S. pombe telomere defects. Our study identifies known and novel gene deletions affecting the fitness of strains with telomere defects. The interactions we identify may be relevant in human cells.

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1.

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1(BLM). How Exo1 and Dna2-Sgs1(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.

Scoring and manipulating gene position and dynamics using FROS in budding yeast.

The spatial organization of the genome within the nucleus is now seen as a key contributor to genome function. Studying chromatin dynamics in living cells has been rendered possible by the development of fast microscopy coupled with fluorescent repressor operator systems (FROS). In these systems, arrays of protein-binding sites integrated at specific loci by homologous recombination are monitored through the fluorescence of tagged DNA-binding proteins. In the budding yeast, where homologous recombination is efficient, this technique, combined with targeting assay and genetic analysis, has been extremely powerful for studying the determinants and function of chromatin dynamics in living cells. However, issues have been recurrently raised in different species regarding the use of these systems. Here we discuss the different uses of gene tagging with FROS and their limitations, focusing in budding yeast as a model organism.

The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin.

SUMOylation promotes targeting of HP1α to pericentric heterochromatin. Here we identify the SUMO-specific protease SENP7 in mouse as a maintenance factor for HP1α accumulation at this location. SENP7 interacts directly with HP1α, localizes at HP1-enriched pericentric domains and can deconjugate SUMOylated HP1α in vivo. Depletion of SENP7 delocalizes HP1α from pericentric heterochromatin without affecting H3K9me3 levels. We propose that following targeting of HP1α, a subsequent deSUMOylation event enables HP1α retention at these domains.

Tight protein-DNA interactions favor gene silencing.

The heterochromatin-like structure formed by the yeast silent information regulator complex (SIR) represses transcription at the silent mating type loci and telomeres. Here, we report that tight protein-DNA complexes induce ectopic recruitment of the SIR complex, promoting gene silencing and changes in subnuclear localization when cis-acting elements are nearby. Importantly, lack of the replication fork-associated helicase Rrm3 enhances this induced gene repression. Additionally, Sir3 and Sir4 are enriched genome-wide at natural replication pause sites, including tRNA genes. Consistently, inserting a tRNA gene promotes SIR-mediated silencing of a nearby gene. These results reveal that replication stress arising from tight DNA-protein interactions favors heterochromatin formation.

Tip1/CLIP-170 protein is required for correct chromosome poleward movement in fission yeast.

The plus-end microtubule binding proteins (+TIPs) play an important role in the regulation of microtubule stability and cell polarity during interphase. In S. pombe, the CLIP-170 like protein Tip1, together with the kinesin Tea2, moves along the microtubules towards their plus ends. Tip1 also requires the EB1 homolog Mal3 to localize to the microtubule tips. Given the requirement for Tip1 for microtubule stability, we have investigated its role during spindle morphogenesis and chromosome movement. Loss of Tip1 affects metaphase plate formation and leads to the activation of the spindle assembly checkpoint. In the absence of Tip1 we also observed the appearance of lagging chromosomes, which do not influence the normal rate of spindle elongation. Our results suggest that S. pombe Tip1/CLIP170 is directly or indirectly required for correct chromosome poleward movement independently of Mal3/EB1.

Re-positioning genes to the nuclear envelope in mammalian cells: impact on transcription.

The spatial organization of the genome within the nucleus is thought to contribute to genome functions. A key component of the nuclear architecture is the nuclear envelope, which is often associated with inactive chromatin. Studies in budding yeast indicate that nuclear position can directly affect gene function. However, the causal relationship between gene position and gene activity in mammalian cells has been more elusive. Several groups recently addressed this issue by tethering genes to the inner nuclear membrane. Their studies show that the nuclear periphery is not refractory to gene transcription, but can modulate the activity of certain genes. The 3D organization of the genome might, thus, provide an additional level of regulation necessary for fine-tuning gene expression.