Reverse transcriptase meets DNA, again: Possible roles for transposable elements in host DNA repair.

Retrotransposons are selfish genetic elements that encode an enzyme, reverse transcriptase (RT), which converts the element-encoded RNA into DNA prior to or during genomic integration. New studies provide compelling evidence that a bacterial group II intron-like RT has adapted enzymatic activities associated with RTs to function in host DNA repair.

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.

Bur1 functions with TORC1 for vacuole-mediated cell cycle progression.

The vacuole/lysosome plays essential roles in the growth and proliferation of many eukaryotic cells via the activation of target of rapamycin complex 1 (TORC1). Moreover, the yeast vacuole/lysosome is necessary for progression of the cell division cycle, in part via signaling through the TORC1 pathway. Here, we show that an essential cyclin-dependent kinase, Bur1, plays a critical role in cell cycle progression in cooperation with TORC1. A mutation in BUR1 combined with a defect in vacuole inheritance shows a synthetic growth defect. Importantly, the double mutant, as well as a bur1-267 mutant on its own, has a severe defect in cell cycle progression from G1 phase. In further support that BUR1 functions with TORC1, mutation of bur1 alone results in high sensitivity to rapamycin, a TORC1 inhibitor. Mechanistic insight for Bur1 function comes from the findings that Bur1 directly phosphorylates Sch9, a target of TORC1, and that both Bur1 and TORC1 are required for the activation of Sch9. Together, these discoveries suggest that multiple signals converge on Sch9 to promote cell cycle progression.

Co-transcriptional splicing efficiencies differ within genes and between cell types.

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII) which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of co-transcriptional splicing in human cells. In this study, we used Bru-seq and BruChase-seq to determine the co-transcriptional splicing efficiencies of 17,000 introns expressed across 6 human cell lines. We found that less than half of all introns across these 6 cell lines were co-transcriptionally spliced. Splicing efficiencies for individual introns showed variations across cell lines, suggesting that splicing may be regulated in a cell-type specific manner. Moreover, the splicing efficiency of introns varied within genes. The efficiency of co-transcriptional splicing did not correlate with gene length, intron position, splice site strengths, or the intron/neighboring exons GC content. However, we identified binding signals from multiple RNA binding proteins (RBPs) that correlated with splicing efficiency, including core spliceosomal machinery components-such as SF3B4, U2AF1 and U2AF2 showing higher binding signals in poorly spliced introns. In addition, multiple RBPs, such as BUD13, PUM1 and SND1, showed preferential binding in exons that flank introns with high splicing efficiencies. The nascent RNA splicing patterns presented here across multiple cell types add to our understanding of the complexity in RNA splicing, wherein RNA-binding proteins may play important roles in determining splicing outcomes in a cell type- and intron-specific manner.

Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo.

Resection of the 5'-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5' ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60-70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

Locus-specific transcription silencing at the FHIT gene suppresses replication stress-induced copy number variant formation and associated replication delay.

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.

Frequency of DNA end joining in trans is not determined by the predamage spatial proximity of double-strand breaks in yeast.

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.

Double-strand breaks in motion: implications for chromosomal rearrangement.

DNA double-strand breaks (DSBs) must be rejoined properly to prevent the occurrence of serious genomic rearrangements associated with many human diseases. Non-homologous end joining (NHEJ) is a DSB repair mechanism known to protect genomic integrity that is also implicated in creating genomic translocations, inversions, deletions, and insertions. We recently investigated the impact of the pre-damage spatial proximity of DSB-bearing loci on the frequency of trans repair by NHEJ and surprisingly found no correlation between them. In this review, we consider various models that might account for these unexpected results. While DSB movement is necessary to explain our findings, many questions remain about the nature and timing of that motion.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics.

Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining.

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.

Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications.

The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation.

Pooled segregant sequencing reveals genetic determinants of yeast pseudohyphal growth.

The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (Σ1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In Σ1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.

Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 (YPEL5) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit (PPP1CB) in CLL. Two of seven index cases (28%) harbored the reciprocal RNA chimeras in our initial screening. Using quantitative real-time PCR (q real-time PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were detected in 97 of 103 CLL samples (95%) but not in paired normal samples, benign lymphocytes, or various unrelated cancers. Whole-genome sequencing and Southern blotting demonstrated no evidence for a genomic fusion between YPEL5 and PPP1CB. YPEL5/PPP1CB chimera, when introduced into mammalian cells, expressed a truncated PPP1CB protein that demonstrated diminished phosphatase activity. PPP1CB silencing resulted in enhanced proliferation and colony formation of MEC1 and JVM3 cells, implying a role in the pathogenesis of mature B-cell leukemia. These studies uncover a potential role for recurrent RNA chimeras involving phosphatases in the pathogenesis of a common form of leukemia.

Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.

De novo CNV formation in mouse embryonic stem cells occurs in the absence of Xrcc4-dependent nonhomologous end joining.

Spontaneous copy number variant (CNV) mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and de novo pathogenic CNVs, features recapitulated in experimental systems in which CNVs are induced by exogenous replication stress. To test whether the canonical nonhomologous end joining (NHEJ) pathway of double-strand break (DSB) repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ-deficient Xrcc4(-/-) mouse embryonic stem (ES) cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4(-/-) cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.

Hydroxyurea induces de novo copy number variants in human cells.

Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. Spontaneous CNVs are also a major cause of genetic and developmental disorders and arise frequently in cancer cells. As with all mutation classes, genetic and environmental factors almost certainly increase the risk for new and deleterious CNVs. However, despite the importance of CNVs, there is limited understanding of these precipitating risk factors and the mechanisms responsible for a large percentage of CNVs. Here we report that low doses of hydroxyurea, an inhibitor of ribonucleotide reductase and an important drug in the treatment of sickle cell disease and other diseases induces a high frequency of de novo CNVs in cultured human cells that resemble pathogenic and aphidicolin-induced CNVs in size and breakpoint structure. These CNVs are distributed throughout the genome, with some hotspots of de novo CNV formation. Sequencing revealed that CNV breakpoint junctions are characterized by short microhomologies, blunt ends, and short insertions. These data provide direct experimental support for models of replication-error origins of CNVs and suggest that any agent or condition that leads to replication stress has the potential to induce deleterious CNVs. In addition, they point to a need for further study of the genomic consequences of the therapeutic use of hydroxyurea.