DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.

Instability throughout the Saccharomyces cerevisiae genome resulting from Pms1 endonuclease deficiency.

The endonuclease activity of Pms1 directs mismatch repair by generating a nick in the newly replicated DNA strand. Inactivating Pms2, the human homologue of yeast Pms1, increases the chances of colorectal and uterine cancers. Here we use whole genome sequencing to show that loss of this endonuclease activity, via the pms1-DE variant, results in strong mutator effects throughout the Saccharomyces cerevisiae genome. Mutation rates are strongly increased for mutations resulting from all types of single-base substitutions and for a wide variety of single- and multi-base indel mutations. Rates for these events are further increased in strains combining pms1-DE with mutator variants of each of the three major leading and lagging strand replicases. In all cases, mutation rates, spectra, biases, and context preferences are statistically indistinguishable from strains with equivalent polymerases but lacking initial mismatch recognition due to deletion of MSH2. This implies that, across the nuclear genome, strand discrimination via the Pms1 endonuclease is as important for MMR as is initial mismatch recognition by Msh2 heterodimers.

Widespread transcriptional pausing and elongation control at enhancers.

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans.

Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents.

Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Correction: Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

[This corrects the article DOI: 10.1371/journal.pbio.3000464.].

Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks.

The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin.

Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.

A post-transcriptional regulon controlled by TtpA, the single tristetraprolin family member expressed in Dictyostelium discoideum.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol Î´) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol Î´ are the observations that Pol Î´ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

Vanadium Pentoxide Exposure Causes Strain-Dependent Changes in Mitochondrial DNA Heteroplasmy, Copy Number, and Lesions, but Not Nuclear DNA Lesions.

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure.

Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells.

H/ACA snoRNA levels are regulated during stem cell differentiation.

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

Mutation signatures specific to DNA alkylating agents in yeast and cancers.

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (SN1 and SN2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that SN1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of SN1-type agents was conserved across yeast, mice and human cancers. Conversely, SN2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to SN2-type alkylating chemicals. The estimates of mutation loads associated with the SN2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the SN2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers.

Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals.

Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.

Investigation of the adolescent female breast transcriptome and the impact of obesity.

BACKGROUND: Early life environmental exposures affect breast development and breast cancer risk in adulthood. The breast is particularly vulnerable during puberty when mammary epithelial cells proliferate exponentially. In overweight/obese (OB) women, inflammation increases breast aromatase expression and estrogen synthesis and promotes estrogen-receptor (ER)-positive breast cancer. In contrast, recent epidemiological studies suggest that obesity during childhood decreases future breast cancer risk. Studies on environmental exposures and breast cancer risk have thus far been limited to animal models. Here, we present the first interrogation of the human adolescent breast at the molecular level and investigate how obesity affects the immature breast. METHODS: We performed RNA-seq in 62 breast tissue samples from adolescent girls/young women (ADOL; mean age 17.8 years) who underwent reduction mammoplasty. Thirty-one subjects were non-overweight/obese (NOB; mean BMI 23.4 kg/m2) and 31 were overweight/obese (OB; BMI 32.1 kg/m2). We also compared our data to published mammary transcriptome datasets from women (mean age 39 years) and young adult mice, rats, and macaques. RESULTS: The ADOL breast transcriptome showed limited (30%) overlap with other species, but 88% overlap with adult women for the 500 most highly expressed genes in each dataset; only 43 genes were shared by all groups. In ADOL, there were 120 differentially expressed genes (DEG) in OB compared with NOB samples (padj < 0.05). Based on these DEG, Ingenuity Pathway Analysis (IPA) identified the cytokines CSF1 and IL-10 and the chemokine receptor CCR2 as among the most highly activated upstream regulators, suggesting increased inflammation in the OB breast. Classical ER targets (e.g., PR, AREG) were not differentially expressed, yet IPA identified the ER and PR and growth factors/receptors (VEGF, HGF, HER3) and kinases (AKT1) involved in hormone-independent ER activation as activated upstream regulators in OB breast tissue. CONCLUSIONS: These studies represent the first investigation of the human breast transcriptome during late puberty/young adulthood and demonstrate that obesity is associated with a transcriptional signature of inflammation which may augment estrogen action in the immature breast microenvironment. We anticipate that these studies will prompt more comprehensive cellular and molecular investigations of obesity and its effect on the breast during this critical developmental window.

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks.

The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.

ORIO (Online Resource for Integrative Omics): a web-based platform for rapid integration of next generation sequencing data.

Established and emerging next generation sequencing (NGS)-based technologies allow for genome-wide interrogation of diverse biological processes. However, accessibility of NGS data remains a problem, and few user-friendly resources exist for integrative analysis of NGS data from different sources and experimental techniques. Here, we present Online Resource for Integrative Omics (ORIO; https://orio.niehs.nih.gov/), a web-based resource with an intuitive user interface for rapid analysis and integration of NGS data. To use ORIO, the user specifies NGS data of interest along with a list of genomic coordinates. Genomic coordinates may be biologically relevant features from a variety of sources, such as ChIP-seq peaks for a given protein or transcription start sites from known gene models. ORIO first iteratively finds read coverage values at each genomic feature for each NGS dataset. Data are then integrated using clustering-based approaches, giving hierarchical relationships across NGS datasets and separating individual genomic features into groups. In focusing its analysis on read coverage, ORIO makes limited assumptions about the analyzed data; this allows the tool to be applied across data from a variety of experiments and techniques. Results from analysis are presented in dynamic displays alongside user-controlled statistical tests, supporting rapid statistical validation of observed results. We emphasize the versatility of ORIO through diverse examples, ranging from NGS data quality control to characterization of enhancer regions and integration of gene expression information. Easily accessible on a public web server, we anticipate wide use of ORIO in genome-wide investigations by life scientists.