Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

Double-Stranded RNA Structural Elements Holding the Key to Translational Regulation in Cancer: The Case of Editing in RNA-Binding Motif Protein 8A

Mesothelioma is an aggressive cancer associated with asbestos exposure. RNA-binding motif protein 8a (RBM8A) mRNA editing increases in mouse tissues upon asbestos exposure. The aim of this study was to further characterize the role of RBM8A in mesothelioma and the consequences of its mRNA editing. RBM8A protein expression was higher in mesothelioma compared to mesothelial cells. Silencing RBM8A changed splicing patterns in mesothelial and mesothelioma cells but drastically reduced viability only in mesothelioma cells. In the tissues of asbestos-exposed mice, editing of Rbm8a mRNA was associated with increased protein immunoreactivity, with no change in mRNA levels. Increased adenosine deaminase acting on dsRNA (ADAR)-dependent editing of Alu elements in the RBM8A 3′UTR was observed in mesothelioma cells compared to mesothelial cells. Editing stabilized protein expression. The unedited RBM8A 3′UTR had a stronger interaction with Musashi (MSI) compared to the edited form. The silencing of MSI2 in mesothelioma or overexpression of Adar2 in mesothelial cells resulted in increased RBM8A protein levels. Therefore, ADAR-dependent editing contributes to maintaining elevated RBM8A protein levels in mesothelioma by counteracting MSI2-driven downregulation. A wider implication of this mechanism for the translational control of protein expression is suggested by the editing of similarly structured Alu elements in several other transcripts.

MeX pipeline for analysis of mobile genetic elements in cancer genome

Background: Mobile genetic elements (MGEs) comprise a major portion of the human genome and are essential for genetic diversity. These elements are known to have the capability to induce mutations in the human genome. To date, there are several MGE insertions which have been reported to be associated with cancer. We aim to use genome next-generation sequencing data and appropriate bioinformatics tools to accurately identify the insertion sites of MGEs in the human genome.Results: Herein, we introduce the MeX pipeline for the localization and annotation of MGEs in paired-end sequencing data. It requires the reference genome sequence, MGE sequences and paired-end sequencing reads. We evaluated MeX on high depth (>75×) Illumina HiSeq data produced at the Broad Institute (NA12878) against human genome 38-built (including only chromosome 1, 2 and 3) and Alu elements. We could identify 78 reference and 1 non-reference Alu insertions in the NA12878 sample. Upon annotation, it was found that the non-reference Alu element was in the 3' UTR region of the RNF2 gene. Out of 78 reference insertions, 42 were in the intronic region, 7 in the upstream region, 5 in the downstream region, 1 in the 3’ UTR region and the rest were not associated with any gene. MeX showed high performance for the identification and annotation of MGEs in genome samples.Conclusion: This study showed that MeX is a robust and powerful tool for the identification and annotation of MGE insertions. It may also serve as a valuable tool to study the phenotypic changes resulting from transpositional events in cancer genomics.

The Mutational Dynamics of Short Tandem Repeats in Large, Multigenerational Families

Short tandem repeats (STRs) are tandemly repeated sequences of 1-6 bp motifs. STRs compose approximately 3% of the genome, and mutations at STR loci have been linked to dozens of human diseases including amyotrophic lateral sclerosis, Friedreich ataxia, Huntington disease, and fragile X syndrome. Improving our understanding of these mutations would increase our knowledge of the mutational dynamics of the genome and may uncover additional loci that contribute to disease. Here, to estimate the genome-wide pattern of mutations at STR loci, we analyzed blood-derived whole-genome sequencing data for 544 individuals from 29 three-generation CEPH pedigrees. These pedigrees contain both sets of grandparents, the parents, and an average of 9 grandchildren per family. Using HipSTR we identified de novo STR mutations in the 2nd generation of these pedigrees. Analyzing ~1.6 million STR loci, we estimate the empircal de novo STR mutation rate to be 5.24*10-5 mutations per locus per generation. We find that perfect repeats mutate ~2x more often than imperfect repeats. De novo STRs are significantly enriched in Alu elements (p < 2.2e-16). Approximately 30% of STR mutations occur within Alu elements, which compose only ~11% of the genome, and ~10% are found in LINE-1 insertions, which compose ~17% of the genome. Phasing these de novo mutations to the parent of origin shows that parental transmission biases vary among families. We estimate the average number of de novo genome-wide STR mutations per individual to be ~85, which is similar to the average number of observed de novo single nucleotide variants.

Alu repeats at the boundaries of regulatory elements shape the epigenetic environment of immune genes in T cells

Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates essentially all H3K4me3 marks, while excluding DNA methylation. In contrast, enhancer-enriched H3K4me1 and H3K27ac marks eventually cross the first-Alu limit, consistent with enhancer-annotation occasionally overlapping with Alu elements. Remarkably, the average length of TSS-to-first Alu intervals greatly varies in-between tissues, being longer in stem- and shorter in immune-cells. Shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with serendipitous RNA polymerase II transcription and accumulation of H3K4me1 signal at the first-Alu. At several T-cell first-Alus, the DNA methylation was further found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alu upstream of TSSs functions as a dynamic boundary for regulatory elements, initiating the upstream DNA-methylation landscape, while also participating in the recording of immune gene transcriptional events.

Stochastic Effects in Retrotransposon Dynamics Revealed by Modeling under Competition for Cellular Resources

Transposons are genomic elements that can relocate within a host genome using a ‘cut’- or ‘copy-and-paste’ mechanism. They make up a significant part of many genomes, serve as a driving force for genome evolution, and are linked with Mendelian diseases and cancers. Interactions between two specific retrotransposon types, autonomous (e.g., LINE1/L1) and nonautonomous (e.g., Alu), may lead to fluctuations in the number of these transposons in the genome over multiple cell generations. We developed and examined a simple model of retrotransposon dynamics under conditions where transposon replication machinery competed for cellular resources: namely, free ribosomes and available energy (i.e., ATP molecules). Such competition is likely to occur in stress conditions that a malfunctioning cell may experience as a result of a malignant transformation. The modeling revealed that the number of actively replicating LINE1 and Alu elements in a cell decreases with the increasing competition for resources; however, stochastic effects interfere with this simple trend. We stochastically simulated the transposon dynamics in a cell population and showed that the population splits into pools with drastically different transposon behaviors. The early extinction of active Alu elements resulted in a larger number of LINE1 copies occurring in the first pool, as there was no competition between the two types of transposons in this pool. In the other pool, the competition process remained and the number of L1 copies was kept small. As the level of available resources reached a critical value, both types of dynamics demonstrated an increase in noise levels, and both the period and the amplitude of predator–prey oscillations rose in one of the cell pools. We hypothesized that the presented dynamical effects associated with the impact of the competition for cellular resources inflicted on the dynamics of retrotransposable elements could be used as a characteristic feature to assess a cell state, or to control the transposon activity.

Intronic Architecture Links DNA Methylation to Gene Expression and Helps Drive Subtype-Specific Transcriptional Landscapes in DNMT3A- and IDH1/2-Mutant Acute Myeloid Leukemias (AML)

Mutations in DNMT3A and IDH1/2 are each found in ~20% of AML patients. 10-15% of AMLs carry mutations in both genes (herein, double mutants), resulting in a unique methylation landscape and upregulation of a signaling signature. In murine models, the presence of both mutations results in greater leukemogenic potential. However, the specific mechanism through which DNA methylation (DNAme) drives gene expression programs in double mutants remains unclear. We hypothesized that the link between DNAme and gene expression would be explained by more than simple proximity, and that the genomic architecture of the affected genes would play a key role. To test this, we first performed an unbiased correlation analysis of gene expression with DNAme at all CpG sites (mCs) located within the same topologically associated domain (TAD). We identified 406 genes with significant (FDR&gt; 5% and absolute rho &gt; 0.5) expression-methylation correlations with mCs proximal to the respective genes (herein the E-M gene set). In addition, another 2,088 genes (the L E-M set) were identified with long-range correlations (&gt;2Kb from the gene body) with mCs in the respective TAD (median distance = 451 Kb). As a set, the E-M genes significantly overlapped (P &lt; 10 -2) with genes identified as either differentially expressed (DE; n=890) or differentially methylated (DM; n= 4,006) between IDH1/2 and DNMT3A mutant AMLs. Notably, a simple overlap analysis of DE and DM genes showed no significant overlap between them, thus demonstrating that correlation analysis performed better in bridging the epigenome with the transcriptome. DAVID and Gene Set Enrichment Analysis on the genes ranked by correlation strength revealed that signaling, fructose and lipid metabolism pathways are enriched in the E-M gene set (FDR &lt; 5%) but not in the L E-M set. Analysis of transcription factor (TF) binding profiles did not reveal a common set of TF(s) binding to the mCs proximal to the genes of the identified pathways. Thus, we hypothesized that the E-M genes have other structural characteristics in common that drive regulation through DNAme, for which we focused on their genomic architecture. This analysis revealed that introns of genes in both the E-M and L E-M sets are significantly denser in Mammalian Interspersed Repeats (MIR) than expected by random chance (P &lt; 10 -2). Additionally, E-M genes were significantly sparser in endogenous retroviruses (ERVL) and primate-specific Alu elements. mCs with significant correlations were also enriched at MIR and depleted from Alu elements (P &lt; 10 -2), thus creating a regulatory network between mCs and genes with MIR sequences as the common denominator. Genome-wide, CpGs within retrotransposons that were differentially methylated among the three AML subtypes were enriched at enhancer regions or coding genes, particularly the E-M genes. Furthermore, the Dnmt3a knock-out (KO) or Idh2 R140Q knock-in mouse models display the same architectural biases at genes correlated with DNAme as the E-M genes identified in the human samples. Next, we sought to put our findings in the context of normal hematopoiesis and found that genes upregulated during normal hematopoietic differentiation are significantly denser in MIR elements and sparser of Alu elements than expected (P &lt; 10 -2). Alignment of the leukemic samples within normal differentiation trajectories revealed that double mutants resembled differentiated cell types more closely, while DNMT3A and IDH1/2 single mutants resembled hematopoietic stem cells. The E-M and L E-M sets significantly overlapped (P &lt; 10 -2) with those genes upregulated during myeloid but not erythroid or lymphoid differentiation, demonstrating that genes regulated by DNAme are at the core of the biology of these AMLs. In summary, our integrative work sheds light on a novel mechanism in which epigenetic modifications can regulate gene expression through MIR sequences within introns of hematopoietic-relevant genes and we posit that overlapping CpG dinucleotides may act as recruiters or substrates of DNMT3A and/or TET proteins. This mechanism seems to also be active in normal hematopoiesis and thus, is hijacked by leukemic cells. Therefore, our findings identify retrotransposons as a missing link in the understanding of epigenetic regulation of gene expression, reveal a previously uncharacterized role for these elements in leukemogenesis, and point to different cells of origin for each AML subtype. Disclosures No relevant conflicts of interest to declare.

Identification and Characterization of New Alu Element Insertion in the BRCA1 Exon 14 Associated with Hereditary Breast and Ovarian Cancer

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant cancer predisposition syndrome characterized by an increased risk of breast and ovarian cancers. Germline pathogenic variants in BRCA1 are found in about 7–10% of all familial breast cancers and 10% of ovarian cancers. Alu elements are the most abundant mobile DNA element in the human genome and are known to affect the human genome by different mechanisms leading to human disease. We report here the detection, by next-generation sequencing (NGS) analysis coupled with a suitable bioinformatics pipeline, of an AluYb8 element in exon 14 of the BRCA1 gene in a family with HBOC history first classified as BRCA-negative by Sanger sequencing and first NGS analysis. The c.4475_c.4476insAluYb8 mutation impacts splicing and induces the skipping of exon 14. As a result, the produced mRNA contains a premature stop, leading to the production of a short and likely non-functional protein (pAla1453Glyfs*10). Overall, our study allowed us to identify a novel pathogenic variant in BRCA1 and showed the importance of bioinformatics tool improvement and versioning.

Reduced RNA editing in the failing human heart mediates alternative circular RNA splicing

Background and purpose Post-transcriptional RNA editing is an important mechanism in the development of human diseases. RNA editing can affect RNA stability and alternative splicing. The aim of our study was to characterize RNA editing and its impact on alternative RNA splicing in the healthy and failing human heart. Methods and results Human heart samples of heart failure (HF) patients (n=20) and controls (n=10) were analyzed using RNA sequencing with subsequent analysis of RNA editing. We identified adenosine-to-inosine (A-to-I) editing as the major form of RNA editing in human hearts, being reduced in HF patients. Consistently, we found the editing enzyme ADAR2 reduced in HF patients. A-to-I RNA editing predominantly occurred in intronic regions of protein-coding genes, specifically in repetitive, primate-specific Alu elements which can affect RNA splicing. Indeed, we found 173 circular RNAs (circRNAs) regulated by alternative mRNA splicing in the failing heart. Loss of ADAR2 led to reduced RNA editing concomitant with an increase of circRNA, while overexpression reduced circRNA expression and enhanced RNA editing. Conclusion A-to-I editing is the major type of RNA editing in the human heart, being reduced in HF. We demonstrate a primate-specific alternative RNA splicing mechanism mediated by RNA editing in human hearts. The findings may be relevant to diseases with reduced RNA editing such as cancer, neurological and cardiac diseases. FUNDunding Acknowledgement Type of funding sources: None.

CircHIPK3 Plays Vital Roles in Cardiovascular Disease

Circular RNAs (circRNAs) are covalently closed RNAs that function in various physiological and pathological processes. CircRNAs are widely involved in the development of cardiovascular disease (CVD), one of the leading causes of morbidity and mortality worldwide. CircHIPK3 is generated from the second exon of the HIPK3 gene, a corepressor of homeodomain transcription factors. As an exonic circRNA (ecRNA), circHIPK3 is produced through intron-pairing driven circularization facilitated by Alu elements. In the past 5 years, a growing number of studies have revealed the multifunctional roles of circHIPK3 in different diseases, such as cancer and CVD. CircHIPK3 mainly participates in CVD pathogenesis through interacting with miRNAs. This paper summarizes the current literature on the biogenesis and functions of circHIPK3, elucidates the role of circHIPK3 in different CVD patterns, and explores future perspectives.