Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor ß (TGF-ß), is reduced; that then leads to the activation of the TGF-ß pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.

A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.

RNA abasic sites in yeast and human cells.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.

Common genetic variants account for differences in gene expression among ethnic groups.

Variation in DNA sequence contributes to individual differences in quantitative traits, but in humans the specific sequence variants are known for very few traits. We characterized variation in gene expression in cells from individuals belonging to three major population groups. This quantitative phenotype differs significantly between European-derived and Asian-derived populations for 1,097 of 4,197 genes tested. For the phenotypes with the strongest evidence of cis determinants, most of the variation is due to allele frequency differences at cis-linked regulators. The results show that specific genetic variation among populations contributes appreciably to differences in gene expression phenotypes. Populations differ in prevalence of many complex genetic diseases, such as diabetes and cardiovascular disease. As some of these are probably influenced by the level of gene expression, our results suggest that allele frequency differences at regulatory polymorphisms also account for some population differences in prevalence of complex diseases.

In silico method for inferring genotypes in pedigrees.

Our genotype inference method combines sparse marker data from a linkage scan and high-resolution SNP genotypes for several individuals to infer genotypes for related individuals. We illustrate the method's utility by inferring over 53 million SNP genotypes for 78 children in the Centre d'Etude du Polymorphisme Humain families. The method can be used to obtain high-density genotypes in different family structures, including nuclear families commonly used in complex disease gene mapping studies.

A quantitative model of normal Caenorhabditis elegans embryogenesis and its disruption after stress.

The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allowroutine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increase in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.

Isoform Switching Regulates the Response to Ionizing Radiation Through SRSF1.

PURPOSE: This study investigated how isoform switching affects the cellular response to ionizing radiation (IR), an understudied area despite its relevance to radiation therapy in cancer treatment. We aimed to identify changes in transcript isoform expression post-IR exposure and the proteins mediating these changes, with a focus on their potential to modulate radiosensitivity. METHODS AND MATERIALS: Using RNA sequencing, we analyzed the B-cell lines derived from 10 healthy individuals at 3 timepoints, applying the mixture of isoforms algorithm to quantify alternative splicing. We examined RNA binding protein motifs within the sequences of IR-responsive isoforms and validated the serine/arginine-rich splicing factor 1 (SRSF1) as a predominant mediator through RNA immunoprecipitation. We further investigated the effects of SRSF1 on radiosensitivity by RNA interference and by analyzing publicly available data on patients with cancer. RESULTS: We identified ∼1900 radiation-responsive alternatively spliced isoforms. Many isoforms were differentially expressed without changes in their overall gene expression. Over a third of these transcripts underwent exon skipping, while others used proximal last exons. These IR-responsive isoforms tended to be shorter transcripts missing vital domains for preventing apoptosis and promoting cell division but retaining those necessary for DNA repair. Our combined computational, genetic, and molecular analyses identified the proto-oncogene SRSF1 as a mediator of these radiation-induced isoform-switching events that promote apoptosis. After exposure to DNA double-strand break-inducing agents, SRSF1 expression decreased. A reduction in SRSF1 increased radiosensitivity in vitro and among patients with cancer. CONCLUSIONS: We establish a pivotal role for isoform switching in the cellular response to IR and propose SRSF1 as a promising biomarker for assessing radiation therapy effectiveness.

Deconvolution of gene expression from cell populations across the C. elegans lineage.

BACKGROUND: Knowledge of when and in which cells each gene is expressed across multicellular organisms is critical in understanding both gene function and regulation of cell type diversity. However, methods for measuring expression typically involve a trade-off between imaging-based methods, which give the precise location of a limited number of genes, and higher throughput methods such as RNA-seq, which include all genes, but are more limited in their resolution to apply to many tissues. We propose an intermediate method, which estimates expression in individual cells, based on high-throughput measurements of expression from multiple overlapping groups of cells. This approach has particular benefits in organisms such as C. elegans where invariant developmental patterns make it possible to define these overlapping populations of cells at single-cell resolution. RESULT: We implement several methods to deconvolve the gene expression in individual cells from population-level data and determine the accuracy of these estimates on simulated data from the C. elegans embryo. CONCLUSION: These simulations suggest that a high-resolution map of expression in the C. elegans embryo may be possible with expression data from as few as 30 cell populations.

Nanopore-based direct sequencing of RNA transcripts with 10 different modified nucleotides reveals gaps in existing technology.

RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current "RNA-sequencing" methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods.

Guanine quadruplexes mediate mitochondrial RNA polymerase pausing.

The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in cells. Guanine-rich sequences form structures called guanine quadruplexes (G4) that result from non-canonical base pairing between guanine residues. These stable structures are enriched in gene promoters and have been correlated with the locations of RNA polymerase II pausing (Pol II). While promoter-proximal RNA polymerase pausing regulates gene expression, the effects of guanine quadruplexes on gene transcription have been less clear. We determined the pattern of mitochondrial RNA polymerase (mtRNAP) pausing in human fibroblasts and found that it pauses over 400 times on the mitochondrial genome. We identified quadruplexes as a mediator of mtRNAP pausing and show that stabilization of quadruplexes impeded transcription by mtRNAP. Gene products encoded by the mitochondrial genome are required for oxidative phosphorylation and the decreased transcription by mtRNAP resulted in lower expression of mitochondrial genes and significantly reduced ATP generation. Energy from mitochondria is essential for transport function in renal epithelia, and impeded mitochondrial transcription inhibits transport function in renal proximal tubule cells. These results link formation of guanine quadruplex structures to regulation of mtRNAP elongation and mitochondrial function.

Monozygotic twins reveal germline contribution to allelic expression differences.

Variation in the level of gene expression is a major determinant of a cell's function and characteristics. Common allelic variants of genes can be expressed at different levels and thus contribute to phenotypic diversity. We have measured allelic expression differences at heterozygous loci in monozygotic twins and in unrelated individuals. We show that the extent of differential allelic expression is highly similar within monozygotic twin pairs for many loci, implying that allelic differences in gene expression are under genetic control. We also show that even subtle departures from equal allelic expression are often genetically determined.

Mapping determinants of human gene expression by regional and genome-wide association.

To study the genetic basis of natural variation in gene expression, we previously carried out genome-wide linkage analysis and mapped the determinants of approximately 1,000 expression phenotypes. In the present study, we carried out association analysis with dense sets of single-nucleotide polymorphism (SNP) markers from the International HapMap Project. For 374 phenotypes, the association study was performed with markers only from regions with strong linkage evidence; these regions all mapped close to the expressed gene. For a subset of 27 phenotypes, analysis of genome-wide association was performed with >770,000 markers. The association analysis with markers under the linkage peaks confirmed the linkage results and narrowed the candidate regulatory regions for many phenotypes with strong linkage evidence. The genome-wide association analysis yielded highly significant results that point to the same locations as the genome scans for about 50% of the phenotypes. For one candidate determinant, we carried out functional analyses and confirmed the variation in cis-acting regulatory activity. Our findings suggest that association studies with dense SNP maps will identify susceptibility loci or other determinants for some complex traits or diseases.

The Lipocalin LPR-1 Cooperates with LIN-3/EGF Signaling To Maintain Narrow Tube Integrity in Caenorhabditis elegans.

Lipocalins are secreted cup-shaped glycoproteins that bind sterols, fatty acids, and other lipophilic molecules. Lipocalins have been implicated in a wide array of processes related to lipophilic cargo transport, sequestration, and signaling, and several are used as biomarkers for human disease, but the functions of most lipocalins remain poorly understood. Here we show that the Caenorhabditis elegans lipocalin LPR-1 is required to maintain apical membrane integrity and a continuous lumen in two narrow unicellular tubes, the excretory duct and pore, during a period of rapid lumen elongation. LPR-1 fusion protein is expressed by the duct and pore and accumulates both intracellularly and in apical extracellular compartments, but it can also function cell nonautonomously when provided from outside of the excretory system. lpr-1 mutant defects can be rescued by increased signaling through the epidermal growth factor (EGF)-Ras-extracellular signal regulated kinase (ERK) pathway, which promotes the more elongated duct vs. less elongated pore tube fate. Spatial and temporal rescue experiments indicate that Ras signaling acts within the duct and pore tubes during or prior to cell fate determination to bypass the requirement for LPR-1 lpr-1 mutations did not disrupt LIN-3/EGF-dependent duct-fate specification, prevent functioning of any specific LIN-3/EGF isoform, or alter LET-23/EGFR localization, and reduced signaling did not phenocopy or enhance lpr-1 mutant defects. These data suggest that LPR-1 protects lumen integrity through a LIN-3/EGF-independent mechanism, but that increased signaling upregulates some target(s) that can compensate for lpr-1 absence.

Polymorphic variation in human meiotic recombination.

In this study, our phenotype of interest is meiotic recombination. Using genotypes of approximately 6,000 SNP markers in members of the Centre d'Etude du Polymorphisme Humain Utah pedigrees, we found extensive individual variation in the number of female and male recombination events. The locations and frequencies of these recombination events vary along the genome. In both female and male meiosis, the regions with the most recombination events are found at the ends of the chromosomes. Our analysis also shows that there are polymorphic differences among individuals in the activity of the recombination "jungles"; these preferred sites of meiotic recombination differ greatly among individuals. These findings have important implications for understanding genetic disorders that result from improper chromosome segregation.

Gene expression phenotype in heterozygous carriers of ataxia telangiectasia.

The defining characteristic of recessive diseases is the absence of a phenotype in the heterozygous carriers. Nonetheless, subtle manifestations may be detectable by new methods, such as expression profiling. Ataxia telangiectasia (AT) is a typical recessive disease, and individual carriers cannot be reliably identified. As a group, however, carriers of an AT disease allele have been reported to have a phenotype that distinguishes them from normal control individuals: increased radiosensitivity and risk of cancer. We show here that the phenotype is also detectable, in lymphoblastoid cells from AT carriers, as changes in expression level of many genes. The differences are manifested both in baseline expression levels and in response to ionizing radiation. Our findings show that carriers of a recessive disease may have an "expression phenotype." In the particular case of AT, this suggests a new approach to the identification of carriers and enhances understanding of their increased cancer risk. More generally, we demonstrate that genomic technologies offer the opportunity to identify and study unaffected carriers, who are hundreds of times more common than affected patients.