Anna Vilborg

Widespread Inducible Transcription Downstream of Human Genes

Pervasive transcription of the human genome generates RNAs whose mode of formation and functions are largely uncharacterized. Here, we combine RNA-seq with detailed mechanistic studies to describe a transcript type derived from protein-coding genes. The resulting RNAs, which we call DoGs for downstream of gene containing transcripts, possess long non-coding regions (often >45 kb) and remain chromatin bound. DoGs are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoG levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. We detect DoG transcription in several human cell lines and provide evidence for thousands of DoGs genome wide.

Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

Significance Cells and organisms live in constantly changing environments. Therefore, cells have evolved complex mechanisms to cope with physiological and environmental stresses. Many of these mechanisms involve transcriptional responses facilitating survival and adaptation. Recent evidence documents extensive transcriptional readthrough beyond annotated gene ends in response to stress, but the role and regulation of these downstream of gene-containing transcripts (DoGs) remain elusive. Here we report that induction of transcriptional readthrough is a hallmark of the mammalian stress response. We explore its causes and consequences in a genome-wide fashion, identifying thousands of readthrough transcripts that are induced in three different stress conditions. Our results suggest potential roles for this class of transcripts in the maintenance of open chromatin under stress. Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.

Readthrough transcription: How are DoGs made and what do they do?

ABSTRACT In recent years, the realization that most of the genome is transcribed has transformed the study of mammalian gene expression. Much effort has gone into investigating how this pervasive transcription is regulated and what the functions of the resulting transcripts are, if any. We recently discovered that stress-induced transcriptional readthrough generates very long downstream of gene containing transcripts (DoGs), which may explain up to 20% of intergenic transcription. DoGs are induced by osmotic stress at the level of transcription by a mechanism that depends on calcium release from the endoplasmic reticulum mediated by IP3 receptors. Here, we discuss DoG induction and function in the context of the literature, with special focus on 2 outstanding questions. First, we discuss possible molecular mechanisms underlying DoG induction through reduced transcription termination. Second, we explore how DoGs may function in maintaining euchromatin after nuclear scaffold stress. In short, we review important aspects of DoG biogenesis and function, and provide an outlook for continued DoG study.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification—this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.Cancer diagnostics: Targeted DNA sequencing for low-quality tumor samplesA new DNA sequencing technology enables comprehensive genetic analyses of poor-quality tumor samples. Hanlee Ji from Stanford University in California, USA, together with colleagues from a company he cofounded called TOMA Biosciences, tested the performance of a targeted sequencing assay known as oligonucleotide-selective sequencing (OS-Seq). They used the “in-solution” version of OS-Seq, which involves a pre-processing step to remove any damaged DNA and then sequences target regions of the genome to look for duplications, insertions or deletions of DNA segments. Using archival specimens (which often contain low quantities of degraded DNA) from patients with lung and colorectal cancer, the researchers showed they could detect sequence variants in a panel of 130 cancer-related genes. The findings suggest the OS-Seq assay could help inform treatment decisions for cancer patients, even with clinical specimens of low quality.

Abstract 422: Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications

Next Generation Sequencing is increasingly used in translational cancer research and as a diagnostic test. Most tumor specimens available for testing are formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE-derived DNA is typically damaged, causing difficulties for standard library preparation methods. Frayed ends prevent blunt-ended double-stranded adapter ligation, used for whole genome sequencing (WGS) and targeted bait hybridization library preparation. Fragmented DNA and modified bases interfere with PCR, which underlies amplification based targeted sequencing. We present a single-stranded library preparation method especially forgiving for low quality FFPE-derived DNA. By excising damaged bases and using single-stranded adapter ligation, we omit repair aimed at generating blunt-ended double-stranded DNA, and eliminate the need for whole genome PCR. After ligation of the first adapter, libraries can either undergo second adapter ligation to generate WGS libraries, or be used as input to oligo-selective sequencing (OS-Seq) target capture. We show that our FFPE-derived PCR-free WGS libraries exhibit coverage depth and uniformity highly similar to double-stranded PCR-free libraries of matched cell line (non-FFPE) materials. Additionally, we present performance of our WGS library preparation on clinical FFEP samples. We further demonstrate reproducible high and uniform coverage for reference standards and clinical FFPE samples in targeted capture. We show a lack of correlation between DNA quality and sequencing metrics such as on-target rate (R 2 =0.005), uniformity (R 2 =0.0002), and coverage (R 2 =0.06), with successful removal of C>T FFPE artifacts comparable to standard repair methods. We demonstrate sensitive copy number alteration detection through analysis of coverage depth, feasible due to the absence of pre-capture PCR. In conclusion, our single-stranded library preparation method is forgiving for sample quality and provides uniform and high coverage as the basis for somatic variant calling. Citation Format: Anna Vilborg. Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 422.

Abstract 4019: A PCR-bias free capture-based library preparation platform permitting highly accurate and sensitive CNA detection in tumor molecular profiling and liquid biopsy

Next Generation Sequencing is increasingly implemented as a diagnostic test to identify actionable mutations in cancer patient samples. However, for routine diagnostics, tumor DNA is extracted from formalin-fixed, paraffin-embedded (FFPE) samples, which yields low quantity of damaged DNA. Inability to accurately repair the ends of these DNA fragments impairs adapter ligation by standard double stranded ligation methods. The resulting low yield of adapter-ligated DNA introduces the need for whole-genome PCR amplification prior to target capture. The drawback of such PCR amplification is the introduction of PCR biases, causing reduced sensitivity in the detection of copy number alterations (CNAs), an important biomarker for targeted therapy. To address the need for a library preparation platform that performs well with low quality and quantity DNA, and without relying on massive PCR amplification, we developed an improved, in-solution, version the OS-Seq targeted enrichment assay. OS-Seq circumvents the reliance on PCR amplification by using a single-stranded adapter ligation approach. Damaged bases induced by formalin fixation are removed by excision instead of attempting repair, and then DNA is denatured prior to adapter ligation. This method of adapter ligation result in yields of ~50% for low quality samples, eliminating the need for whole genome PCR. OS-Seq directly uses the adapter-ligated DNA in a linear targeted primer-extension, followed by low-cycle post-capture PCR expansion with Illumina bridge-PCR primers prior to library sequencing. We investigated the PCR duplication rate of the OS-Seq libraries by including an 11-mer random barcode to track unique molecules. We found that most input molecules were present in the sequencing reads at only one copy. Further, we demonstrate a linear correlation between the amount of DNA input (ranging from 1 to 600 ng) and the number of unique molecules sequenced (R2=0.94). Importantly, we show that this low PCR bias allows OS-Seq to detect CNAs in Coriell and Horizon Diagnostic cell lines highly concordant to digital PCR detection (R2=0.96). Further, we present CNA calling on cell line DNA sonicated to 200 bp fragments at 10 ng DNA input, mimicking cell-free DNA. In addition to CNA detection, OS-Seq detects SNVs with a sensitivity of 92-97% and a specificity of 100% down to 5% VAF. In conclusion, the OS-Seq library preparation method relies on single stranded adapter ligation and in-solution target capture, which generates uniform coverage with minimal PCR requirement, resulting in highly sensitive CNA calling. Note: This abstract was not presented at the meeting. Citation Format: Anna Vilborg, Yosr Bouhlal, Ryan Koheler, Daniel Mendoza, Federico Goodsaid, Yannick Pouliot, Austin So, Francisco De La Vega, Hanlee Ji. A PCR-bias free capture-based library preparation platform permitting highly accurate and sensitive CNA detection in tumor molecular profiling and liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2017-4019

Calcium signaling and transcription: elongation, DoGs, and eRNAs

The calcium ion (Ca2+) is a key intracellular signaling molecule with far-reaching effects on many cellular processes. One of the most important such Ca2+ regulated processes is transcription. A body of literature describes the effect of Ca2+ signaling on transcription initiation as occurring mainly through activation of gene-specific transcription factors by Ca2+-induced signaling cascades. However, the reach of Ca2+ extends far beyond the first step of transcription. In fact, Ca2+ can regulate all phases of transcription, with additional effects on transcription-associated events such as alternative splicing. Importantly, Ca2+ signaling mediates reduced transcription termination in response to certain stress conditions. This reduction allows readthrough transcription, generating a highly inducible and diverse class of downstream of gene containing transcripts (DoGs) that we have recently described.

Abstract IA14: Noncoding RNAs of viral and cellular origin: Links to oncogenesis

EBER2 is a highly abundant nuclear noncoding RNA expressed by Epstein-Barr virus (EBV). Probing its possible chromatin localization by Capture Hybridization Analysis of RNA Targets (CHART) revealed EBER29s presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the important B-cell transcription factor PAX5. EBER2 interacts with and is required for PAX5 localization to the TRs. Knockdown of EBER2 or PAX5 decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication, which is known to be linked to the onset of EBV-associated tumors. Recruitment of the EBER2-PAX5 complex is mediated by base pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence in both EBER2 and the TRs. Using base pairing with nascent transcripts to guide an interacting transcription factor to its DNA target site in chromatin is a previously undescribed function for a trans-acting noncoding RNA. We have uncovered a novel transcript type derived from human protein-coding genes that is represented by thousands of examples genome-wide. These RNAs, which we call DoGs for downstream of gene containing transcripts, are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoGs possess long non-coding regions (often >45 kb) and remain chromatin bound. Their levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. DoGs may play a role in reinforcing the nuclear scaffold after stress. 1. Lee, N., Moss, W., Yario, T., Steitz, J.A. (2015). An EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell 160, 607-618. 2. Vilborg, A., Passarelli, M.C., Yario, T.A., Tycowski, K.T. and Steitz, J.A. (2014). Widespread inducible transcription downstream of human genes. Mol. Cell. 59, 449-461. Citation Format: Joan A. Steitz, Nara Lee, Walter N. Moss, Therese A. Yario, Anna Vilborg, Maria C. Passarelli, Kazimierz T. Tycowski. Noncoding RNAs of viral and cellular origin: Links to oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA14.