ABSTRACT
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ABSTRACT
Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , RNA, Circular/analysis , RNA, Messenger/analysis , RNA, Ribosomal/analysis , Single-Cell Analysis/methods , Benchmarking , Cell Line, Tumor , Gene Library , Humans , Microfluidic Analytical Techniques , Poly A/genetics , Poly A/metabolism , RNA, Circular/genetics , RNA, Messenger/genetics , RNA, Ribosomal/genetics , Sequence Analysis, RNA/statistics & numerical dataABSTRACT
Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.
Subject(s)
Neuroblastoma/genetics , RNA, Long Noncoding/genetics , Cell Line, Tumor , Gene Drive Technology/methods , Gene Expression Profiling/methods , Humans , Neural Stem Cells/physiology , Sequence Analysis, RNA/methods , Signal Transduction/genetics , Transcription Factors/geneticsABSTRACT
Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
Subject(s)
Brain Neoplasms/genetics , Forkhead Box Protein M1/genetics , Gene Expression Regulation, Neoplastic , Kv Channel-Interacting Proteins/genetics , N-Myc Proto-Oncogene Protein/genetics , Neuroblastoma/genetics , Repressor Proteins/genetics , T-Box Domain Proteins/genetics , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , DNA Copy Number Variations , Epigenesis, Genetic , Forkhead Box Protein M1/metabolism , HEK293 Cells , Histones/genetics , Histones/metabolism , Humans , Kv Channel-Interacting Proteins/metabolism , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Neuroblastoma/pathology , Organoids/drug effects , Organoids/metabolism , Organoids/pathology , Panobinostat/pharmacology , Phenylenediamines/pharmacology , Pyrimidines/pharmacology , Repressor Proteins/metabolism , Signal Transduction , T-Box Domain Proteins/metabolism , Triazoles/pharmacology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
BACKGROUND: Reconstructing transcript models from RNA-sequencing (RNA-seq) data and establishing these as independent transcriptional units can be a challenging task. Current state-of-the-art tools for long non-coding RNA (lncRNA) annotation are mainly based on evolutionary constraints, which may result in false negatives due to the overall limited conservation of lncRNAs. RESULTS: To tackle this problem we have developed the Zipper plot, a novel visualization and analysis method that enables users to simultaneously interrogate thousands of human putative transcription start sites (TSSs) in relation to various features that are indicative for transcriptional activity. These include publicly available CAGE-sequencing, ChIP-sequencing and DNase-sequencing datasets. Our method only requires three tab-separated fields (chromosome, genomic coordinate of the TSS and strand) as input and generates a report that includes a detailed summary table, a Zipper plot and several statistics derived from this plot. CONCLUSION: Using the Zipper plot, we found evidence of transcription for a set of well-characterized lncRNAs and observed that fewer mono-exonic lncRNAs have CAGE peaks overlapping with their TSSs compared to multi-exonic lncRNAs. Using publicly available RNA-seq data, we found more than one hundred cases where junction reads connected protein-coding gene exons with a downstream mono-exonic lncRNA, revealing the need for a careful evaluation of lncRNA 5'-boundaries. Our method is implemented using the statistical programming language R and is freely available as a webtool.
