Non-coding RNA LEVER sequestration of PRC2 can mediate long range gene regulation.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA, LEVER, which mapped 236 kb upstream of the ß-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and ß-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.

Repurposing RNA sequencing for discovery of RNA modifications in clinical cohorts.

The study of RNA modifications in large clinical cohorts can reveal relationships between the epitranscriptome and human diseases, although this is especially challenging. We developed ModTect (https://github.com/ktan8/ModTect), a statistical framework to identify RNA modifications de novo by standard RNA-sequencing with deletion and mis-incorporation signals. We show that ModTect can identify both known (N 1-methyladenosine) and previously unknown types of mRNA modifications (N 2,N 2-dimethylguanosine) at nucleotide-resolution. Applying ModTect to 11,371 patient samples and 934 cell lines across 33 cancer types, we show that the epitranscriptome was dysregulated in patients across multiple cancer types and was additionally associated with cancer progression and survival outcomes. Some types of RNA modification were also more disrupted than others in patients with cancer. Moreover, RNA modifications contribute to multiple types of RNA-DNA sequence differences, which unexpectedly escape detection by Sanger sequencing. ModTect can thus be used to discover associations between RNA modifications and clinical outcomes in patient cohorts.

Myeloid lncRNA LOUP mediates opposing regulatory effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here, we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA and DNA interactions with the broadly expressed Runt-related transcription factor 1 (RUNX1), we identified the long noncoding RNA (lncRNA) originating from the upstream regulatory element of PU.1 (LOUP). This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia (AML), wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein, RUNX1-ETO, limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell-type-specific RNAs and transcription factors, as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.

Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression.

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.

CSI NGS Portal: An Online Platform for Automated NGS Data Analysis and Sharing.

Next-generation sequencing (NGS) has been a widely-used technology in biomedical research for understanding the role of molecular genetics of cells in health and disease. A variety of computational tools have been developed to analyse the vastly growing NGS data, which often require bioinformatics skills, tedious work and a significant amount of time. To facilitate data processing steps minding the gap between biologists and bioinformaticians, we developed CSI NGS Portal, an online platform which gathers established bioinformatics pipelines to provide fully automated NGS data analysis and sharing in a user-friendly website. The portal currently provides 16 standard pipelines for analysing data from DNA, RNA, smallRNA, ChIP, RIP, 4C, SHAPE, circRNA, eCLIP, Bisulfite and scRNA sequencing, and is flexible to expand with new pipelines. The users can upload raw data in FASTQ format and submit jobs in a few clicks, and the results will be self-accessible via the portal to view/download/share in real-time. The output can be readily used as the final report or as input for other tools depending on the pipeline. Overall, CSI NGS Portal helps researchers rapidly analyse their NGS data and share results with colleagues without the aid of a bioinformatician. The portal is freely available at: https://csibioinfo.nus.edu.sg/csingsportal.

CAV1 - GLUT3 signaling is important for cellular energy and can be targeted by Atorvastatin in Non-Small Cell Lung Cancer.

Background: The development of molecular targeted therapies, such as EGFR-TKIs, has positively impacted the management of EGFR mutated NSCLC. However, patients with innate and acquired resistance to EGFR-TKIs still face limited effective therapeutic options. Statins are the most frequently prescribed anti-cholesterol agents and have been reported to inhibit the progression of various malignancies, including in lung. However, the mechanism by which statin exerts its anti-cancer effects is unclear. This study is designed to investigate the anti-proliferative effects and identify the mechanism-of-action of statins in NSCLC. Methods: In this study, the anti-tumoral properties of Atorvastatin were investigated in NSCLC utilizing cell culture system and in vivo models. Results: We demonstrate a link between elevated cellular cholesterol and TKI-resistance in NSCLC, which is independent of EGFR mutation status. Atorvastatin suppresses growth by inhibiting Cav1 expression in tumors in cell culture system and in in vivo models. Subsequent interrogations demonstrate an oncogenic physical interaction between Cav1 and GLUT3, and glucose uptake found distinctly in TKI-resistant NSCLC and this may be due to changes in the physical properties of Cav1 favoring GLUT3 binding in which significantly stronger Cav1 and GLUT3 physical interactions were observed in TKI-resistant than in TKI-sensitive NSCLC cells. Further, the differential effects of atorvastatin observed between EGFR-TKI resistant and sensitive cells suggest that EGFR mutation status may influence its actions. Conclusions: This study reveals the inhibition of oncogenic role of Cav1 in GLUT3-mediated glucose uptake by statins and highlights its potential impact to overcome NSCLC with EGFR-TKI resistance.

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

BACKGROUND & AIMS: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. METHODS: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. RESULTS: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP. CONCLUSIONS: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

DNMT3B shapes the mCA landscape and regulates mCG for promoter bivalency in human embryonic stem cells.

DNMT3B is known as a de novo DNA methyltransferase. However, its preferential target sites for DNA methylation are largely unknown. Our analysis on ChIP-seq experiment in human embryonic stem cells (hESC) revealed that DNMT3B, mCA and H3K36me3 share the same genomic distribution profile. Deletion of DNMT3B or its histone-interacting domain (PWWP) demolished mCA in hESCs, suggesting that PWWP domain of DNMT3B directs the formation of mCA landscape. In contrast to the common presumption that PWWP guides DNMT3B-mediated mCG deposition, we found that deleting PWWP does not affect the mCG landscape. Nonetheless, DNMT3B knockout led to the formation of 2985 de novo hypomethylated regions at annotated promoter sites. Upon knockout, most of these promoters gain the bivalent marks, H3K4me3 and H3K27me3. We call them spurious bivalent promoters. Gene ontology analysis associated spurious bivalent promoters with development and cell differentiation. Overall, we found the importance of DNMT3B for shaping the mCA landscape and for maintaining the fidelity of the bivalent promoters in hESCs.

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide.

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable.

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non-small cell lung cancer.

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16-C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI-resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA-approved anti-obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI-resistant EGFR mutant NSCLC patients.

Purifying selection shapes the coincident SNP distribution of primate coding sequences.

Genome-wide analysis has observed an excess of coincident single nucleotide polymorphisms (coSNPs) at human-chimpanzee orthologous positions, and suggested that this is due to cryptic variation in the mutation rate. While this phenomenon primarily corresponds with non-coding coSNPs, the situation in coding sequences remains unclear. Here we calculate the observed-to-expected ratio of coSNPs (coSNPO/E) to estimate the prevalence of human-chimpanzee coSNPs, and show that the excess of coSNPs is also present in coding regions. Intriguingly, coSNPO/E is much higher at zero-fold than at nonzero-fold degenerate sites; such a difference is due to an elevation of coSNPO/E at zero-fold degenerate sites, rather than a reduction at nonzero-fold degenerate ones. These trends are independent of chimpanzee subpopulation, population size, or sequencing techniques; and hold in broad generality across primates. We find that this discrepancy cannot fully explained by sequence contexts, shared ancestral polymorphisms, SNP density, and recombination rate, and that coSNPO/E in coding sequences is significantly influenced by purifying selection. We also show that selection and mutation rate affect coSNPO/E independently, and coSNPs tend to be less damaging and more correlated with human diseases than non-coSNPs. These suggest that coSNPs may represent a "signature" during primate protein evolution.

NCLscan: accurate identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA) with a good balance between sensitivity and precision.

Analysis of RNA-seq data often detects numerous 'non-co-linear' (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts (trans-spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method ('NCLscan'), which utilized a stepwise alignment strategy to almost completely eliminate false calls (>98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans-spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans-spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome.

Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency.

Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platform-dependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA ("tsRMST"). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineage-specific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization.