Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation.

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We perform saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identify 3432 functionally abnormal variants, in three distinct classes. We train a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants can account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.

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.

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for saturation genome editing and other deep mutational scanning experiments.

MOTIVATION: CRISPR/Cas9-based technology allows for the functional analysis of genetic variants at single nucleotide resolution whilst maintaining genomic context. This approach, known as saturation genome editing (SGE), a form of deep mutational scanning, systematically alters each position in a target region to explore its function. SGE experiments require the design and synthesis of oligonucleotide variant libraries which are introduced into the genome. This technology is applicable to diverse fields such as disease variant identification, drug development, structure-function studies, synthetic biology, evolutionary genetics and host-pathogen interactions. Here, we present the Variant Library Annotation Tool (VaLiAnT) which can be used to generate variant libraries from user-defined genomic coordinates and standard input files. The software can accommodate user-specified species, reference sequences and transcript annotations. RESULTS: Coordinates for a genomic range are provided by the user to retrieve a corresponding oligonucleotide reference sequence. A user-specified range within this sequence is then subject to systematic, nucleotide and/or amino acid saturating mutator functions. VaLiAnT provides a novel way to retrieve, mutate and annotate genomic sequences for oligonucleotide library generation. Specific features for SGE library generation can be employed. In addition, VaLiAnT is configurable, allowing for cDNA and prime editing saturation library generation, with other diverse applications possible. AVAILABILITY AND IMPLEMENTATION: VaLiAnT is a command line tool written in Python. Source code, testing data, example input and output files and executables are available (https://github.com/cancerit/VaLiAnT) in addition to a detailed user manual (https://github.com/cancerit/VaLiAnT/wiki). VaLiAnT is licensed under AGPLv3. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

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.

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

Derivation of Transgene-Free Induced Pluripotent Stem Cells from a Single Drop of Blood.

Human-induced pluripotent stem cells (hiPSCs) have great potential for future use in therapeutic regenerative medicine. Based on the current protocol for deriving hiPSCs, invasive procedures such as skin biopsies and venipuncture are required for obtaining donor samples. Herein, we present a detailed protocol for deriving hiPSCs from human finger-prick (FP) blood. In this method, the transgene-free hiPSCs can be easily generated from only 10 µl of FP blood. The finger-pricked iPSCs (FPiPSCs) show all the pluripotency markers and can be easily differentiated into various cell lineages. The time required for deriving the FPiPSCs is relatively short-10 to 15 days for FP blood expansion and 20 to 30 days for reprogramming. This method can be easily adapted for setting up a large scale iPSC bank as it requires only 10 µl of the donor FP blood, which can be easily collected. © 2016 by John Wiley & Sons, Inc.

Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a "do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.

Interference of intrinsic curvature of DNA by DNA-intercalating agents.

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope.