The anticancer potential of the CLK kinases inhibitors 1C8 and GPS167 revealed by their impact on the epithelial-mesenchymal transition and the antiviral immune response.

The diheteroarylamide-based compound 1C8 and the aminothiazole carboxamide-related compound GPS167 inhibit the CLK kinases, and affect the proliferation of a broad range of cancer cell lines. A chemogenomic screen previously performed with GPS167 revealed that the depletion of components associated with mitotic spindle assembly altered sensitivity to GPS167. Here, a similar screen performed with 1C8 also established the impact of components involved in mitotic spindle assembly. Accordingly, transcriptome analyses of cells treated with 1C8 and GPS167 indicated that the expression and RNA splicing of transcripts encoding mitotic spindle assembly components were affected. The functional relevance of the microtubule connection was confirmed by showing that subtoxic concentrations of drugs affecting mitotic spindle assembly increased sensitivity to GPS167. 1C8 and GPS167 impacted the expression and splicing of transcripts in pathways relevant to tumor progression, including MYC targets and the epithelial mesenchymal transition (EMT). Finally, 1C8 and GPS167 altered the expression and alternative splicing of transcripts involved in the antiviral immune response. Consistent with this observation, depleting the double-stranded RNA sensor DHX33 suppressed GPS167-mediated cytotoxicity on HCT116 cells. Our study uncovered molecular mechanisms through which 1C8 and GPS167 affect cancer cell proliferation as well as processes critical for metastasis.

PCSK7: A novel regulator of apolipoprotein B and a potential target against non-alcoholic fatty liver disease.

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration.

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

The Tug1 lncRNA locus is essential for male fertility.

BACKGROUND: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. RESULTS: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. CONCLUSIONS: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

Diverging RNPs: Toward Understanding lncRNA-Protein Interactions and Functions.

RNA-protein interactions are essential to a variety of biological processes. The realization that mammalian genomes are pervasively transcribed brought a tidal wave of tens of thousands of newly identified long noncoding RNAs (lncRNAs) and raised questions about their purpose in cells. The vast majority of lncRNAs have yet to be studied, and it remains to be determined to how many of these transcripts a function can be ascribed. However, results gleaned from studying a handful of these macromolecules have started to reveal common themes of biological function and mechanism of action involving intricate RNA-protein interactions. Some lncRNAs were shown to regulate the chromatin and transcription of distant and neighboring genes in the nucleus, while others regulate the translation or localization of proteins in the cytoplasm. Some lncRNAs were found to be crucial during development, while mutations and aberrant expression of others have been associated with several types of cancer and a plethora of diseases. Over the last few years, the establishment of new technologies has been key in providing the tools to decode the rules governing lncRNA-protein interactions and functions. This chapter will highlight the general characteristics of lncRNAs, their function, and their mode of action, with a special focus on protein interactions. It will also describe the methods at the disposition of scientists to help them cross this next frontier in our understanding of lncRNA biology.

An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

In Vivo Characterization of Linc-p21 Reveals Functional cis-Regulatory DNA Elements.

The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, in vivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements.

Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain.

Long noncoding RNAs (lncRNAs) have been implicated in numerous cellular processes including brain development. However, the in vivo expression dynamics and molecular pathways regulated by these loci are not well understood. Here, we leveraged a cohort of 13 lncRNAnull mutant mouse models to investigate the spatiotemporal expression of lncRNAs in the developing and adult brain and the transcriptome alterations resulting from the loss of these lncRNA loci. We show that several lncRNAs are differentially expressed both in time and space, with some presenting highly restricted expression in only selected brain regions. We further demonstrate altered regulation of genes for a large variety of cellular pathways and processes upon deletion of the lncRNA loci. Finally, we found that 4 of the 13 lncRNAs significantly affect the expression of several neighboring proteincoding genes in a cis-like manner. By providing insight into the endogenous expression patterns and the transcriptional perturbations caused by deletion of the lncRNA locus in the developing and postnatal mammalian brain, these data provide a resource to facilitate future examination of the specific functional relevance of these genes in neural development, brain function, and disease.

UBAP2L is a novel BMI1-interacting protein essential for hematopoietic stem cell activity.

Multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L.

Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre.

RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.

RNAi screen identifies Jarid1b as a major regulator of mouse HSC activity.

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.

Long noncoding RNAs regulate adipogenesis.

The prevalence of obesity has led to a surge of interest in understanding the detailed mechanisms underlying adipocyte development. Many protein-coding genes, mRNAs, and microRNAs have been implicated in adipocyte development, but the global expression patterns and functional contributions of long noncoding RNA (lncRNA) during adipogenesis have not been explored. Here we profiled the transcriptome of primary brown and white adipocytes, preadipocytes, and cultured adipocytes and identified 175 lncRNAs that are specifically regulated during adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, we have identified numerous lncRNAs that are functionally required for proper adipogenesis.

Differential analysis of gene regulation at transcript resolution with RNA-seq.

Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.

Multiple knockout mouse models reveal lincRNAs are required for life and brain development.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.

Posttranslational regulation of self-renewal capacity: insights from proteome and phosphoproteome analyses of stem cell leukemia.

We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell-enriched leukemia.

A role for GPx3 in activity of normal and leukemia stem cells.

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.

A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia.

In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.

Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer.

Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development.

A functional screen to identify novel effectors of hematopoietic stem cell activity.

Despite tremendous progress made toward the identification of the molecular circuitry that governs cell fate in embryonic stem cells, genes controlling this process in the adult hematopoietic stem cell have proven to be more difficult to unmask. We now report the results of a novel gain-of-function screening approach, which identified a series of 18 nuclear factors that affect hematopoietic stem cell activity. Overexpression of ten of these factors resulted in an increased repopulating activity compared to unmanipulated cells. Interestingly, at least four of the 18 factors, Fos, Tcfec, Hmgb1, and Sfpi1, show non-cell-autonomous functions. The utilization of this screening method together with the creation of a database enriched for potential determinants of hematopoietic stem cell self-renewal will serve as a resource to uncover regulatory networks in these cells.