Wuming Gong

PVT1 dependence in cancer with MYC copy-number increase

‘Gain’ of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent ‘gene desert’ of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.

Integrated siRNA design based on surveying of features associated with high RNAi effectiveness

BackgroundShort interfering RNAs have allowed the development of clean and easily regulated methods for disruption of gene expression. However, while these methods continue to grow in popularity, designing effective siRNA experiments can be challenging. The various existing siRNA design guidelines suffer from two problems: they differ considerably from each other, and they produce high levels of false-positive predictions when tested on data of independent origins.ResultsUsing a distinctly large set of siRNA efficacy data assembled from a vast diversity of origins (the siRecords data, containing records of 3,277 siRNA experiments targeting 1,518 genes, derived from 1,417 independent studies), we conducted extensive analyses of all known features that have been implicated in increasing RNAi effectiveness. A number of features having positive impacts on siRNA efficacy were identified. By performing quantitative analyses on cooperative effects among these features, then applying a disjunctive rule merging (DRM) algorithm, we developed a bundle of siRNA design rule sets with the false positive problem well curbed. A comparison with 15 online siRNA design tools indicated that some of the rule sets we developed surpassed all of these design tools commonly used in siRNA design practice in positive predictive values (PPVs).ConclusionThe availability of the large and diverse siRNA dataset from siRecords and the approach we describe in this report have allowed the development of highly effective and generally applicable siRNA design rule sets. Together with ever improving RNAi lab techniques, these design rule sets are expected to make siRNAs a more useful tool for molecular genetics, functional genomics, and drug discovery studies.

siRecords: an extensive database of mammalian siRNAs with efficacy ratings

UNLABELLED Short interfering RNAs (siRNAs) have been gaining popularity as the gene knock-down tool of choice by many researchers because of the clean nature of their workings as well as the technical simplicity and cost efficiency in their applications. We have constructed siRecords, a database of siRNAs experimentally tested by researchers with consistent efficacy ratings. This database will help siRNA researchers develop more reliable siRNA design rules; in the mean time, siRecords will benefit experimental researchers directly by providing them with information about the siRNAs that have been experimentally tested against the genes of their interest. Currently, more than 4100 carefully annotated siRNA sequences obtained from more than 1200 published siRNA studies are hosted in siRecords. This database will continue to expand as more experimentally tested siRNAs are published. AVAILABILITY The siRecords database can be accessed at http://siRecords.umn.edu/siRecords/

PepCyber:P∼PEP: a database of human protein–protein interactions mediated by phosphoprotein-binding domains

Phosphoprotein-binding domains (PPBDs) mediate many important cellular and molecular processes. Ten PPBDs have been known to exist in the human proteome, namely, 14-3-3, BRCT, C2, FHA, MH2, PBD, PTB, SH2, WD-40 and WW. PepCyber:P∼PEP is a newly constructed database specialized in documenting human PPBD-containing proteins and PPBD-mediated interactions. Our motivation is to provide the research community with a rich information source emphasizing the reported, experimentally validated data for specific PPBD–PPEP interactions. This information is not only useful for designing, comparing and validating the relevant experiments, but it also serves as a knowledge-base for computationally constructing systems signaling pathways and networks. PepCyber:P∼PEP is accessible through the URL, http://www.pepcyber.org/PPEP/. The current release of the database contains 7044 PPBD-mediated interactions involving 337 PPBD-containing proteins and 1123 substrate proteins.

Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages.

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Homeostatic adaptations in brain energy metabolism in mouse models of Huntington disease

Impairment of energy metabolism is a key feature of Huntington disease (HD). Recently, we reported longitudinal neurochemical changes in R6/2 mice measured by in-vivo proton magnetic resonance spectroscopy (1H MRS; Zacharoff et al, 2012). Here, we present similar 1H MRS measurements at an early stage in the milder Q111 mouse model. In addition, we measured the concentration of ATP and inorganic phosphate (Pi), key energy metabolites not accessible with 1H MRS, using 31P MRS both in Q111 and in R6/2 mice. Significant changes in striatal creatine and phosphocreatine were observed in Q111 mice at 6 weeks relative to control, and these changes were largely reversed at 13 weeks. No significant change was detected in ATP concentration, in either HD mouse, compared with control. Calculated values of [ADP], phosphorylation potential, relative rate of ATP synthase (v/Vmax(ATP)), and relative rate of creatine kinase (v/Vmax(CK)) were calculated from the measured data. ADP concentration and v/Vmax(ATP) were increased in Q111 mice at 6 weeks, and returned close to normal at 13 weeks. In contrast, these parameters were normal in R6/2 mice. These results suggest that early changes in brain energy metabolism are followed by compensatory shifts to maintain energetic homeostasis from early ages through manifest disease.

siDRM: An effective and generally applicable online siRNA design tool

Small interfering RNAs (siRNAs) have become an indispensable tool for the investigation of gene functions. Most existing siRNA design tools were trained on datasets assembled from confined origins, incompatible with the diverse siRNA laboratory practice to which these tools will ultimately be applied. We have performed an updated analysis using the disjunctive rule merging (DRM) approach on a large and diverse dataset compiled from siRecords, and implemented the resulting rule sets in siDRM, a new online siRNA design tool. siDRM also implements a few high-sensitivity rule sets and fast rule sets, links to siRecords, and uses several filters to check unwanted detrimental effects, including innate immune responses, cell toxic effects and off-target activities in selecting siRNAs. A performance comparison using an independent dataset indicated that siDRM outperforms 19 existing siRNA design tools in identifying effective siRNAs.

siRecords: a database of mammalian RNAi experiments and efficacies.

RNAi-based gene-silencing techniques offer a fast and cost-effective way of knocking down genes’ functions in an easily regulated manner. Exciting progress has been made in recent years in the application of these techniques in basic biomedical research and therapeutic development. However, it remains a difficult task to design effective siRNA experiments with high efficacy and specificity. We present siRecords, an extensive database of mammalian RNAi experiments with consistent efficacy ratings. This database serves two purposes. First, it provides a large and diverse dataset of siRNA experiments. This dataset faithfully represents the general, diverse RNAi experimental practice, and allows more reliable siRNA design tools to be developed with the overfitting problem well curbed. Second, the database helps experimental RNAi researchers directly by providing them with the efficacy and other information about the siRNAs experiments designed and conducted previously against the genes of their interest. The current release of siRecords contains the records of 17 192 RNAi experiments targeting 5086 genes.

Dpath software reveals hierarchical haemato-endothelial lineages of Etv2 progenitors based on single-cell transcriptome analysis

Developmental, stem cell and cancer biologists are interested in the molecular definition of cellular differentiation. Although single-cell RNA sequencing represents a transformational advance for global gene analyses, novel obstacles have emerged, including the computational management of dropout events, the reconstruction of biological pathways and the isolation of target cell populations. We develop an algorithm named dpath that applies the concept of metagene entropy and allows the ranking of cells based on their differentiation potential. We also develop self-organizing map (SOM) and random walk with restart (RWR) algorithms to separate the progenitors from the differentiated cells and reconstruct the lineage hierarchies in an unbiased manner. We test these algorithms using single cells from Etv2-EYFP transgenic mouse embryos and reveal specific molecular pathways that direct differentiation programmes involving the haemato-endothelial lineages. This software program quantitatively assesses the progenitor and committed states in single-cell RNA-seq data sets in a non-biased manner.

The Etv2-miR-130a Network Regulates Mesodermal Specification

MicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.