Network propagation-based prioritization of long tail genes in 17 cancer types.

BACKGROUND: The diversity of genomic alterations in cancer poses challenges to fully understanding the etiologies of the disease. Recent interest in infrequent mutations, in genes that reside in the "long tail" of the mutational distribution, uncovered new genes with significant implications in cancer development. The study of cancer-relevant genes often requires integrative approaches pooling together multiple types of biological data. Network propagation methods demonstrate high efficacy in achieving this integration. Yet, the majority of these methods focus their assessment on detecting known cancer genes or identifying altered subnetworks. In this paper, we introduce a network propagation approach that entirely focuses on prioritizing long tail genes with potential functional impact on cancer development. RESULTS: We identify sets of often overlooked, rarely to moderately mutated genes whose biological interactions significantly propel their mutation-frequency-based rank upwards during propagation in 17 cancer types. We call these sets "upward mobility genes" and hypothesize that their significant rank improvement indicates functional importance. We report new cancer-pathway associations based on upward mobility genes that are not previously identified using driver genes alone, validate their role in cancer cell survival in vitro using extensive genome-wide RNAi and CRISPR data repositories, and further conduct in vitro functional screenings resulting in the validation of 18 previously unreported genes. CONCLUSION: Our analysis extends the spectrum of cancer-relevant genes and identifies novel potential therapeutic targets.

Generation of Pluripotent Stem Cells Using Somatic Cell Nuclear Transfer and Induced Pluripotent Somatic Cells from African Green Monkeys.

Patient-specific stem cells derived from somatic cell nuclear transfer (SCNT) embryos or from induced pluripotent stem cells (iPSCs) could be used to treat various diseases with minimal immune rejection. Many studies using these cells have been conducted in rats and mice; however, there exist numerous dissimilarities between the rodents and humans limiting the clinical predictive power and experimental utility of rodent experiments alone. Nonhuman primates (NHPs) share greater homology to human than rodents in all respects, including genomics, physiology, biochemistry, and the immune system. Thus, experimental data obtained from monkey studies would be more predictive for designing an effective cell replacement therapy in humans. Unfortunately, there are few iPSC lines and even fewer SCNT lines that have been derived in NHPs, hampering broader studies in regenerative medicine. One promising potential therapy would be the replacement of dopamine neurons that are lost in Parkinson's disease. After dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the African green monkey (Chlorocebus sabaeus) shows the most complete model of Parkinsonism compared with other species and brain pathology and behavioral changes are almost identical to those in humans after accidental exposure to MPTP. Therefore, we have developed a SCNT procedure to generate multiple pluripotent stem cell lines in this species for studies of possible treatment of Parkinsonism and for comparing with cells derived from iPSCs. Using 24 female monkeys as egg donors and 7 somatic cell donor monkeys, we have derived 11 SCNT embryonic stem cell lines that expressed typical stemness genes and formed all three germ layer derivatives. We also derived two iPSC lines using an episome-mediated reprogramming factor delivery system. This report describes the process for deriving these cell lines and proving their pluripotency for differentiation into various potentially therapeutic cells.

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors.

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.

Extended lifespan and reduced adiposity in mice lacking the FAT10 gene.

The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation. Although FAT10 is induced by inflammatory mediators and implicated in immunity, the physiological functions of FAT10 are poorly defined. We report the discovery that FAT10 regulates lifespan through pleiotropic actions on metabolism and inflammation. Median and overall lifespan are increased 20% in FAT10ko mice, coincident with elevated metabolic rate, preferential use of fat as fuel, and dramatically reduced adiposity. This phenotype is associated with metabolic reprogramming of skeletal muscle (i.e., increased AMP kinase activity, ß-oxidation and -uncoupling, and decreased triglyceride content). Moreover, knockout mice have reduced circulating glucose and insulin levels and enhanced insulin sensitivity in metabolic tissues, consistent with elevated IL-10 in skeletal muscle and serum. These observations suggest novel roles of FAT10 in immune metabolic regulation that impact aging and chronic disease.

Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line.

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34- cells, using RNA-Seq. We subsequently identified the target genes bound by TCF7, using ChIP-Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34- state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.

Something old, something new: plant innate immunity and autophagy.

Autophagy performs a variety of established functions during plant growth and development. Recently, autophagy has been further implicated in the regulation of programmed cell death induced during the plant innate immune response. In this chapter we describe specific mechanisms through which autophagy may contribute to a successful defense against pathogen invasion. Accumulating evidence shows that the plant immune system utilizes the chloroplasts as primary sites for the regulation of cell death programs. Viruses also appear to utilize the chloroplast as a site of replication and accumulation, potentially inactivating chloroplast defense signaling in the process. Autophagy-like mechanisms have been observed to target the chloroplast, which we refer to as "chlorophagy," potentially targeting invasive viruses for degradation or regulating chloroplast-based signaling during the immune response. We hypothesize that chlorophagy is significant for the execution of plant immune defenses, during both basal and effector-triggered immunity.

Autophagy takes its TOLL on innate immunity.

Toll-like receptors (TLRs) activate a complimentary set of defense responses that protect cells during microbial infection. In the recent issue of Immunity, Xu et al. (2007) elucidate a molecular pathway that connects TLR4-mediated innate immune signaling to autophagy, a process of cytoplasmic sequestration and subsequent recycling or degradation.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays.

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities.

Autophagy and plant innate immunity.

Plant innate immunity is often associated with specialized programmed cell death at or near the site of pathogen infection. Despite the isolation of several lesion mimic mutants, the molecular mechanisms that regulate cell death during an immune response remain obscure. Recently, autophagy, an evolutionarily conserved process of bulk protein and organelle turnover, was shown to play an important role in limiting cell death initiated during plant innate immune responses. Consistent with its role in plants, several studies in animals also demonstrate that the autophagic machinery is involved in innate as well as adaptive immunities. Here, we review the role of autophagy in plant innate immunity. Because autophagy is observed in healthy and dying plant cells, we will also examine whether autophagy plays a protective or a destructive role during an immune response.

Life after death: are autophagy genes involved in cell death and survival during plant innate immune responses?

It has not escaped the attention of the plant disease resistance community that the vacuole is rapidly emerging as a central player in the execution of cell death. On the one hand the targeted destruction of the vacuole-from the inside out-by vacuolar processing enzymes (VPE) is required to induce PCD in pathogen-infected cells. On the other hand, an intact vacuole is vital for a functional autophagic response to ensure survival of uninfected cells. At face value, the two responses seem to represent distinct resistance mechanisms that operate at divergent branch points and their use of the vacuole merely coincidental. However, closer examination has led us to propose an interesting hypothesis that accounts for these two opposing roles of the vacuole in both VPE-mediated PCD and autophagydependent cell survival. During initial infection, we propose a mechanism similar to the CPY transport pathway in yeast wherein a select set of genes, including several which encode a phosphatidylinositol 3-kinase complex that is needed for autophagy, are needed for VPE transport, vacuolar processing and initiation of PCD. Later during infection, autophagyspecific genes are needed for autophagosome formation, sequestration of VPE preproteins and VPE degradation.