Modeling familial cancer with induced pluripotent stem cells.

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis.

Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3-/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3-/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.

NetExplore: a web server for modeling small network motifs.

MOTIVATION: Quantitative and qualitative assessment of biological data often produces small essential recurrent networks, containing 3-5 components called network motifs. In this context, model solutions for small network motifs represent very high interest. RESULTS: Software package NetExplore has been created in order to generate, classify and analyze solutions for network motifs including up to six network components. NetExplore allows plotting and visualization of the solution's phase spaces and bifurcation diagrams. AVAILABILITY AND IMPLEMENTATION: The current version of NetExplore has been implemented in Perl-CGI and is accessible at the following locations: http://line.bioinfolab.net/nex/NetExplore.htm and http://nex.autosome.ru/nex/NetExplore.htm.

Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.

Clusters of temporal discordances reveal distinct embryonic patterning mechanisms in Drosophila and anopheles.

Evolutionary innovations can be driven by spatial and temporal changes in gene expression. Several such differences have been documented in the embryos of lower and higher Diptera. One example is the reduction of the ancient extraembryonic envelope composed of amnion and serosa as seen in mosquitoes to the single amnioserosa of fruit flies. We used transcriptional datasets collected during the embryonic development of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, to search for whole-genome changes in gene expression underlying differences in their respective embryonic morphologies. We found that many orthologous gene pairs could be clustered based on the presence of coincident discordances in their temporal expression profiles. One such cluster contained genes expressed specifically in the mosquito serosa. As shown previously, this cluster is re-deployed later in development at the time of cuticle synthesis. In addition, there is a striking difference in the temporal expression of a subset of maternal genes. Specifically, maternal transcripts that exhibit a sharp reduction at the time of the maternal-zygotic transition in Drosophila display sustained expression in the Anopheles embryo. We propose that gene clustering by local temporal discordance can be used for the de novo identification of the gene batteries underlying morphological diversity.

FOXO3-mTOR metabolic cooperation in the regulation of erythroid cell maturation and homeostasis.

Ineffective erythropoiesis is observed in many erythroid disorders including ß-thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3(-/-) erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of ß-thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism-mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis.

Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo.

Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs.

Significant alterations in signaling pathways and transcriptional regulatory programs together represent major hallmarks of many cancers. These, among all, include the reactivation of stemness, which is registered by the expression of pathways that are active in the embryonic stem cells (ESCs). Here, we assembled gene sets that reflect the stemness and proliferation signatures and used them to analyze a large panel of RNA-seq data from The Cancer Genome Atlas (TCGA) Consortium in order to specifically assess the expression of stemness-related and proliferation-related genes across a collection of different tumor types. We introduced a metric that captures the collective similarity of the expression profile of a tumor to that of ESCs, which showed that stemness and proliferation signatures vary greatly between different tumor types. We also observed a high degree of intertumoral heterogeneity in the expression of stemness- and proliferation-related genes, which was associated with increased hazard ratios in a fraction of tumors and mirrored by high intratumoral heterogeneity and a remarkable stemness capacity in metastatic lesions across cancer cells in single cell RNA-seq datasets. Taken together, these results indicate that the expression of stemness signatures is highly heterogeneous and cannot be used as a universal determinant of cancer. This calls into question the universal validity of diagnostic tests that are based on stem cell markers.

Evolution of the ventral midline in insect embryos.

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D. mel.) and the mosquito Anopheles gambiae (A. gam.), the midline is narrow and spans just 1-2 cells. However, in the honeybee, Apis mellifera (A. mel.), the ventral midline is broad and encompasses 5-6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D. mel., but it is activated solely by Twi in A. mel. We suggest that the Twi-only mode of regulation--and the broad ventral midline--represents the ancestral form of CNS patterning in Holometabolous insects.

Temporal waves of coherent gene expression during Drosophila embryogenesis.

MOTIVATION: Animal development depends on localized patterns of gene expression. Whole-genome methods permit the global identification of differential expression patterns. However, most gene-expression-clustering methods focus on the analysis of entire expression profiles, rather than temporal segments or time windows. RESULTS: In the current study, local clustering of temporal time windows was applied to developing embryos of the fruitfly, Drosophila melanogaster. Large-scale developmental events, involving temporal activation of hundreds of genes, were identified as discrete gene clusters. The time-duration analysis revealed six temporal waves of coherent gene expression during Drosophila embryogenesis. The most powerful expression waves preceded major morphogenetic movements, such as germ band elongation and dorsal closure. These waves of gene expression coincide with the inhibition of maternal transcripts during early development, the specification of ectoderm, differentiation of the nervous system, differentiation of the digestive tract, deposition of the larval cuticle and the reorganization of the cytoskeleton during global morphogenetic events. We discuss the implications of these findings with respect to the gene regulatory networks governing Drosophila development. AVAILABILITY: Data and software are available from the UC Berkeley web resource http://flydev.berkeley.edu/cgi-bin/GTEM/dmap_dm-ag/index_dmap.htm

Stripe formation in the early fly embryo: principles, models, and networks.

Early development of animal embryos begins from spatially distributed products of gene expression, i.e., gradients. While maternal and early zygotic genes form broad and/or terminal gradients, their direct targets appear later on as relatively narrow stripes, which foreshadow presumptive germ layers or future segments. Evidently, stripe expression of the zygotic genes is among the key mechanisms of embryo patterning. In this paper, known qualitative and quantitative models for the stripe formation are considered on the example of early embryogenesis of Drosophila. The current model analysis emphasizes the role of spatial information flow in development. Discussion is given on frequent network motifs, pointing to spatial stripe formation solutions.

Organization of developmental enhancers in the Drosophila embryo.

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. Here, we investigate the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. We also present evidence that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. We conclude that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo.

Dual regulation by the Hunchback gradient in the Drosophila embryo.

The regulation of segmentation gene expression is investigated by computational modeling using quantitative expression data. Previous tissue culture assays and transgene analyses raised the possibility that Hunchback (Hb) might function as both an activator and repressor of transcription. At low concentrations, Hb activates gene expression, whereas at high concentrations it mediates repression. Under the same experimental conditions, transcription factors encoded by other gap genes appear to function as dedicated repressors. Models based on dual regulation suggest that the Hb gradient can be sufficient for establishing the initial Kruppel (Kr) expression pattern in central regions of the precellular embryo. The subsequent refinement of the Kr pattern depends on the combination of Hb and the Giant (Gt) repressor. The dual-regulation models developed for Kr also explain some of the properties of the even-skipped (eve) stripe 3+7 enhancer. Computational simulations suggest that repression results from the dimerization of Hb monomers on the DNA template.

How the Dorsal gradient works: insights from postgenome technologies.

Gradients of extracellular signaling molecules and transcription factors are used in a variety of developmental processes, including the patterning of the Drosophila embryo, the establishment of diverse neuronal cell types in the vertebrate neural tube, and the anterior-posterior patterning of vertebrate limbs. Here, we discuss how a gradient of the maternal transcription factor Dorsal produces complex patterns of gene expression across the dorsal-ventral (DV) axis of the early Drosophila embryo. The identification of 60-70 Dorsal target genes, along with the characterization of approximately 35 associated regulatory DNAs, suggests that there are at least six different regulatory codes driving diverse DV expression profiles.

A self-organizing system of repressor gradients establishes segmental complexity in Drosophila.

Gradients of regulatory factors are essential for establishing precise patterns of gene expression during development; however, it is not clear how patterning information in multiple gradients is integrated to generate complex body plans. Here we show that opposing gradients of two Drosophila transcriptional repressors, Hunchback (Hb) and Knirps (Kni), position several segments by differentially repressing two distinct regulatory regions (enhancers) of the pair-rule gene even-skipped (eve). Computational and in vivo analyses suggest that enhancer sensitivity to repression is controlled by the number and affinity of repressor-binding sites. Because the kni expression domain is positioned between two gradients of Hb, each enhancer directs expression of a pair of symmetrical stripes, one on each side of the kni domain. Thus, only two enhancers are required for the precise positioning of eight stripe borders (four stripes), or more than half of the whole eve pattern. Our results show that complex developmental expression patterns can be generated by simple repressor gradients. They also support the utility of computational analyses for defining and deciphering regulatory information contained in genomic DNA.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

Hematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Time warping of evolutionary distant temporal gene expression data based on noise suppression.

BACKGROUND: Comparative analysis of genome wide temporal gene expression data has a broad potential area of application, including evolutionary biology, developmental biology, and medicine. However, at large evolutionary distances, the construction of global alignments and the consequent comparison of the time-series data are difficult. The main reason is the accumulation of variability in expression profiles of orthologous genes, in the course of evolution. RESULTS: We applied Pearson distance matrices, in combination with other noise-suppression techniques and data filtering to improve alignments. This novel framework enhanced the capacity to capture the similarities between the temporal gene expression datasets separated by large evolutionary distances. We aligned and compared the temporal gene expression data in budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast, which are separated by more then approximately 400 myr of evolution. We found that the global alignment (time warping) properly matched the duration of cell cycle phases in these distant organisms, which was measured in prior studies. At the same time, when applied to individual ortholog pairs, this alignment procedure revealed groups of genes with distinct alignments, different from the global alignment. CONCLUSION: Our alignment-based predictions of differences in the cell cycle phases between the two yeast species were in a good agreement with the existing data, thus supporting the computational strategy adopted in this study. We propose that the existence of the alternative alignments, specific to distinct groups of genes, suggests presence of different synchronization modes between the two organisms and possible functional decoupling of particular physiological gene networks in the course of evolution.

Computational models for neurogenic gene expression in the Drosophila embryo.

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.

Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila.

The Drosophila compound eye consists of approximately 750 independently functioning ommatidia, each containing two photoreceptor subpopulations. The outer photoreceptors participate in motion detection, while the inner photoreceptors contribute to color vision. Although the inner photoreceptors, R7 and R8, terminally differentiate into functionally related cells, they differ in their molecular and morphological makeup. Our data indicates that several aspects of R7 versus R8 cell fate determination are regulated by the transcription factor Prospero (Pros). pros is specifically expressed in R7 cells, and R7 cells mutant for pros derepress R8 rhodopsins, lose R7 rhodopsins and acquire an R8-like morphology. This suggests that R7 inner photoreceptor cell fate is acquired from a default R8-like fate that is regulated, in part, via the direct transcriptional repression of R8 rhodopsins in R7 cells. Furthermore, this study provides transcriptional targets for pros that may lend insight into its role in regulating neuronal development in flies and vertebrates.