Characterization Of Gene Regulatory Networks Research Articles

Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Robust discovery of gene regulatory networks from single-cell gene expression data by Causal Inference Using Composition of Transactions.

Gene regulatory networks (GRNs) drive organism structure and functions, so the discovery and characterization of GRNs is a major goal in biological research. However, accurate identification of causal regulatory connections and inference of GRNs using gene expression datasets, more recently from single-cell RNA-seq (scRNA-seq), has been challenging. Here we employ the innovative method of Causal Inference Using Composition of Transactions (CICT) to uncover GRNs from scRNA-seq data. The basis of CICT is that if all gene expressions were random, a non-random regulatory gene should induce its targets at levels different from the background random process, resulting in distinct patterns in the whole relevance network of gene-gene associations. CICT proposes novel network features derived from a relevance network, which enable any machine learning algorithm to predict causal regulatory edges and infer GRNs. We evaluated CICT using simulated and experimental scRNA-seq data in a well-established benchmarking pipeline and showed that CICT outperformed existing network inference methods representing diverse approaches with many-fold higher accuracy. Furthermore, we demonstrated that GRN inference with CICT was robust to different levels of sparsity in scRNA-seq data, the characteristics of data and ground truth, the choice of association measure and the complexity of the supervised machine learning algorithm. Our results suggest aiming at directly predicting causality to recover regulatory relationships in complex biological networks substantially improves accuracy in GRN inference.

Characterization of Gene Regulatory Networks in Plants Using New Methods and Data Types.

A major question in plant biology is to understand how plant growth, development, and environmental responses are controlled and coordinated by the activities of regulatory factors. Gene regulatory network (GRN) analyses require integrated approaches that combine experimental approaches with computational analyses. A wide range of experimental approaches and tools are now available, such as targeted perturbation of gene activities, quantitative and cell-type specific measurements of dynamic gene activities, and systematic analysis of the molecular 'hard-wiring' of the systems. At the computational level, different tools and databases are available to study regulatory sequences, including intuitive visualizations to explore data-driven gene regulatory networks in different plant species. Furthermore, advanced data integration approaches have recently been developed to efficiently leverage complementary regulatory data types and learn context-specific networks.

A genome-wide computational approach to define microRNA-Polycomb/trithorax gene regulatory circuits in Drosophila

Characterization of gene regulatory networks is fundamental to understanding homeostatic development. This process can be simplified by analyzing relatively simple genomes such as the genome of Drosophila melanogaster. In this work we have developed a computational framework in Drosophila to explore for the presence of gene regulatory circuits between two large groups of transcriptional regulators: the epigenetic group of the Polycomb/trithorax (PcG/trxG) proteins and the microRNAs (miRNAs). We have searched genome-wide for miRNA targets in PcG/trxG transcripts as well as for Polycomb Response Elements (PREs) in miRNA genes. Our results show that 10% of the analyzed miRNAs could be controlling PcG/trxG gene expression, while 40% of those miRNAs are putatively controlled by the selected set of PcG/trxG proteins. The integration of these analyses has resulted in the predicted existence of 3 classes of miRNA-PcG/trxG crosstalk interactions that define potential regulatory circuits. In the first class, miRNA-PcG circuits are defined by miRNAs that reciprocally crosstalk with PcG. In the second, miRNA-trxG circuits are defined by miRNAs that reciprocally crosstalk with trxG. In the third class, miRNA-PcG/trxG shared circuits are defined by miRNAs that crosstalk with both PcG and trxG regulators. These putative regulatory circuits may uncover a novel mechanism in Drosophila for the control of PcG/trxG and miRNAs levels of expression. The computational framework developed here for Drosophila melanogaster can serve as a model case for similar analyses in other species. Moreover, our work provides, for the first time, a new and useful resource for the Drosophila community to consult prior to experimental studies investigating the epigenetic regulatory networks of miRNA-PcG/trxG mediated gene expression.

CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

SHORT-ROOT Controls Cell Elongation in the Etiolated Arabidopsis Hypocotyl.

Transcriptional regulation, a core component of gene regulatory networks, plays a key role in controlling individual organism’s growth and development. To understand how plants modulate cellular processes for growth and development, the identification and characterization of gene regulatory networks are of importance. The SHORT-ROOT (SHR) transcription factor is known for its role in cell divisions in Arabidopsis (Arabidopsis thaliana). However, whether SHR is involved in hypocotyl cell elongation remains unknown. Here, we reveal that SHR controls hypocotyl cell elongation via the transcriptional regulation of XTH18, XTH22, and XTH24, which encode cell wall remodeling enzymes called xyloglucan endotransglucosylase/hydrolases (XTHs). Interestingly, SHR activates transcription of the XTH genes, independently of its partner SCARECROW (SCR), which is different from the known mode of action. In addition, overexpression of the XTH genes can promote cell elongation in the etiolated hypocotyl. Moreover, confinement of SHR protein in the stele still induces cell elongation, despite the aberrant organization in the hypocotyl ground tissue. Therefore, it is likely that SHR-mediated growth is uncoupled from SHR-mediated radial patterning in the etiolated hypocotyl. Our findings also suggest that intertissue communication between stele and endodermis plays a role in coordinating hypocotyl cell elongation of the Arabidopsis seedling. Taken together, our study identifies SHR as a new crucial regulator that is necessary for cell elongation in the etiolated hypocotyl.

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells

BackgroundRobust methods to culture primary airway epithelial cells were developed several decades ago and these cells provide the model of choice to investigate many diseases of the human lung. However, the molecular signature of cells from different regions of the airway epithelium has not been well characterized. MethodsWe utilize DNase-seq and RNA-seq to examine the molecular signatures of primary cells derived from human tracheal and bronchial tissues, as well as healthy and diseased (cystic fibrosis (CF)) donor lung tissue. ResultsOur data reveal an airway cell signature that is divergent from other epithelial cell types and from common airway epithelial cell lines. The differences between tracheal and bronchial cells are clearly evident as are common regulatory features. Only minor variation is seen between bronchial cells from healthy or CF donors. ConclusionsThese data are a valuable resource for functional genomics analysis of airway epithelial tissues in human disease.

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells

Abstract AS04: Epigenomic characterization of gene regulatory networks in human ovarian cancer stem cells

Abstract Ovarian cancer is the fifth leading cause of cancer mortality among women in the United States. Despite aggressive surgery and chemotherapy, most patients relapse and develop drug-resistant tumors, leading to the <40% chance of 5 year survival in ovarian cancer patients. Evidence suggests that a small subpopulation of cells in the tumor, ovarian cancer stem cells (OvCSCs), are responsible for tumor relapse and drug resistance. OvCSCs have enhanced tumorigenicity, inherent chemoresistance, and stem-like properties such as the ability to self-renew or divide asymmetrically. These latter characteristics give CSCs an analogous role in tumors that healthy stem cells have in developing organs (adult stem cells) or entire organisms (embryonic stem cells).The goal of this study is to determine how the cancer stem cell state is regulated by annotating the gene regulatory network of OvCSCs. In doing so, we will uncover commonalities that exist between OvCSCs and embryonic or progenitor stem cells. The epigenome provides the structural framework for cell-type specific gene regulation. Chromatin modifications that uniquely mark gene regulatory elements, like promoters and enhancers, are easily identified throughout the genome using ChIP-seq. ChIP-seq performed in pluripotent stem cells has revealed many epigenetic features that distinguish them from differentiated cells. In an effort to better understand CSC gene regulatory networks, we are generating comprehensive transcriptomic maps and globally identifying enhancers marked by histone 3, lysine 4 monomethylation (via RNAseq and H3K4me1 ChIP-seq respectively) in OvCSCs and their daughter tumor cells. We are also comparing OvCSCs to previously generated data in human embryonic stem cells (hESCs) to identify shared stemness traits. OvCSCs were acquired from patient biopsies and detailed characterization identified a CD133+ CSC population within the tumor. We are using a mouse xenograft model to expand tumors in vivo. Harvested tumors are depleted of blood lineage cells and CD133+ cells (CSCs) are isolated from the remaining cells in the tumor. Our analysis of RNA-seq and enhancer maps have revealed OvCSC signatures that are absent in tumor cells but present in embryonic stem cells. Many important embryonic stem cell transcription factors are expressed in OvCSCs and have almost identical chromatin enhancer profiles. We identified over 7000 H1 hESC enhancers that have at least 95% overlap with OvCSC enhancers implying a shared regulatory network in these cell types. Motif analysis of nucleosome-free regions within enhancers reveals enrichment for many ESC transcription factors that are also expressed in OvCSCs. Our integrative analysis indicates that OvCSCs share common features of the ESC regulatory network and is beginning to provide novel insight on why CSCs have stem cell properties. We are continuing to build maps for additional histone modifications and genome-wide DNA methylation in OvCSCs to gain a comprehensive view of how the epigenome regulates the cancer stem cell state. Citation Format: Stephanie L. Battle, Antti Larjo, Joling Liao, Harri Lähdesmäki, Andre Lieber, R. David Hawkins. Epigenomic characterization of gene regulatory networks in human ovarian cancer stem cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS04.

Development of a feeding trochophore in the polychaete Hydroides elegans.

Hydroides elegans is an indirectly developing polychaete with equal spiral cleavage, gastrulation by invagination, and a feeding trochophore. Expression of several transcription factors and differentiation genes has been characterized. Comparative analysis reveals evolutionarily conserved roles. For example, the synexpression of transcription factors FoxA and Brachyury suggests homology of primary and secondary gut openings in protostomes and deuterostomes, and the expression of Sall suggests similar regulatory controls in the posterior growth zone of bilaterians. Differences in gene expression suggest regulatory differences control gastrulation by invagination in polychaetes with a feeding trochophore and gastrulation by epiboly in polychaetes without a feeding trochophore. Association of histone variant H2A.Z with transcriptional potency and its expression suggest a developmental role during both embryogenesis and the larva-to-adult transformation. Methods are being developed for experimental exploration of the gene regulatory networks involved in trochophore development in Hydroides. It is unknown if polychaete feeding trochophores evolved from a larval stage already present in the life cycle of the last common ancestor of protostomes and deuterostomes. Previous evolutionary scenarios about larval origins overemphasize the discontinuity between larval and adult development and require the early evolution of undifferentiated and transcriptionally potent "set aside" cells. Indirect development may proceed by developmental remodeling of differentiated cells and could have evolved after gradual transformation of juveniles into larvae; undifferentiated and transcriptionally potent cells would have evolved secondarily. Comprehensive characterization of gene regulatory networks for feeding trochophore development may help resolve these major evolutionary questions.

An embryonic stem cell‐based system for rapid analysis of transcriptional enhancers

With the growing use of genome-wide screens for cis-regulatory elements, there is a pressing need for platforms that enable fast and cost-effective experimental validation of identified hits in relevant developmental and tissue contexts. Here, we describe a murine embryonic stem cell (ESC)-based system that facilitates rapid analysis of putative transcriptional enhancers. Candidate enhancers are targeted with high efficiency to a defined genomic locus via recombinase-mediated cassette exchange. Targeted ESCs are subsequently differentiated in vitro into desired cell types, where enhancer activity is monitored by reporter gene expression. As a proof of principle, we analyzed a previously characterized, Sonic hedgehog (Shh)-dependent, V3 interneuron progenitor (pV3)-specific enhancer for the Nkx2.2 gene, and observed highly specific enhancer activity. Given the broad potential of ESCs to generate a spectrum of cell types, this system can serve as an effective platform for the characterization of gene regulatory networks controlling cell fate specification and cell function.

Ventralization of an indirect developing hemichordate by NiCl2 suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms)

One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl2), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl2 disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior–posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl2 sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl2 sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan.

Gene regulatory networks and the role of robustness and stochasticity in the control of gene expression

In any given cell, thousands of genes are expressed and work in concert to ensure the cell's function, fitness, and survival. Each gene, in turn, must be expressed at the proper time and in the proper amounts to ensure the appropriate functional outcome. The regulation and expression of some genes are highly robust; their expression is controlled by invariable expression programs. For instance, developmental gene expression is extremely similar in a given cell type from one individual to another. The expression of other genes is more variable: Their levels are noisy and are different from cell to cell and from individual to individual. This can be highly beneficial in physiological responses to outside cues and stresses. Recent advances have enabled the analysis of differential gene expression at a systems level. Gene regulatory networks (GRNs) involving interactions between large numbers of genes and their regulators have been mapped onto graphic diagrams that are used to visualize the regulatory relationships. The further characterization of GRNs has already uncovered global principles of gene regulation. Together with synthetic network biology, such studies are starting to provide insights into the transcriptional mechanisms that cause robust versus stochastic gene expression and their relationships to phenotypic robustness and variability. Here, we discuss GRNs and their topological properties in relation to transcriptional and phenotypic outputs in development and organismal physiology.

A Gene Signature-Based Approach Identifies mTOR as a Regulator of p73

Although genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcription factors has been more challenging. In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73. We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling. The p73 signature was linked to corresponding signatures produced by drug candidates, using the in silico Connectivity Map resource, to identify drugs that would induce p73 activity. Of the pharmaceutical agents identified, there was enrichment for direct or indirect inhibitors of mammalian Target of Rapamycin (mTOR) signaling. Treatment of both primary cells and cancer cell lines with rapamycin, metformin, and pyrvinium resulted in an increase in p73 levels, as did RNA interference-mediated knockdown of mTOR. Further, a subset of genes associated with insulin response or autophagy exhibited mTOR-mediated, p73-dependent expression. Thus, downstream gene signatures can be used to identify upstream regulators of transcription factor activity, and in doing so, we identified a new link between mTOR, p73, and p73-regulated genes associated with autophagy and metabolic pathways.