Gene Networks of Fully Connected Triads with Complete Auto-Activation Enable Multistability and Stepwise Stochastic Transitions

Fully-connected triads (FCTs), such as the Oct4-Sox2-Nanog triad, have been implicated as recurring transcriptional motifs embedded within the regulatory networks that specify and maintain cellular states. To explore the possible connections between FCT topologies and cell fate determinations, we employed computational network screening to search all possible FCT topologies for multistability, a dynamic property that allows the rise of alternate regulatory states from the same transcriptional network. The search yielded a hierarchy of FCTs with various potentials for multistability, including several topologies capable of reaching eight distinct stable states. Our analyses suggested that complete auto-activation is an effective indicator for multistability, and, when gene expression noise was incorporated into the model, the networks were able to transit multiple states spontaneously. Different levels of stochasticity were found to either induce or disrupt random state transitioning with some transitions requiring layovers at one or more intermediate states. Using this framework we simulated a simplified model of induced pluripotency by including constitutive overexpression terms. The corresponding FCT showed random state transitioning from a terminal state to the pluripotent state, with the temporal distribution of this transition matching published experimental data. This work establishes a potential theoretical framework for understanding cell fate determinations by connecting conserved regulatory modules with network dynamics. Our results could also be employed experimentally, using established developmental transcription factors as seeds, to locate cell lineage specification networks by using auto-activation as a cipher

Gene Networks of Fully Connected Triads with Complete Auto-Activation Enable Multistability and Stepwise Stochastic Transitions

abstract: Fully-connected triads (FCTs), such as the Oct4-Sox2-Nanog triad, have been implicated as recurring transcriptional motifs embedded within the regulatory networks that specify and maintain cellular states. To explore the possible connections between FCT topologies and cell fate determinations, we employed computational network screening to search all possible FCT topologies for multistability, a dynamic property that allows the rise of alternate regulatory states from the same transcriptional network. The search yielded a hierarchy of FCTs with various potentials for multistability, including several topologies capable of reaching eight distinct stable states. Our analyses suggested that complete auto-activation is an effective indicator for multistability, and, when gene expression noise was incorporated into the model, the networks were able to transit multiple states spontaneously. Different levels of stochasticity were found to either induce or disrupt random state transitioning with some transitions requiring layovers at one or more intermediate states. Using this framework we simulated a simplified model of induced pluripotency by including constitutive overexpression terms. The corresponding FCT showed random state transitioning from a terminal state to the pluripotent state, with the temporal distribution of this transition matching published experimental data. This work establishes a potential theoretical framework for understanding cell fate determinations by connecting conserved regulatory modules with network dynamics. Our results could also be employed experimentally, using established developmental transcription factors as seeds, to locate cell lineage specification networks by using auto-activation as a cipher.The article is published at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.010287

Knockdown of Brm and Baf170, Components of Chromatin Remodeling Complex, Facilitates Reprogramming of Somatic Cells

© Copyright 2015, Mary Ann Liebert, Inc. 2015. The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells, esBAF contains Brg1 and Baf155, while their homologs, Brm and Baf170, are present in BAF of somatic cells. In this study, we sought to determine whether Brm and Baf170 play any roles in induced pluripotent stem cell (iPSC) reprogramming by using shRNA-mediated knockdown studies in the mouse model. We found that knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improve the numbers of iPSC colonies formed. We further showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed somatic cells (pre-iPSCs). Finally, we found that the expression of Brm and Baf170 during reprogramming was regulated by Jak/Stat3 activity. Taken together, these data suggest that inhibiting somatic BAF improves complete reprogramming by facilitating the activation of the pluripotency circuitry

From Endogenous to Synthetic microRNA-Mediated Regulatory Circuits: An Overview

MicroRNAs are short non-coding RNAs that are evolutionarily conserved and are pivotal post-transcriptional mediators of gene regulation. Together with transcription factors and epigenetic regulators, they form a highly interconnected network whose building blocks can be classified depending on the number of molecular species involved and the type of interactions amongst them. Depending on their topology, these molecular circuits may carry out specific functions that years of studies have related to the processing of gene expression noise. In this review, we first present the different over-represented network motifs involving microRNAs and their specific role in implementing relevant biological functions, reviewing both theoretical and experimental studies. We then illustrate the recent advances in synthetic biology, such as the construction of artificially synthesised circuits, which provide a controlled tool to test experimentally the possible microRNA regulatory tasks and constitute a starting point for clinical applications

Synthetic Regulation of Eukaryotic Gene Expression by Noncoding RNA

Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions. For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming. We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control. Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.</p

The Human Heterochromatin Landscape: Genomic Subtypes, Bound Proteins, And Contributions To Cell Identity

Large portions of mammalian genomes are packaged into structurally compact heterochromatin, which protects genome integrity and suppresses transcription of lineage-inappropriate genes. Characterization of heterochromatic regions has relied on genomic mapping of associated histone modifications, such as H3K9me3 and H3K27me3, and purification of proteins interacting with these modifications. Heterochromatic regions marked by H3K9me3 have been shown to impede gene activation during reprogramming to pluripotency, and I find that H3K9me3 domains can similarly impede conversion of fibroblasts to hepatocytes. However, both H3K9me3 and H3K27me3 can be found in transcriptionally active chromatin, limiting the accuracy of histone marks alone for identifying heterochromatin domains or bound proteins that impede reprogramming. I developed a biophysical method to purify heterochromatic regions, using sucrose gradients to isolate chromatin fragments that are resistant to sonication. Sequencing of the purified material (Gradient-seq) revealed the genomic landscape of structural heterochromatin in human fibroblasts, which is transcribed at low levels and contains largely distinct H3K9me3 and H3K27me3 domains, as well as unmarked regions. Gradient-seq also uncovered subtypes of H3K9me3 and H3K27me3 domains that are structurally euchromatic, a distinction corroborated by increased gene transcription, hypomethylation at CpG islands, decreased association with the nuclear lamina, and increased activation during hepatic reprogramming. Using quantitative proteomics, we found 172 proteins associated with heterochromatin after gradient sedimentation and H3K9me3-directed IP. The identified proteins include known transcriptional repressors and are enriched for proteins shown to impede reprogramming to pluripotency. We show that the RNA-binding protein RBMX, one of the proteins most enriched by gradient sedimentation and H3K9me3 IP, is a functional regulator of heterochromatin. RBMX and the related protein RBMXL1 are required for silencing of select heterochromatinized genes, and depletion of these proteins in fibroblasts renders H3K9me3-marked hepatocyte genes more competent for activation during reprogramming to the hepatic lineage. Thus, our biophysical method for heterochromatin isolation has allowed us to create a genome-wide map of chromatin compaction in human cells, to identify chromatin domain subtypes that impede conversion between differentiated lineages, and to discover novel heterochromatin proteins that contribute to this reprogramming barrier

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Balancing macrophage activation in health and disease:The epigenetic, transcriptional and immunometabolic insights

Macrophages are crucial components of the innate immune system. Macrophages manifest extreme heterogeneity in response to the local milieu. The plasticity and diversity of macrophages are pivotal for host defense against pathogenic insults and the maintenance of tissue homeostasis. However, dysregulated macrophage activation can lead to acute and chronic inflammatory disorders, such as COVID-19, atherosclerosis, and inflammatory bowel diseases. The activation states of macrophages are shaped by various mechanisms, such as epigenetic modifications, transcriptional regulation, and metabolic alterations. This dissertation provides comprehensive transcriptomic and metabolomic profiles of widely used macrophage models. We build a macrophage activation classifier and identify key regulatory network modules constructed by unbiased approaches. Furthermore, our data delivers insights into targeting metabolic pathways, kinases, and epigenetic enzymes for therapeutic development against various diseases. These findings advance our knowledge in macrophage activation and pathogenesis of different diseases and disorders that can support future studies from basic science to translational medicine and from bench to bedside

High-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration

Human cells and tissues are key systems to study human biology and physiology, and to develop new strategies and targeting drugs for human diseases. Since the study and testing on human beings may not be acceptable due to exposure to risks and practical and ethical concerns, in vitro strategies are of paramount importance to rely on human organism and avoid non-fully predictive animal models. The demand of research in clinical and industrial fields for effective, representative and affordable strategies is undoubtedly increasing. Conventional cell culture systems and drug discovery are normally performed in vessels with a characteristic dimension in the order of centimeters. Nutrients are delivered to cells through liquid media containing balanced saline buffers and oligo-elements. A reasonable amount of medium is necessary to homogeneously cover a cell layer and must exchanged with fresh media to maintain a proper amount of available nutrients and remove released waste products. Many studies and applications require expensive reagents and are subjected to limited data throughput. The discovery of reprogramming process by 2012 Nobel Prize Yamanaka opened breakthrough new perspective on research and clinical applications. Basically, from a patient’s skin biopsy it is now possible to derive induced pluripotent stem cells (iPSC) and to obtain new tissues for an ad hoc self-repair. So far, human iPSC (hiPSC) have not been applied to clinics due to some unexplored aspects on their derivation, non clinical-grade methods and the significative cost of hiPSC derivation per patient. The down-scale of reprogramming process could provide an unique opportunity to derive cost-effective hiPSC and obtain valuable human in vitro tissues. The aim of this thesis is the development of a comprehensive platform for the reprogramming of human cells at the microscale. To this end, we focused on the development of cell microenvironment which is composed by both soluble and solid components. During this thesis, synthetic and biodegradable hydrogels were developed. The large-scale production of mechanically-tunable poly-acrylamide-based substrates were fundamental to reveal the interaction occurring between substrate stiffness and cell behavior and fate. Engineering of biodegradable hydrogels has revealed the potential to develop in vitro functional tissues and to integrate them at a later stage in patients. Chemical modifications were transferred to topological substrate control and in turn in microfluidic platforms. Microfluidic chip environment and management was designed in order to allow long-term adhesion, culture and biologically relevant cell behaviors. Adhesion proteins fundamental for cell attachment and growth were modified and integrated with the micronized substrates. Since medium for microfluidic cell culture relies on perfusion, continuous or periodic flow could be applied. Thus, we studied the management of media delivery in order to determine the best strategy for long-term cell cultures. The achievements obtained with both substrate and microfluidic cell culture development was applied to the generation of a new platform for hiPSC derivation, differentiation and testing at the microscale. For the first time, it is possible to obtain human iPSC clones in microfluidics with a remarked reduction of minimum requirements (materials, reagents, overall expenses). The production of cost effective hiPSC can lead to a mass production of characterized and functional tissues that can be either integrated in 3D developed constructs and serve as valuable tissue source derivation for drug development. Our platform opens new perspectives in studying and treating both abundant and rare diseases involving both scientists and entrepreneur