Mutual Repression Research Articles

Impacts of the feedback loop between sense-antisense RNAs in regulating circadian rhythms

Antisense transcripts are a unique group of non-coding RNAs and play regulatory roles in a variety of biological processes, including circadian rhythms. Per2AS is an antisense transcript to the sense core clock gene Period2 (Per2) in mouse and its expression is rhythmic and antiphasic to Per2. To understand the impact of Per2AS-Per2 interaction, we developed a new mathematical model that mechanistically described the mutually repressive relationship between Per2 and Per2AS. This mutual repression can regulate both amplitude and period of circadian oscillation by affecting a negative feedback regulation of Per2. Simulations from this model also fit with experimental observations that could not be fully explained by our previous model. Our revised model can not only serve as a foundation to build more detailed models to better understand the impact of Per2AS-Per2 interaction in the future, but also be used to analyze other sense-antisense RNA pairs that mutually repress each other.

The genetic determination of alternate stages in polyphenic insects.

Molt-based transitions in form are a central feature of insect life that have enabled adaptation to diverse and changing environments. The endocrine regulation of these transitions is well established, but an understanding of their genetic regulation has only recently emerged from insect models. The pupal and adult stages of metamorphosing insects are determined by the stage specifying transcription factors broad-complex (br) and Ecdysone inducible protein 93 (E93), respectively. A probable larval determinant, chronologically inappropriate metamorphosis (chinmo), has just recently been characterized. Expression of these three transcription factors in the metamorphosing insects is regulated by juvenile hormone with ecdysteroid hormones, and by mutual repression between the stage-specific transcription factors. This review explores the hypothesis that variations in the onset, duration, and tissue-specific expression of chinmo, br, and E93 underlie other polyphenisms that have arisen throughout insects, including the castes of social insects, aquatic stages of mayflies, and the neoteny of endoparasites. The mechanisms that constrain how chinmo, br, and E93 expression may vary will also constrain the ways that insect life history may evolve. I find that four types of expression changes are associated with novel insect forms: (1) heterochronic shift in the turnover of expression, (2) expansion or contraction of expression, (3) tissue-specific expression, and (4) redeployment of stage-specific expression. While there is more to be learned about chinmo, br, and E93 function in diverse insect taxa, the studies outlined here show that insect stages are modular units in developmental time and a substrate for evolutionary forces to act upon.

MKL/SRF and Bcl6 mutual transcriptional repression safeguards the fate and positioning of neocortical progenitor cells mediated by RhoA.

During embryogenesis, multiple intricate and intertwined cellular signaling pathways coordinate cell behavior. Their slightest alterations can have dramatic consequences for the cells and the organs they form. The transcriptional repressor Bcl6 was recently found as important for brain development. However, its regulation and integration with other signals is unknown. Using in vivo functional approaches combined with molecular mechanistic analysis, we identified a reciprocal regulatory loop between B cell lymphoma 6 (Bcl6) and the RhoA-regulated transcriptional complex megakaryoblastic leukemia/serum response factor (MKL/SRF). We show that Bcl6 physically interacts with MKL/SRF, resulting in a down-regulation of the transcriptional activity of both Bcl6 and MKL/SRF. This molecular cross-talk is essential for the control of proliferation, neurogenesis, and spatial positioning of neural progenitors. Overall, our data highlight a regulatory mechanism that controls neuronal production and neocortical development and reveal an MKL/SRF and Bcl6 interaction that may have broader implications in other physiological functions and in diseases.

Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration.

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, we investigate how spatial patterns of signaling pathways and repair behaviors arise. We find that Eiger expression, which drives JNK/AP-1 signaling, transiently arrests proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, we demonstrate that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for our conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments.

1275 A mutual repression between MITF and neural transcription factors specifies the fate of schwann cell precursors

1275 A mutual repression between MITF and neural transcription factors specifies the fate of schwann cell precursors

Unbalanced response to growth variations reshapes the cell fate decision landscape.

The global regulation of cell growth rate on gene expression perturbs the performance of gene networks, which would impose complex variations on the cell-fate decision landscape. Here we use a simple synthetic circuit of mutual repression that allows a bistable landscape to examine how such global regulation would affect the stability of phenotypic landscape and the accompanying dynamics of cell-fate determination. We show that the landscape experiences a growth-rate-induced bifurcation between monostability and bistability. Theoretical and experimental analyses reveal that this bifurcating deformation of landscape arises from the unbalanced response of gene expression to growth variations. The path of growth transition across the bifurcation would reshape cell-fate decisions. These results demonstrate the importance of growth regulation on cell-fate determination processes, regardless of specific molecular signaling or regulation.

Manipulation of Signal Gradient and Transcription Factors Recapitulates: Multiple Hypothalamic Identities.

During development, the hypothalamus emerges from the ventral diencephalon and is regionalized into several distinct functional domains. Each domain is characterized by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signaling and its downstream transcription network are required for hypothalamic regionalization and establishment.

Emergent dynamics of a three-node regulatory network explain phenotypic switching and heterogeneity: a case study of Th1/Th2/Th17 cell differentiation.

Naïve helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a “master regulator”—T-bet (Th1), GATA3 (Th2), and RORγT (Th17)—that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotype, Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17, and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remain unclear. Here through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any master regulator can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/RORγT.

Signal processing capacity of the cellular sensory machinery regulates the accuracy of chemotaxis under complex cues

SummaryChemotaxis is ubiquitous in many biological processes, but it still remains elusive how cells sense and decipher multiple chemical cues. In this study, we postulate a hypothesis that the chemotactic performance of cells under complex cues is regulated by the signal processing capacity of the cellular sensory machinery. The underlying rationale is that cells in vivo should be able to sense and process multiple chemical cues, whose magnitude and compositions are entangled, to determine their migration direction. We experimentally show that the combination of transforming growth factor-β and epidermal growth factor suppresses the chemotactic performance of cancer cells using independent receptors to sense the two cues. Based on this observation, we develop a biophysical framework suggesting that the antagonism is caused by the saturation of the signal processing capacity but not by the mutual repression. Our framework suggests the significance of the signal processing capacity in the cellular sensory machinery.

Coexistence of Hopf-born rotation and heteroclinic cycling in a time-delayed three-gene auto-regulated and mutually-repressed core genetic regulation network

In this work, we study the behavior of a time-delayed mutually repressive auto-activating three-gene system. Delays are introduced to account for the location difference between DNA transcription that leads to production of messenger RNA and its translation that result in protein synthesis. We study the dynamics of the system using numerical simulations, computational bifurcation analysis and mathematical analysis. We find Hopf bifurcations leading to stable and unstable rotation in the system, and we study the rotational behavior as a function of cyclic mutual repression parameter asymmetry between each gene pair in the network. We focus on how rotation co-exists with a stable heteroclinic flow linking the three saddles in the system. We find that this coexistence allows for a transition between two markedly different types of rotation leading to strikingly different phenotypes. One type of rotation belongs to Hopf-induced rotation while the other type, belongs to heteroclinic cycling between three saddle nodes in the system. We discuss the evolutionary and biological implications of our findings.

A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

Long noncoding RNA Gm20319, acting as competing endogenous RNA, regulated GNE expression by sponging miR-7240-5p to involve in deoxynivalenol-induced liver damage in vitro

The regulatory effects of competing endogenous RNA (ceRNA) network have been highlighted on the occurrence and development of diseases. However, the effect of ceRNA network in liver with subchronic deoxynivalenol (DON) exposure has remained unclear so far. Here, lncRNA Gm20319-miR-7240-5p-GNE (glucosamine UDP-N-acetyl-2-epimerase/N-acetylmannosamine kinase) network was identified in DON exposed-liver tissues after DON exposure for 90 days. Subchronic DON exposure induced the mild inflammation in liver tissues. In DON-treated liver tissues and Hepa 1–6 cell line, the expression of Gm20319 and GNE were both downregulated while miR-7240-5p expression was upregulated. The gain- and loss-of-function expression in vitro revealed there was a mutual repression between Gm20319 and miR-7240-5p, and they regulated GNE expression in an opposite direction. Dual luciferase reporter assays showed miR-7240-5p inhibited Gm20319 and GNE expression by directly binding. Co-transfection experiment in vitro revealed Gm20319 and miR-7240-5p could indirectly regulate sialic acid level by directly modulating GNE expression, thereby also influencing the expression of SOD1 and IL-1β. This study revealed Gm20319-miR-7240-5p-GNE network reduced sialic acid level to influence the expression of SOD1 and IL-1β in liver, which might involve in liver damage induced by DON. Gm20319 might be a potential research molecular target for DON-induced liver damage.

Mutual Zonated Interactions of Wnt and Hh Signaling Are Orchestrating the Metabolism of the Adult Liver in Mice and Human

The Hedgehog (Hh) and Wnt/β-Catenin (Wnt) cascades are morphogen pathways whose pronounced influence on adult liver metabolism has been identified in recent years. How both pathways communicate and control liver metabolic functions are largely unknown. Detecting core components of Wnt and Hh signaling and mathematical modeling showed that both pathways in healthy liver act largely complementary to each other in the pericentral (Wnt) and the periportal zone (Hh) and communicate mainly by mutual repression. The Wnt/Hh module inversely controls the spatiotemporal operation of various liver metabolic pathways, as revealed by transcriptome, proteome, and metabolome analyses. Shifting the balance to Wnt (activation) or Hh (inhibition) causes pericentralization and periportalization of liver functions, respectively. Thus, homeostasis of the Wnt/Hh module is essential for maintaining proper liver metabolism and to avoid the development of certain metabolic diseases. With caution due to minor species-specific differences, these conclusions may hold for human liver as well.

Improvement of the memory function of a mutual repression network in a stochastic environment by negative autoregulation

BackgroundCellular memory is a ubiquitous function of biological systems. By generating a sustained response to a transient inductive stimulus, often due to bistability, memory is central to the robust control of many important biological processes. However, our understanding of the origins of cellular memory remains incomplete. Stochastic fluctuations that are inherent to most biological systems have been shown to hamper memory function. Yet, how stochasticity changes the behavior of genetic circuits is generally not clear from a deterministic analysis of the network alone. Here, we apply deterministic rate equations, stochastic simulations, and theoretical analyses of Fokker-Planck equations to investigate how intrinsic noise affects the memory function in a mutual repression network.ResultsWe find that the addition of negative autoregulation improves the persistence of memory in a small gene regulatory network by reducing stochastic fluctuations. Our theoretical analyses reveal that this improved memory function stems from an increased stability of the steady states of the system. Moreover, we show how the tuning of critical network parameters can further enhance memory.ConclusionsOur work illuminates the power of stochastic and theoretical approaches to understanding biological circuits, and the importance of considering stochasticity when designing synthetic circuits with memory function.

Reciprocal transrepression between FOXF2 and FOXQ1 controls basal‐like breast cancer aggressiveness

FOXF2 and FOXQ1, forkhead box transcription factor superfamily members, are encoded by neighboring genes located on human chromosome 6p25.3 and play opposite roles in epithelial-mesenchymal transition (EMT) and metastasis in basal-like breast cancer (BLBC). However, the relationship between FOXF2 and FOXQ1 in cancer remains unknown. Here, we found mutual transcriptional repression between FOXF2 and FOXQ1, and the reciprocal negative feedback loop controlled EMT, aggressiveness, and chemoresistance in BLBC cells. We further demonstrated that FOXF2 recruited nuclear receptor corepressor 1 and histone deacetylase 3 to the FOXQ1 promoter to inhibit its transcription in BLBC cells, but FOXQ1 did not exert such an effect on FOXF2. Our findings reveal novel mechanisms underlying the determination of BLBC aggressiveness and the transrepressive function of FOXF2 in a basal-like cell subtype-specific manner. Therefore, blocking the vicious cycle of the abnormal reciprocal feedback loop between FOXF2 and FOXQ1 to induce cell differentiation and restore tissue homeostasis is a promising strategy for the treatment of aggressive BLBC.-Kang, L.-J., Yu, Z.-H., Cai, J., He, R., Lu, J.-T., Hou, C., Wang, Q.-S., Li, X.-Q., Zhang, R., Feng, Y.-M. Reciprocal transrepression between FOXF2 and FOXQ1 controls basal-like breast cancer aggressiveness.

Landscape reveals critical network structures for sharpening gene expression boundaries

BackgroundSpatial pattern formation is a critical issue in developmental biology. Gene expression boundary sharpening has been observed from both experiments and modeling simulations. However, the mechanism to determine the sharpness of the boundary is not fully elucidated.ResultsWe investigated the boundary sharpening resulted by three biological motifs, interacting with morphogens, and uncovered their probabilistic landscapes. The landscape view, along with calculated average switching time between attractors, provides a natural explanation for the boundary sharpening behavior relying on the noise induced gene state switchings. To possess boundary sharpening potential, a gene network needs to generate an asymmetric bistable state, i.e. one of the two stable states is less stable than the other. We found that the mutual repressed self-activation model displays more robust boundary sharpening ability against parameter perturbation, compared to the mutual repression or the self-activation model. This is supported by the results of switching time calculated from the landscape, which indicate that the mutual repressed self-activation model has shortest switching time, among three models. Additionally, introducing cross gradients of morphogens provides a more stable mechanism for the boundary sharpening of gene expression, due to a two-way switching mechanism.ConclusionsOur results reveal the underlying principle for the gene expression boundary sharpening, and pave the way for the mechanistic understanding of cell fate decisions in the pattern formation processes of development.

Two Nucleolar Proteins, GDP1 and OLI2, Function As Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial-Abaxial Patterning in Arabidopsis thaliana.

Leaf abaxial–adaxial patterning is dependent on the mutual repression of leaf polarity genes expressed either adaxially or abaxially. In Arabidopsis thaliana, this process is strongly affected by mutations in ribosomal protein genes and in ribosome biogenesis genes in a sensitized genetic background, such as asymmetric leaves2 (as2). Most ribosome-related mutants by themselves do not show leaf abaxialization, and one of their typical phenotypes is the formation of pointed rather than rounded leaves. In this study, we characterized two ribosome-related mutants to understand how ribosome biogenesis is linked to several aspects of leaf development. Previously, we isolated oligocellula2 (oli2) which exhibits the pointed-leaf phenotype and has a cell proliferation defect. OLI2 encodes a homolog of Nop2 in Saccharomyces cerevisiae, a ribosome biogenesis factor involved in pre-60S subunit maturation. In this study, we found another pointed-leaf mutant that carries a mutation in a gene encoding an uncharacterized protein with a G-patch domain. Similar to oli2, this mutant, named g-patch domain protein1 (gdp1), has a reduced number of leaf cells. In addition, gdp1 oli2 double mutants showed a strong genetic interaction such that they synergistically impaired cell proliferation in leaves and produced markedly larger cells. On the other hand, they showed additive phenotypes when combined with several known ribosomal protein mutants. Furthermore, these mutants have a defect in pre-rRNA processing. GDP1 and OLI2 are strongly expressed in tissues with high cell proliferation activity, and GDP1-GFP and GFP-OLI2 are localized in the nucleolus. These results suggest that OLI2 and GDP1 are involved in ribosome biogenesis. We then examined the effects of gdp1 and oli2 on adaxial–abaxial patterning by crossing them with as2. Interestingly, neither gdp1 nor oli2 strongly enhanced the leaf polarity defect of as2. Similar results were obtained with as2 gdp1 oli2 triple mutants although they showed severe growth defects. These results suggest that the leaf abaxialization phenotype induced by ribosome-related mutations is not merely the result of a general growth defect and that there may be a sensitive process in the ribosome biogenesis pathway that affects adaxial–abaxial patterning when compromised by a mutation.

Landscape of gene networks for random parameter perturbation.

Landscape approaches have been exploited to study the stochastic dynamics of gene networks. However, how to calculate the landscape with a wide range of parameter variations and how to investigate the influence of the network topology on the global properties of gene networks remain to be elucidated. Here, I developed an approach for the landscape of random parameter perturbation (LRPP) to address this issue. Based on a self-consistent approximation approach, by making perturbations to parameters in a given range, I obtained the landscape for gene network systems. I applied this approach to two biological models, one for the mutual repression model and the other for the embryonic stem (ES) cell differentiation network. For the mutual repression model, my results confirm quantitatively that positive feedback promotes the robustness of multistability. For the ES cell differentiation model, I identify three cell states, representing the ES cell, the differentiation cell, and the intermediate state cell, respectively. I propose that the intermediate states and the wide range of parameter values coming from inhomogeneous cellular environments provide possible explanations for the heterogeneity observed in single cell experiments. I also offer a counterintuitive result that noise could reduce heterogeneity and promote the stability of cell states. These results support that the network topology determines the operating principles of the genetic networks, reflected by the representative landscapes from LRPP. This work provides a new route to obtain the potential landscape for a gene network system given a wide range of parameter values and study the influences of the network topology on the global properties of the system.

Assessing regulatory information in developmental gene regulatory networks

Gene regulatory networks (GRNs) provide a transformation function between the static genomic sequence and the primary spatial specification processes operating development. The regulatory information encompassed in developmental GRNs thus goes far beyond the control of individual genes. We here address regulatory information at different levels of network organization, from single node to subcircuit to large-scale GRNs and discuss how regulatory design features such as network architecture, hierarchical organization, and cis-regulatory logic contribute to the developmental function of network circuits. Using specific subcircuits from the sea urchin endomesoderm GRN, for which both circuit design and biological function have been described, we evaluate by Boolean modeling and in silico perturbations the import of given circuit features on developmental function. The examples include subcircuits encoding positive feedback, mutual repression, and coherent feedforward, as well as signaling interaction circuitry. Within the hierarchy of the endomesoderm GRN, these subcircuits are organized in an intertwined and overlapping manner. Thus, we begin to see how regulatory information encoded at individual nodes is integrated at all levels of network organization to control developmental process.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells

Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3′ enhancer of Hoxa1. This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.