Delay In Gene Expression Research Articles

Feedforward Loops: Evolutionary Conserved Network Motifs Redesigned for Synthetic Biology Applications

Feedforward loops (FFLs) are relatively simple network motifs, made of three interacting genes, that have been found in a large number in E. coli and S. cerevisiae. More recently, they have also been discovered in multicellular eukaryotes. FFLs are evolutionary favored motifs because they enable cells to survive critical environmental conditions. Among the eight types of possible FFLs, the so-called coherent 1 and incoherent 1 FFL are the most abundant. The former carries out a sign-sensitive delay in gene expression; the latter is a pulse generator and a response time accelerator. So far, only few synthetic FFLs have been engineered, either in cell-free systems or in vivo. In this work, we review the main experimental works published on FFLs, with particular focus on novel designs for synthetic FFLs. They are, indeed, quite different from the natural ones that arose during the course of evolution.

Feed-forward regulation adaptively evolves via dynamics rather than topology when there is intrinsic noise

In transcriptional regulatory networks (TRNs), a canonical 3-node feed-forward loop (FFL) is hypothesized to evolve to filter out short spurious signals. We test this adaptive hypothesis against a novel null evolutionary model. Our mutational model captures the intrinsically high prevalence of weak affinity transcription factor binding sites. We also capture stochasticity and delays in gene expression that distort external signals and intrinsically generate noise. Functional FFLs evolve readily under selection for the hypothesized function but not in negative controls. Interestingly, a 4-node “diamond” motif also emerges as a short spurious signal filter. The diamond uses expression dynamics rather than path length to provide fast and slow pathways. When there is no idealized external spurious signal to filter out, but only internally generated noise, only the diamond and not the FFL evolves. While our results support the adaptive hypothesis, we also show that non-adaptive factors, including the intrinsic expression dynamics, matter.

Noise in the Vertebrate Segmentation Clock Is Boosted by Time Delays but Tamed by Notch Signaling

SUMMARYTaming cell-to-cell variability in gene expression is critical for precise pattern formation during embryonic development. To investigate the source and buffering mechanism of expression variability, we studied a biological clock, the vertebrate segmentation clock, controlling the precise spatiotemporal patterning of the vertebral column. By counting single transcripts of segmentation clock genes in zebrafish, we show that clock genes have low RNA amplitudes and expression variability is primarily driven by gene extrinsic sources, which is suppressed by Notch signaling. We further show that expression noise surprisingly increases from the posterior progenitor zone to the anterior segmentation and differentiation zone. Our computational model reproduces the spatial noise profile by incorporating spatially increasing time delays in gene expression. Our results, suggesting that expression variability is controlled by the balance of time delays and cell signaling in a vertebrate tissue, will shed light on the accuracy of natural clocks in multi-cellular systems and inspire engineering of robust synthetic oscillators.

Characterisation of Postia placenta colonisation during 36 weeks in acetylated southern yellow pine sapwood at three acetylation levels including genomic DNA and gene expression quantification of the fungus

Abstract One way to protect timber in service against basidiomycete deterioration is by means of acetylation via reaction with acetic anhydride. The reason why acetylated wood (WAc) is resistant against decay fungi is still not exactly understood. The aim of this study was to contribute to this field of science, and Postia placenta colonisation after 4, 12, 20, 28 and 36 weeks was observed at three acetylation levels of Pinus spp. sapwood. Mass loss (ML) and wood moisture content (MC) data reflected the acetylation levels. The initial equilibrium MC (EMC) proved to be a good indicator of subsequent ML. Genomic DNA quantification showed P. placenta colonisation in all samples, also in samples where no ML were detectable. The number of expressed gene transcripts was limited, but the findings supported the results of previous studies: WAc seems to have some resistance against oxidative mechanisms, which are part of the metabolism of P. placenta. This leads to a delay in decay initiation, a delay in expression of genes involved in enzymatic depolymerisation, and a slower decay rate. The magnitudes of these effects are presented for each acetylation level. The data also imply that there is no absolute decay threshold at high acetylation levels, but instead a significant delay of decay initiation and a slower decay rate.

Adeno‐associated viral delivery of TrkB after upper cervical spinal hemisection enhances recovery (1091.6)

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury.Grant Funding Source: Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine

Gene therapy in status epilepticus

Gene therapy in human disease has expanded rapidly in recent years with the development of safer and more effective viral vectors, and presents a novel approach to the treatment of epilepsy. Studies in animals models have demonstrated that overexpression of inhibitory peptides can modify seizure threshold, prevent the development of epilepsy, and modify established epilepsy. More recently there has been a flurry of studies using optogenetics in which light-activated channels expressed in neurons can transiently change neuronal excitability on exposure to light, thereby enabling the development of closed loop systems to detect and stop seizure activity. The treatment of status epilepticus presents its own challenges. Because of both the delay in gene expression following transfection and also the necessity of using focal transfection, there are a limited number of situations in which gene therapy can be used in status epilepticus. One such condition is epilepsia partialis continua (EPC). We have used gene therapy in a model of EPC and have shown that we can "cure" the condition. Recent evidence suggesting that gene therapy targeting subcortical regions can modify generalized or more diffuse epilepsies, indicates that the range of situations in status epilepticus in which gene therapy could be used will expand.

The Herpes Simplex Virus 2 UL21 Protein Is Essential for Virus Propagation

Herpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ΔUL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ΔUL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ΔUL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ΔUL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ΔUL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ΔUL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.

Stability of delay induced oscillations in gene expression of Hes1 protein model

Mathematical analysis of the Hes1 genetic oscillator system is presented. The considered model is a modification of the model proposed by Monk [N.A. Monk, Oscillatory expression of Hes1, p53, and NF- B driven by transcriptional time delays, Curr. Biol. 13 (2003) 1409–1413]. In this paper we model separately time delays connected with transcription and translation processes. Global existence, uniqueness and non-negativity of solutions are proved. The existence of a unique positive steady state is shown. The stability of the steady state is studied and it is shown that it depends on the sum of time delays. Conditions guaranteeing the occurrence of the Hopf bifurcation as well as its stability are proved. Presented analysis partially confirms the results obtained numerically by Jensen et al. [M. Jensen, K. Sneppen, G. Tiana, Sustained oscillations and time delays in gene expression of protein Hes1, FEBS Lett. 541 (2003) 176–177].

Stochasticity and time delays in gene expression and evolutionary game theory

We discuss effects of stochasticity and time delays in simple models of population dynamics. In social-type models, where individuals react to the information concerning the state of the population at some earlier time, sufficiently large time delays may cause oscillations. In biological-type models, where some changes already take place in the population at an earlier time, oscillations might not be present for any time delay. We illustrate this idea in models of delayed random walks, gene expression, and population dynamics of evolutionary game theory.

Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells

This study investigated the combined function of the Autographa californica multiple nucleopolyhedrovirus overlapping genes ac16 (BV/ODV-E26, DA26) and ac17. Ac17 is a late gene and the promoter is within the ac16 open reading frame. A double ac16 – ac17 knockout virus was generated to assess the function of each gene independently or together. Loss of ac17 did not affect viral DNA synthesis but budded virus (BV) production was reduced. Deletion of both ac16 – ac17 resulted in reduced viral DNA synthesis and a further reduction in BV production. In BV infected Sf9 cells, viral gene expression was delayed up to 12 h in the absence of both AC16 and AC17 but not if either gene was present. Cells infected by transfecting viral DNA, by-passing the BV particle, exhibited no delay in gene expression from the double knockout virus. AC16 and AC17 are therefore required for rapid viral gene expression in cells infected by BV.

Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.

Effects of p38 MAP Kinase Inhibitors on the Differentiation and Maturation of Erythroid Progenitors

In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation.

LEARNING GENE NETWORK USING TIME-DELAYED BAYESIAN NETWORK

Exact determination of a gene network is required to discover the higher-order structures of an organism and to interpret its behavior. Most research work in learning gene networks either assumes that there is no time delay in gene expression or that there is a constant time delay. This paper shows how Bayesian Networks can be applied to represent multi-time delay relationships as well as directed loops. The intractability of the network learning algorithm is handled by using an improved mutual information criterion. Also, a new structure learning algorithm, "Learning By Modification", is proposed to learn the sparse structure of a gene network. The experimental results on synthetic data and real data show that our method is more accurate in determining the gene structure as compared to the traditional methods. Even transcriptional loops spanning over the whole cell can be detected by our algorithm.

Gene Expression Time Delays and Turing Pattern Formation Systems

The incorporation of time delays can greatly affect the behaviour of partial differential equations and dynamical systems. In addition, there is evidence that time delays in gene expression due to transcription and translation play an important role in the dynamics of cellular systems. In this paper, we investigate the effects of incorporating gene expression time delays into a one-dimensional putative reaction diffusion pattern formation mechanism on both stationary domains and domains with spatially uniform exponential growth. While oscillatory behaviour is rare, we find that the time taken to initiate and stabilise patterns increases dramatically as the time delay is increased. In addition, we observe that on rapidly growing domains the time delay can induce a failure of the Turing instability which cannot be predicted by a naive linear analysis of the underlying equations about the homogeneous steady state. The dramatic lag in the induction of patterning, or even its complete absence on occasions, highlights the importance of considering explicit gene expression time delays in models for cellular reaction diffusion patterning.