scholarly journals TuNR: Orthogonal Control of Mean and Variability of Endogenous Genes in a Human Cell Line

2020 ◽  
Author(s):  
Alain R. Bonny ◽  
João Pedro Fonseca ◽  
Jesslyn E. Park ◽  
Hana El-Samad
Keyword(s):  
Mammalian Cells ◽  
Human Cell Line ◽  
Transcriptional Level ◽  
Clear Understanding ◽  
Gene Expression Noise ◽  
Endogenous Gene ◽  
Basal Expression ◽  
Stochastic Fluctuations ◽  
Human Genes ◽  
Synthetic Circuit

AbstractStochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineered a synthetic circuit to independently modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

scholarly journals Orthogonal control of mean and variability of endogenous genes in a human cell line

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alain R. Bonny ◽  
João Pedro Fonseca ◽  
Jesslyn E. Park ◽  
Hana El-Samad
Keyword(s):  
Mammalian Cells ◽  
Human Cell Line ◽  
Transcriptional Level ◽  
Clear Understanding ◽  
Gene Expression Noise ◽  
Endogenous Gene ◽  
Basal Expression ◽  
Stochastic Fluctuations ◽  
Human Genes ◽  
Synthetic Circuit

AbstractStochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals EMERGENT NETWORK STRUCTURE, EVOLVABLE ROBUSTNESS, AND NONLINEAR EFFECTS OF POINT MUTATIONS IN AN ARTIFICIAL GENOME MODEL

2009 ◽  
Vol 12 (03) ◽  
pp. 293-310 ◽  
Author(s):  
THIMO ROHLF ◽  
CHRISTOPHER R. WINKLER
Keyword(s):  
Evolutionary Dynamics ◽  
Genetic Regulation ◽  
Point Mutations ◽  
Nonlinear Effects ◽  
High Rate ◽  
Stabilizing Selection ◽  
Clear Understanding ◽  
Stochastic Fluctuations ◽  
Neutral Mutations ◽  
Genome Model

Genetic regulation is a key component in development, but a clear understanding of the structure and dynamics of genetic networks is not yet at hand. In this paper we investigate these properties within an artificial genome model originally introduced by Reil [Proc. 5th European Conf. Artificial Life (Springer, 1999), pp. 457–466]. We analyze statistical properties of randomly generated genomes both on the sequence and network level, and show that this model correctly predicts the frequency of genes in genomes as found in experimental data. Using an evolutionary algorithm based on stabilizing selection for a phenotype, we show that dynamical robustness against single base mutations, as against random changes in initial states of regulatory dynamics that mimic stochastic fluctuations in environmental conditions, can emerge in parallel. Point mutations at the sequence level can have strongly nonlinear effects on network wiring, including structurally neutral mutations and simultaneous rewiring of multiple connections, which occasionally lead to strong reorganization of the attractor landscape and metastability of evolutionary dynamics. Similar to real genomes, evolved artificial genomes exhibit both highly conserved regions, as well as regions that are characterized by a high rate of accepted base substitutions.

scholarly journals The Antisense Transcriptomes of Human Cells

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1855-1857 ◽  
Author(s):  
Yiping He ◽  
Bert Vogelstein ◽  
Victor E. Velculescu ◽  
Nickolas Papadopoulos ◽  
Kenneth W. Kinzler
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Human Cells ◽  
Antisense Transcripts ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Dna Strand ◽  
The Relationship ◽  
Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

scholarly journals Regulation of heat shock protein synthesis by quercetin in human erythroleukaemia cells

1994 ◽  
Vol 300 (1) ◽  
pp. 201-209 ◽  
Author(s):  
G Elia ◽  
M G Santoro
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Elevated Temperatures ◽  
K562 Cells ◽  
Hsp70 Expression ◽  
Transcriptional Level ◽  
Hsp70 Mrna ◽  
Shock Proteins ◽  
Hsp70 Synthesis

Synthesis of heat-shock proteins (HSPs) is universally induced in eukaryotic and prokaryotic cells by exposure to elevated temperatures or to other types of environmental stress. In mammalian cells, HSPs belonging to the 70 kDa family (HSP70) have a regulatory role in several cellular processes, and have been shown to be involved in the control of cell proliferation and differentiation. Although many types of HSP70 inducers have been identified, only a few compounds, all belonging to the flavonoid group, have been shown to inhibit HSP70 induction. Because inhibitors of HSP70 synthesis could be an important tool with which to study the function of this protein, we have investigated the effect of quercetin, a flavonoid with antiproliferative activity which is widely distributed in nature, on HSP70 synthesis in human K562 erythroleukaemia cells after treatment with severe or mild heat shock and with other inducers. Quercetin was found to affect HSP70 synthesis at more than one level, depending on the conditions used. Indeed, after severe heat shock (45 degrees C for 20 min) treatment with quercetin, at non-toxic concentrations, was found to inhibit HSP70 synthesis for a period of 3-4 h. This block appeared to be exerted at the post-transcriptional level and to be cell-mediated, as the addition of quercetin during translation of HSP70 mRNA in vitro had no effect. After prolonged (90 min) exposure at 43 degrees C, however, quercetin was found to inhibit also HSP70 mRNA transcription. Pretreatment of K562 cells with quercetin had no effect on HSP70 expression, and quercetin needed to be present during induction to be effective. Under all conditions tested, the quercetin-induced block of HSP70 synthesis was found to be transient and, after an initial delay, synthesis of HSP70 reached the control rate and continued at the same level for several hours after the time at which HSP70 synthesis had been turned off in control cells. Finally, inhibition of HSP70 synthesis by quercetin appeared to be dependent on the temperature used and on the type of stressor.

scholarly journals Induction by prostaglandin A1 of haem oxygenase in myoblastic cells: an effect independent of expression of the 70 kDa heat shock protein

1995 ◽  
Vol 308 (2) ◽  
pp. 455-463 ◽  
Author(s):  
A Rossi ◽  
M G Santoro
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Present Report ◽  
Oxidant Stress ◽  
Stress Protein ◽  
Hsp70 Expression ◽  
Transcriptional Level ◽  
Protective Mechanisms ◽  
Haem Oxygenase ◽  
Prostaglandin A1

Prostaglandins of the A type (PGA) induce the synthesis of 70 kDa heat shock proteins (hsp70) in a large variety of mammalian cells. Induction of hsp70 has been associated with a cytoprotective effect of PGA1 after virus infection or thermal injury. In the present report we provide evidence that, in murine myoblasts, PGA1 is not able to induce hsp70 expression, whereas it increases the synthesis of the constitutive protein, hsc70, and dramatically induces the synthesis of a 32 kDa protein (p32). The p32 protein has been identified as haem oxygenase. PGA1 acts at the transcriptional level by inducing haem oxygenase mRNA synthesis, and the signal for induction appears to be associated with decreased intracellular GSH levels. Haem oxygenase, a low-molecular-mass stress protein induced in mammalian cells by oxidant stress, is known to be part of a general inducible antioxidant defence pathway. The fact that prostaglandin synthesis is stimulated in muscle during contraction and in the heart in response to ischaemia raises the possibility that induction of haem oxygenase by PGA in myoblasts could be part of a protective mechanisms in operation during stress and hypoxia.

Tissue glutathione, nutrition, and oxidative stress

1993 ◽  
Vol 71 (9) ◽  
pp. 746-751 ◽  
Author(s):  
Tammy M. Bray ◽  
Carla G. Taylor
Keyword(s):  
Oxidative Stress ◽  
Mammalian Cells ◽  
Immune Defense ◽  
Clear Understanding ◽  
Oxygen Species ◽  
Dietary Regulation ◽  
Defense Systems ◽  
Nonprotein Thiol ◽  
Potential Strategy ◽  
And Oxidative Stress

Glutathione, a cysteine-containing tripeptide, is the most abundant nonprotein thiol in mammalian cells. Glutathione plays an important role in the detoxification of xenobiotic compounds and in the antioxidation of reactive oxygen species and free radicals. Because of its multiple functions in various tissues and its involvement in many diseases and malnutrition, a clear understanding of the interrelationships among tissue glutathione, nutrition, and oxidative stress is clinically relevant. The focus of this review is to discuss the regulation of tissue glutathione concentration by diet and nutritional status, and to apply this information to those diseases and malnutrition in which decreased tissue glutathione and increased oxidative stress are implicated. A potential strategy to rapidly restore glutathione for both antioxidant and immune defense systems before therapeutic treatment in malnourished patients is discussed.Key words: glutathione, dietary regulation, oxidative stress, malnutrition.

scholarly journals Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice.

1994 ◽  
Vol 14 (4) ◽  
pp. 2809-2821 ◽  
Author(s):  
M I Ramirez ◽  
D Karaoglu ◽  
D Haro ◽  
C Barillas ◽  
R Bashirzadeh ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Transgenic Mice ◽  
Bile Acids ◽  
Transcriptional Activation ◽  
Fusion Gene ◽  
Proximal Promoter ◽  
Transcriptional Level ◽  
Endogenous Gene ◽  
Hydroxylase Gene

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level.

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