scholarly journals Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Pavithran T. Ravindran ◽  
Maxwell Z. Wilson ◽  
Siddhartha G. Jena ◽  
Jared E. Toettcher
Keyword(s):  
Intracellular Signaling ◽  
Target Genes ◽  
Immediate Early Genes ◽  
Tissue Level ◽  
Early Gene ◽  
Immediate Early ◽  
Early Genes ◽  
Natural Target ◽  
Context Specific ◽  
External Stimuli

AbstractMany cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input–output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.

scholarly journals Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

2019 ◽  
Author(s):  
Pavithran T. Ravindran ◽  
Maxwell Z. Wilson ◽  
Siddhartha G. Jena ◽  
Jared E. Toettcher
Keyword(s):  
Growth Factor ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Target Genes ◽  
Immediate Early Genes ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Specific Target ◽  
Early Genes

AbstractFor tissues to grow and function properly, cells must coordinate actions such as proliferation, differentiation and apoptosis. This coordination is achieved in part by the activation of intracellular signaling pathways that trigger the expression of context-specific target genes. While the function of these natural circuits has been actively studied, synthetic biology provides additional powerful tools for deconstructing, repurposing, and designing novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (synIEGs), target genes of the Erk signaling pathway that implement complex, user-defined regulation and can be monitored through the use of live-cell biosensors to track transcription and translation. We demonstrate the power and flexibility of this approach by confirming Erk duration-sensing by the FOS immediate-early gene, elucidating how the BTG2 gene is regulated by transcriptional activation and translational repression after growth-factor stimulation, and by designing a synthetic immediate-early gene that responds with AND-gate logic to the combined presence of growth factor and DNA damage stimuli. Our work paves the way to defining the molecular circuits that link signaling pathways to specific target genes, highlighting an important role for post-transcriptional regulation in signal decoding that may be masked by analyses of RNA abundance alone.

scholarly journals A novel 7-nucleotide motif located in 3' untranslated sequences of the immediate-early gene set mediates platelet-derived growth factor induction of the JE gene.

1992 ◽  
Vol 12 (12) ◽  
pp. 5288-5300 ◽  
Author(s):  
R R Freter ◽  
J C Irminger ◽  
J A Porter ◽  
S D Jones ◽  
C D Stiles
Keyword(s):  
Growth Factor ◽  
Immediate Early Genes ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Platelet Derived Growth Factor ◽  
Immediate Early ◽  
Control Element ◽  
Gene Set ◽  
Cis Acting ◽  
Early Genes

A cohort of the serum and growth factor regulated immediate-early gene set is induced with slower kinetics than c-fos. Two of the first immediate-early genes characterized as such, c-myc and JE, are contained within this subset. cis-acting genomic elements mediating induction of the slower responding subset of immediate-early genes have never been characterized. Herein we characterize two widely separated genomic elements which are together essential for induction of the murine JE gene by platelet-derived growth factor, serum, interleukin-1, and double-stranded RNA. One of these elements is novel in several regards. It is a 7-mer, TTTTGTA, found in the proximal 3' sequences downstream of the JE stop codon. The 3' element is position dependent and orientation independent. It does not function in polyadenylation, splicing, or destabilization of the JE transcript. Copies of the 7-mer or its inverse are found at comparable 3' sites in 25 immediate-early genes that encode transcription factors or cytokines. Given its general occurrence, the 7-mer may be a required cis-acting control element mediating induction of the immediate-early gene set.

Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4

1985 ◽  
Vol 5 (8) ◽  
pp. 1997-2008 ◽  
Author(s):  
N A DeLuca ◽  
P A Schaffer
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immediate Early Genes ◽  
Early Gene ◽  
Immediate Early ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Early Genes ◽  
Simplex Virus

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.

scholarly journals LINC00473 as an Immediate Early Gene under the Control of the EGR1 Transcription Factor

2020 ◽  
Vol 6 (4) ◽  
pp. 46
Author(s):  
Vincenza Aliperti ◽  
Emilia Vitale ◽  
Francesco Aniello ◽  
Aldo Donizetti
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Immediate Early Genes ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Secondary Response ◽  
Early Genes ◽  
Regulatory Cascade ◽  
Non Coding Rnas

Immediate early genes play an essential role in cellular responses to different stimuli. Many of them are transcription factors that regulate the secondary response gene expression. Non-coding RNAs may also be involved in this regulatory cascade. In fact, they are emerging as key actors of gene expression regulation, and evidence suggests that their dysregulation may underly pathological states. We previously took a snapshot of both coding and long non-coding RNAs differentially expressed in neuronal cells after brain-derived neurotrophic factor stimulation. Among these, the transcription factor EGR1 (a well-known immediate early gene) and LINC00473 (a primate-specific long non-coding RNA) that has emerged as an interesting RNA candidate involved in neuronal function and in cancer. In this work, we demonstrated that LINC00473 gene expression kinetics resembled that of immediate early genes in SH-SY5Y and HEK293T cells under different cell stimulation conditions. Moreover, we showed that the expression of LINC00473 is under the control of the transcription factor EGR1, providing evidence for an interesting functional relationship in neuron function.

Expression of the protooncogene c-jun is maintained during myogenic differentiation in rat L6 myoblasts

1993 ◽  
Vol 71 (5-6) ◽  
pp. 260-269 ◽  
Author(s):  
Gopal Thinakaran ◽  
Jnanankur Bag
Keyword(s):  
Myogenic Differentiation ◽  
Constitutive Expression ◽  
Immediate Early Genes ◽  
Binding Activity ◽  
High Serum ◽  
Early Gene ◽  
Immediate Early ◽  
Activator Protein 1 ◽  
L6 Cells ◽  
Early Genes

Skeletal myoblasts undergo terminal differentiation when maintained under low-mitogen conditions. We have examined the expression of c-jun, one of the growth-factor-inducible immediate-early genes, during myogenic differentiation of L6 myoblasts. The steady-state levels of c-jun mRNA, c-Jun polypeptide, and activator protein 1 binding activity were not markedly altered in L6 cells undergoing myogenic differentiation. Although expression of c-jun is induced by serum mitogens in fibroblasts and other cell lines, addition of high serum to proliferating myoblasts resulted in the activation of another immediate early gene junB, but not c-jun mRNA expression. These results indicate that regulation of c-jun may differ from that of other immediate early genes in L6 cells. Manipulation of myogenesis by exposing L6 cells to dimethyl sulfoxide also suggested that expression of myogenin and muscle differentiation could occur in the presence of high levels of c-Jun. Furthermore, expression of c-jun from Moloney murine leukaemia viral long-terminal repeat in transfected L6 cells confirmed that constitutive expression of c-jun does not interfere with myogenesis in L6 myoblasts. Therefore, regulation of c-jun expression in rat L6 cells differs from that in the mouse C2 cell line.Key words: myogenesis, c-jun, protooncogene, differentiation, transcription factor.

Rapid induction in regenerating liver of RL/IF-1 (an I kappa B that inhibits NF-kappa B, RelB-p50, and c-Rel-p50) and PHF, a novel kappa B site-binding complex

1992 ◽  
Vol 12 (6) ◽  
pp. 2898-2908
Author(s):  
M Tewari ◽  
P Dobrzanski ◽  
K L Mohn ◽  
D E Cressman ◽  
J C Hsu ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Liver Regeneration ◽  
Immediate Early Genes ◽  
Early Gene ◽  
Immediate Early ◽  
Hepatic Regeneration ◽  
Regenerating Liver ◽  
Nf Kappa B ◽  
Early Genes ◽  
Site Binding

The liver is one of the few adult tissues that has the capacity to regenerate following hepatectomy or toxic damage. In examining the early growth response during hepatic regeneration, we found that a highly induced immediate-early gene in regenerating liver encodes RL/IF-1 (regenerating liver inhibitory factor) and is the rat homolog of human MAD-3 and probably of chicken pp40. RL/IF-1 has I kappa B activity of broad specificity in that it inhibits the binding of p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50, but not p50 homodimeric NF-kappa B, to kappa B sites. Like RL/IF-1, several members of the NF-kappa B and rel family of transcription factors are immediate-early genes in regenerating liver and mitogen-treated cells. We examined changes in kappa B site binding activity during liver regeneration and discovered a rapidly induced novel kappa B site-binding complex designated PHF [posthepatectomy factor(s)]. PHF is induced over 1,000-fold within minutes posthepatectomy in a protein synthesis-independent manner, with peak activity at 30 min, and is not induced by sham operation. PHF is distinct from p50-p65 NF-kappa B, which is present only in the inactive form in liver posthepatectomy. Although early PHF complexes do not interact strongly with anti-p50 antibodies, PHF complexes present later (3 to 5 h) posthepatectomy react strongly, suggesting that they contain a p50 NF-kappa B subunit. Unlike p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50 complexes, PHF binding to kappa B sites is not inhibited by RL/IF-1. One role of RL/IF-1 in liver regeneration may be to inhibit p50-p65 NF-kappa B activity present in hepatic cells, allowing for the preferential binding of PHF to kappa B sites. Because PHF is induced immediately posthepatectomy in the absence of de novo protein synthesis, PHF could have a role in the regulation of liver-specific immediate-early genes in regenerating liver.

Expression of cyr61, a growth factor-inducible immediate-early gene

1990 ◽  
Vol 10 (7) ◽  
pp. 3569-3577
Author(s):  
T P O'Brien ◽  
G P Yang ◽  
L Sanders ◽  
L F Lau
Keyword(s):  
Growth Factor ◽  
Signal Sequence ◽  
Mrna Level ◽  
Immediate Early Genes ◽  
Early Gene ◽  
Immediate Early ◽  
Early Genes ◽  
Serum Stimulation ◽  
3T3 Fibroblasts ◽  
Kinase C

A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cell growth. We show here that a member of this set of genes, cyr61 (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr61 mRNA is accumulated from the G0/G1 transition through mid-G1. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the G0/G1 transition and in mid-G1 at the same rate. In logarithmically growing cells, the cyr61 mRNA level is constant throughout the cell cycle. Cyr61 contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr61 is synthesized maximally at 1 to 2 h after serum stimulation and has a short half-life within the cell.

A novel 7-nucleotide motif located in 3' untranslated sequences of the immediate-early gene set mediates platelet-derived growth factor induction of the JE gene

1992 ◽  
Vol 12 (12) ◽  
pp. 5288-5300
Author(s):  
R R Freter ◽  
J C Irminger ◽  
J A Porter ◽  
S D Jones ◽  
C D Stiles
Keyword(s):  
Growth Factor ◽  
Immediate Early Genes ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Platelet Derived Growth Factor ◽  
Immediate Early ◽  
Control Element ◽  
Gene Set ◽  
Cis Acting ◽  
Early Genes

A cohort of the serum and growth factor regulated immediate-early gene set is induced with slower kinetics than c-fos. Two of the first immediate-early genes characterized as such, c-myc and JE, are contained within this subset. cis-acting genomic elements mediating induction of the slower responding subset of immediate-early genes have never been characterized. Herein we characterize two widely separated genomic elements which are together essential for induction of the murine JE gene by platelet-derived growth factor, serum, interleukin-1, and double-stranded RNA. One of these elements is novel in several regards. It is a 7-mer, TTTTGTA, found in the proximal 3' sequences downstream of the JE stop codon. The 3' element is position dependent and orientation independent. It does not function in polyadenylation, splicing, or destabilization of the JE transcript. Copies of the 7-mer or its inverse are found at comparable 3' sites in 25 immediate-early genes that encode transcription factors or cytokines. Given its general occurrence, the 7-mer may be a required cis-acting control element mediating induction of the immediate-early gene set.

Immediate-early genes, neuronal plasticity, and memory

1992 ◽  
Vol 70 (9) ◽  
pp. 729-737 ◽  
Author(s):  
H. A. Robertson
Keyword(s):  
Neuronal Plasticity ◽  
Genetic Material ◽  
Physiological Role ◽  
Immediate Early Genes ◽  
Long Term Potentiation ◽  
Early Gene ◽  
Immediate Early ◽  
Early Genes ◽  
Wide Range ◽  
The Brain

The demonstration that the immediate-early gene c-fos is rapidly and transiently expressed in brain following a variety of manipulations has led to intense study of these genes to determine what physiological role they play. The very wide range of stimuli which lead to induction of immediate-early genes (IEGs) in the brain has raised concerns for the specificity of their actions and the suggestion that they might merely be involved in housekeeping functions. On the other hand, there is evidence that these genes may play a role in the transmission of information from cell surface receptors to the genetic material in many instances of neuronal plasticity, including development of seizure susceptibility (kindling), long-term potentiation, drug-induced changes, the phase shift in circadian rhythms, and spreading neuronal depression. In addition to being a putative third (or fourth) messenger involved in transduction of signals to the genetic material, activation of IEGs has proven to be a useful tool for the study of transsynaptic activation of certain neuronal pathways in the brain. Thus, studies on the induction of IEGs are proving to be especially useful in understanding some important functions and properties of the mammalian brain.Key words: immediate-early genes, brain, memory, neuronal plasticity, gene expression.

Intestinal perfusion induces rapid activation of immediate-early genes in weaning rats

2001 ◽  
Vol 281 (4) ◽  
pp. R1274-R1282 ◽  
Author(s):  
Lan Jiang ◽  
Heather Lawsky ◽  
Relicardo M. Coloso ◽  
Mary A. Dudley ◽  
Ronaldo P. Ferraris
Keyword(s):  
Mrna Expression ◽  
Target Genes ◽  
De Novo ◽  
Immediate Early Genes ◽  
Regulatory Elements ◽  
Intestinal Perfusion ◽  
Immediate Early ◽  
Mrna Levels ◽  
Early Genes

C- fos and c- jun are immediate-early genes (IEGs) that are rapidly expressed after a variety of stimuli. Products of these genes subsequently bind to DNA regulatory elements of target genes to modulate their transcription. In rat small intestine, IEG mRNA expression increases dramatically after refeeding following a 48-h fast. We used an in vivo intestinal perfusion model to test the hypothesis that metabolism of absorbed nutrients stimulates the expression of IEGs. Compared with those of unperfused intestines, IEG mRNA levels increased up to 11 times after intestinal perfusion for 0.3–4 h with Ringer solutions containing high (100 mM) fructose (HF), glucose (HG), or mannitol (HM). Abundance of mRNA returned to preperfusion levels after 8 h. Levels of c- fos and c- jun mRNA and proteins were modest and evenly distributed among enterocytes lining the villi of unperfused intestines. HF and HM perfusion markedly enhanced IEG mRNA expression along the entire villus axis. The perfusion-induced increase in IEG expression was inhibited by actinomycin-D. Luminal perfusion induces transient but dramatic increases in c- fos and c- jun expression in villus enterocytes. Induction does not require metabolizable or absorbable nutrients but may involve de novo gene transcription in cells along the villus.

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