scholarly journals Gene activation by a CRISPR-assistant trans enhancer

2019 ◽  
Author(s):  
Xin Hui Xu ◽  
Wei Dai ◽  
Dan Yang Wang ◽  
Jian Wu ◽  
Jin Ke Wang
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
High Efficiency ◽  
Gene Activation ◽  
Biological Research ◽  
Activation Domain ◽  
Activation Efficiency ◽  
New Strategy ◽  
Cmv Enhancer ◽  
Endogenous Genes

AbstractGene activation is essential to the basic biological research and biomedicine. Therefore, various gene activators such as activation domain-ZNF, TALE and CRISPR proteins have been developed for this end, in which the CRISPR protein dead Cas9 (dCas9) is now most widely used. However, the current gene activators are still limited by their inefficient gene activation activity. In this study, we developed a new strategy, CRISPR-assistant trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with a widely used strong enhancer, the CMV enhancer. In this strategy, a trans CMV enhancer DNA was recruited to target gene by dCas9-VP64/sgRNA via annealing between 3’ end of sgRNA and CMV enhancer. The trans enhancer activates gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.

scholarly journals Gene activation by a CRISPR-assisted trans enhancer

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Xinhui Xu ◽  
Jinliang Gao ◽  
Wei Dai ◽  
Danyang Wang ◽  
Jian Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
High Efficiency ◽  
Gene Activation ◽  
Reporter Genes ◽  
Activation Efficiency ◽  
New Strategy ◽  
Cmv Enhancer ◽  
Endogenous Genes ◽  
In Trans

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.

scholarly journals Asymmetric siRNA: New Strategy to Improve Specificity and Reduce Off-Target Gene Expression

2012 ◽  
Vol 23 (5) ◽  
pp. 521-532 ◽  
Author(s):  
Zhongping Yuan ◽  
Xilin Wu ◽  
Chao Liu ◽  
Genxing Xu ◽  
Zhiwei Wu
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Gene Expression ◽  
New Strategy

FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1228-1228
Author(s):  
Yanan Li ◽  
Riddhi M Patel ◽  
Emily Casey ◽  
Jeffrey A. Magee
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Target Gene ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Gene Activation ◽  
Chromatin Accessibility ◽  
Luciferase Reporter ◽  
Enhancer Activity ◽  
Target Gene Expression

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.

scholarly journals RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

2008 ◽  
Vol 28 (11) ◽  
pp. 3623-3638 ◽  
Author(s):  
David E. Nowak ◽  
Bing Tian ◽  
Mohammad Jamaluddin ◽  
Istvan Boldogh ◽  
Leoncio A. Vergara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Gene Activation ◽  
Elongation Factor ◽  
Genetic Network ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Pol Ii ◽  
Target Gene Expression ◽  
Cyclin T1

ABSTRACT NF-κB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-κB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-κB-dependent genes, serine 276 (Ser276)-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-β, interleukin-8 (IL-8), and IκBα. Previous work has shown that TNF-inducible RelA Ser276 phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser276 RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser276 formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser276 RelA is required only for activation of IL-8 and Gro-β, with IκBα being unaffected. These data were confirmed in experiments using RelA−/− murine embryonic fibroblasts reconstituted with a RelA Ser276Ala mutation. In addition, we observe that phospho-Ser276 RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser276 RelA-P-TEFb complex is required for IL-8 and Gro-β gene activation but not for IκBα gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser276 RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-β genes. The second involves a phospho-Ser276 RelA-independent activation of genes preloaded with Pol II, exemplified by the IκBα gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

scholarly journals Gene Activation by Dissociation of an Inhibitor from a Transcriptional Activation Domain

2009 ◽  
Vol 29 (20) ◽  
pp. 5604-5610 ◽  
Author(s):  
Fenglei Jiang ◽  
Benjamin R. Frey ◽  
Margery L. Evans ◽  
Jordan C. Friel ◽  
James E. Hopper
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Gene Activation ◽  
Gene Array ◽  
Strong Support ◽  
Live Cells ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Gene Switch

ABSTRACT Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.

scholarly journals Differential Target Gene Activation by the Staphylococcus aureus Two-Component System saeRS

2009 ◽  
Vol 192 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Markus Mainiero ◽  
Christiane Goerke ◽  
Tobias Geiger ◽  
Christoph Gonser ◽  
Silvia Herbert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Target Gene ◽  
Target Genes ◽  
Gene Activation ◽  
Gene Expression Pattern ◽  
Class Ii ◽  
Class I ◽  
Single Amino Acid ◽  
Single Amino Acid Change

ABSTRACT The saePQRS system of Staphylococcus aureus controls the expression of major virulence factors and encodes a histidine kinase (SaeS), a response regulator (SaeR), a membrane protein (SaeQ), and a lipoprotein (SaeP). The widely used strain Newman is characterized by a single amino acid change in the sensory domain of SaeS (Pro18 in strain Newman [SaeSP], compared with Leu18 in other strains [SaeSL]). SaeSP determines activation of the class I sae target genes (coa, fnbA, eap, sib, efb, fib, sae), which are highly expressed in strain Newman. In contrast, class II target genes (hla, hlb, cap) are not sensitive to the SaeS polymorphism. The SaeSL allele (saeSL ) is dominant over the SaeSP allele, as shown by single-copy integration of saePQRSL in strain Newman, which results in severe repression of class I target genes. The differential effect on target gene expression is explained by different requirements for SaeR phosphorylation. From an analysis of saeS deletion strains and strains with mutated SaeR phosphorylation sites, we concluded that a high level of SaeR phosphorylation is required for activation of class I target genes. However, a low level of SaeR phosphorylation, which can occur independent of SaeS, is sufficient to activate class II target genes. Using inducible saeRS constructs, we showed that the expression of both types of target genes is independent of the saeRS dosage and that the typical growth phase-dependent gene expression pattern is not driven by SaeRS.

scholarly journals Identification of a novel efficient transcriptional activation domain from Chinese fir (Cunninghamia lanceolata)

2020 ◽  
Author(s):  
Tengfei Zhu ◽  
Wenyu Tang ◽  
Delan Chen ◽  
Renhua Zheng ◽  
Jian Li ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
High Efficiency ◽  
Gene Activation ◽  
Chinese Fir ◽  
Luciferase Assay ◽  
Virus Protein ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Flowering Locus

AbstractActivation domains are used as critical components of artificial gene modification tools for genetic breeding. The high efficiency of the activation domain relies on the host plant. However, no activation domain has been identified that originates from Chinese fir (Cunninghamia lanceolate). In this study, a novel strong activator was identified from the whole Chinese fir cDNA library. This plant conserved activator was named TAC 3 (Transcriptional Activation domain from Chinese fir 3). C-terminal 70 amino acids of TAC (TAC3d) have a stronger ability than the commonly used strong activation domain of the virus protein VP16, or the strong plant activation domain, EDLL, in Chinese fir. Through Dual-luciferase assay, phenomic analysis and FT (Flowering Locus T [FT]) quantification, it was shown that, TAC3d can overcome the transcriptional repression of strong plant repressors (Flowering Locus C [FLC]) when fused to its C-terminal domain, thus inhibit the repression of FT expression. In conclusion, for the first time, an activation domain has been identified from Chinese fir. TAC3, which can be used for precise gene activation in Chinese fir in the future, and its function in the plant is more powerful than the commonly used strong activation domain (such as VP16 and EDLL).HighlightTAC3 is the first transcriptional activation domain identified from Chinese fir and its function is more powerful than some commonly used strong transcriptional activators (such as VP16 and EDLL)

scholarly journals Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009039
Author(s):  
Yi Kuang ◽  
Anna Pyo ◽  
Natanel Eafergan ◽  
Brittany Cain ◽  
Lisa M. Gutzwiller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Target Gene ◽  
Gene Activation ◽  
Notch Intracellular Domain ◽  
Repressor Complex ◽  
Notch Activation

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

scholarly journals The BMP signaling gradient is interpreted through concentration thresholds in dorsal–ventral axial patterning

PLoS Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. e3001059
Author(s):  
Hannah Greenfeld ◽  
Jerome Lin ◽  
Mary C. Mullins
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Target Gene ◽  
Target Genes ◽  
Gene Activation ◽  
Spatial Relationship ◽  
Bmp Signaling ◽  
Target Gene Expression ◽  
Gradient Slope ◽  
Distinct Cell

Bone Morphogenetic Protein (BMP) patterns the dorsal–ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

A Method to Construct Gene Regulatory Networks to Estimate and Calculate Time Delays

2013 ◽  
Vol 62 (1) ◽  
Author(s):  
Mohd Saberi Mohamad ◽  
Chai Suk Phin
Keyword(s):  
Gene Expression ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Time Delays ◽  
Time Course ◽  
Target Gene ◽  
Biological Research ◽  
Expression Change ◽  
Gene Regulatory ◽  
Regulator Genes

In general, the motive of this research is to infer gene regulatory network in order to clarify the basis consequences of biological process at the molecular level. Time course gene expression profiling dataset has been widely used in basic biological research, especially in transcription regulation studies since the microarray dataset is a short time course gene expression dataset and have lots of errors, missing value, and noise.  In this research, R library is implemented in this method to construct gene regulatory which aims to estimate and calculate the time delays between genes and transcription factor. Time delay is the parameters of the modeled time delay linear regression models and a time lag during gene expression change of the regulator genes toward target gene expression. The constructed gene regulatory network provided information of time delays between expression change in regulator genes and its target gene which can be applied to investigate important time-related biological process in cells. The result of time delays and regulation patterns in gene regulatory network may contribute into biological research such as cell development, cell cycle, and cell differentiation in any of living cells.

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