Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice

AbstractCRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

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The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms

Eukaryotic Cell ◽

10.1128/ec.00251-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 514-531 ◽

Cited By ~ 20

Author(s):

Barbara Heise ◽

Julia van der Felden ◽

Sandra Kern ◽

Mario Malcher ◽

Stefan Brückner ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcriptional Activation ◽

Binding Sites ◽

Target Genes ◽

Specific Gene ◽

Dependent Manner ◽

A Genome ◽

Regulated Gene Expression

ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

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Tissue-Specific Autoregulation of thestat3 Gene and Its Role in Interleukin-6-Induced Survival Signals in T Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6615-6625.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6615-6625 ◽

Cited By ~ 83

Author(s):

Masahiro Narimatsu ◽

Hisoka Maeda ◽

Shousaku Itoh ◽

Toru Atsumi ◽

Takuya Ohtani ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Interleukin 6 ◽

Target Genes ◽

Gene Activation ◽

Tissue Specific ◽

Direct Target ◽

Responsive Element ◽

Transcription 3

ABSTRACT Signal transducer and activator of transcription 3 (STAT3) mediates signals of various growth factors and cytokines, including interleukin-6 (IL-6). In certain IL-6-responsive cell lines, thestat3 gene is autoregulated by STAT3 through a composite IL-6 response element in its promoter that contains a STAT3-binding element (SBE) and a cyclic AMP-responsive element. To reveal the nature and roles of the stat3 autoregulation in vivo, we generated mice that harbor a mutation in the SBE (stat3 mSBE ). The intact SBE was crucial for IL-6-induced stat3 gene activation in the spleen, especially in the red pulp region, the kidney, and both mature and immature T lymphocytes. The SBE was not required, however, for IL-6-induced stat3 gene activation in hepatocytes. T lymphocytes from the stat3 mSBE/mSBE mice were more susceptible to apoptosis despite the presence of IL-6 than those from wild-type mice. Consistent with this, IL-6-dependent activation of the Pim-1 and junB genes, direct target genes for STAT3, was attenuated in T lymphocytes of thestat3 mSBE/mSBE mice. Thus, the tissue-specific autoregulation of the stat3 gene operates in vivo and plays a role in IL-6-induced antiapoptotic signaling in T cells.

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Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1746 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1746-1758 ◽

Cited By ~ 10

Author(s):

D J DeAngelo ◽

J DeFalco ◽

G Childs

Keyword(s):

Gene Expression ◽

Molecular Weight ◽

Transcriptional Activation ◽

Target Genes ◽

Gene Activation ◽

Upstream Sequence ◽

Blastula Stage ◽

Sequence Element ◽

Enhancer Element ◽

Beta Gene

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

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Phosphorylated forms of GAL4 are correlated with ability to activate transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4623 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4623-4629 ◽

Cited By ~ 31

Author(s):

L M Mylin ◽

M Johnston ◽

J E Hopper

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Polyacrylamide Gel Electrophoresis ◽

Gene Activation ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Transcriptional Activator Protein ◽

Mel Gene ◽

High Level

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

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A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

10.1101/2021.09.07.459151 ◽

2021 ◽

Author(s):

Elena Garcia-Perez ◽

Borja Diego-Martin ◽

Alfredo Quijano-Rubio ◽

Elena Moreno Gimenez ◽

Diego Orzaez ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Nicotiana Benthamiana ◽

Gene Networks ◽

Rna Binding ◽

Gene Activation ◽

Dependent Manner ◽

Copper Binding ◽

Control Of Gene Expression ◽

Guide Rna

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

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Interdependent recruitment of CYC8/TUP1 and the transcriptional activator XYR1 at target promoters is required for induced cellulase gene expression in Trichoderma reesei

PLoS Genetics ◽

10.1371/journal.pgen.1009351 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009351

Author(s):

Lei Wang ◽

Weixin Zhang ◽

Yanli Cao ◽

Fanglin Zheng ◽

Guolei Zhao ◽

...

Keyword(s):

Gene Expression ◽

Trichoderma Reesei ◽

Transcriptional Activation ◽

Target Genes ◽

Gene Activation ◽

Transcriptional Activator ◽

Cellulase Production ◽

Gene Promoters ◽

Cellulase Gene ◽

Histone H4

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.

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p53 drives a transcriptional program that elicits a non-cell-autonomous response and alters cell state in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008474117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23663-23673 ◽

Cited By ~ 1

Author(s):

Sydney M. Moyer ◽

Amanda R. Wasylishen ◽

Yuan Qi ◽

Natalie Fowlkes ◽

Xiaoping Su ◽

...

Keyword(s):

Transcriptional Activation ◽

Cellular Response ◽

Target Genes ◽

Transcriptional Program ◽

Sequencing Data ◽

Tissue Specific ◽

Chip Sequencing ◽

Cell State ◽

P53 Activation

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion ofMdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which includedMdm2but notp21. Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

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Lithium Treatment Potentiates Both in Vitro and in Vivo Retinoic Acid Efficacy in APL.

Blood ◽

10.1182/blood.v120.21.2614.2614 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2614-2614

Author(s):

Fabien Zassadowski ◽

Katka Pokorna ◽

Nicolas Ferre ◽

Laura Llopis ◽

Oussama Chourbagi ◽

...

Keyword(s):

Gene Expression ◽

Retinoic Acid ◽

Mouse Model ◽

Cell Line ◽

Transcriptional Activation ◽

Target Gene ◽

Nb4 Cells ◽

Target Gene Expression ◽

Licl Treatment

Abstract Abstract 2614 We previously demonstrated that although retinoic acid (RA) has targeted efficacy in Acute Promyelocytic Leukemia (APL), heterogeneity exists leading to the appearance of un-targeted clones at the time of relapse. Characterization of these clones is not yet fully unraveled though we and others have previously highlighted the roles of RARα mutations, pharmacogenomics or APL miRNome. We recently identified that the ERK1/2 pathway synergized with RA to restore the transcriptional activity of RA in resistant APL cells, thus restoring RA induced differentiation (Cassinat et al. Mol Cell Biol 2011). These results suggest that targeting interconnected signaling pathways could optimize differentiation therapy efficacy. To this effect, we studied known signaling pathway activators or inhibitors that could potentiate with RA and identified Lithium chloride (LiCl). Treatment of the ATRA sensitive-APL NB4 cell line with LiCl (25mM) decreases proliferation and increases apoptosis (25% and 40% of Annexin V-positive cells at day 1 and 2 respectively) with evidence of caspase 3 cleavage at day 2. Because NB4 cells fully differentiated with RA alone we were unable to observe any synergy when combined with LiCl. Treatment of the RA-resistant APL UF-1 cell line with RA or LiCl alone does not induce differentiation. Combination of RA+LiCl restores differentiation after 3 days of culture (65% CD11b positive and 55% NBT test positive cells). Similar results were obtained with different GSK3 inhibitors, suggesting that the LiCL effects were in part linked to its well characterized GSK3 inhibitory activity. Interestingly, we noted that LiCl treatment induces rapid phosphorylation of ERK1/2 and pretreatment with the MEK/ERK1/2 inhibitor UO126 fully abolished the differentiation induced by the RA+LiCl combination. The combination restores in UF-1 the expression of RA target genes (such as RARα2) to the same levels obtained in NB4 cells treated by RA alone. The level of luciferase activity of an RA responsive element reporter gene was increased with the RA+LiCl combination compared to RA alone. Both target gene expression and luciferase activiy were abolished after inhibition of the MEK/ERK1/2 pathway. Thus, increase in differentiation of UF-1 cells by RA+LiCl is linked to increased RA transcriptional activation. Similar studies in fresh APL patient cells confirmed both the increase in differentiation and level of RA target gene expression and their inhibition by UO126. Finally, to translate these findings in vivo, we used the APL-transplantable mouse model. Plasma lithium levels in treated mice were measured between 0.6 and 1.05 mmol/l, levels reached in humans. When LiCl was combined with RA we repeatedly observed a pronounced survival advantage compared to mice treated by RA alone as evaluated by Kaplan Meier analysis. In this work we demonstrate that LiCl, a well tolerated agent in humans, has the potential, when combined with RA, to restore RA induced transcriptional activation and differentiation in RA resistant APL cells. Furthermore, this combination also increases RA efficacy in an in vivo APL mouse model. Disclosures: Off Label Use: Lithium is a mood modulator administered for bipolar disorders.

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Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform

International Journal of Molecular Sciences ◽

10.3390/ijms21051574 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1574

Author(s):

Sumiyo Morita ◽

Takuro Horii ◽

Mika Kimura ◽

Izuho Hatada

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Viral Vectors ◽

Target Genes ◽

Basic Research ◽

Endogenous Gene ◽

Synergistic Activation ◽

Alternative Approach ◽

Endogenous Genes ◽

Exogenous Genes

Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9–VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9–SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9–SunTag format synergistically activates gene expression using a single sgRNA.

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Molecular bases for the constitutive photomorphogenic phenotypes in Arabidopsis

10.1101/388157 ◽

2018 ◽

Author(s):

Vinh Ngoc Pham ◽

Xiaosa Xu ◽

Enamul Huq

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Target Genes ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Summary Statement ◽

Direct Target ◽

Targeted Gene Expression ◽

Molecular Bases

Summary statementStrikingly similar morphological and gene expression phenotypes among cop1, spaQ and pifQ mutants suggest that the cop phenotype of the cop1 and spaQ mutants might be due in part to a reduced level of PIFsAbstractThe transition from skotomorphogenesis to photomorphogenesis is regulated in part by COP1/SPA complex and PIFs in Arabidopsis. The constitutive photomorphogenic (cop) phenotypes of the cop1 and spaQ mutants were shown to be due to a high abundance of the positively acting transcription factors. Here we show that the four major PIF proteins are unstable in cop1 mutant, and an overexpression of P1F1, P1F3, P1F4 and P1F5 suppresses the cop1 phenotypes in the dark. A comparison of the transcriptome data among cop1, spaQ and pifQ reveals remarkably overlapping gene expression profiles with a preferential regulation of the PIF direct target genes. Additionally, HFR1 strongly inhibits the in vivo binding and transcriptional activation activity of PIF1 in the dark. Taken together, these data suggest that the cop phenotypes of the cop1 and spaQ mutants might be due to a combination of the reduced level of PIFs, increased level of the positive factors (e.g., HY5/HFR1 and others), and the HFR1-mediated inhibition of PIF targeted gene expression in the dark.

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