scholarly journals Multiplexed and tunable transcriptional activation by promoter insertion using nuclease-assisted vector integration

2019 ◽  
Vol 47 (12) ◽  
pp. e67-e67 ◽  
Author(s):  
Alexander Brown ◽  
Jackson Winter ◽  
Michael Gapinske ◽  
Nathan Tague ◽  
Wendy S Woods ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Gene ◽  
Gene Activation ◽  
Precise Control ◽  
Vector Integration ◽  
Synthetic Promoters ◽  
Artificial Transcription Factors ◽  
A Genome ◽  
Control Of Expression ◽  
Line Engineering

Abstract The ability to selectively regulate expression of any target gene within a genome provides a means to address a variety of diseases and disorders. While artificial transcription factors are emerging as powerful tools for gene activation within a natural chromosomal context, current generations often exhibit relatively weak, variable, or unpredictable activity across targets. To address these limitations, we developed a novel system for gene activation, which bypasses native promoters to achieve unprecedented levels of transcriptional upregulation by integrating synthetic promoters at target sites. This gene activation system is multiplexable and easily tuned for precise control of expression levels. Importantly, since promoter vector integration requires just one variable sgRNA to target each gene of interest, this procedure can be implemented with minimal cloning. Collectively, these results demonstrate a novel system for gene activation with wide adaptability for studies of transcriptional regulation and cell line engineering.

Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-based Transcriptional Activation

2021 ◽  
Vol 01 ◽  
Author(s):  
Siti Nor Akmar Abdullah ◽  
Sean Mayes ◽  
Mahdi Moradpour
Keyword(s):  
Genome Sequence ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Gene Activation ◽  
Foxtail Millet ◽  
Setaria Italica ◽  
Waterlogging Tolerance ◽  
Activation Domains ◽  
Functional Studies ◽  
Cereal Species

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely utilized as a gene activation system exploiting its ability to recruit various transcriptional activation domains (TADs) to enhance the expression of the target gene(s). Drought tolerant and resource-efficient crops like millets can mitigate the effects of climate change and strengthen food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence to identify a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring waterlogging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer waterlogging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome to identify and characterize the ortholog from Setaria italica. Softberry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a helpful example of how to advance in genomics research, including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species by utilizing the available genome sequence.

scholarly journals Pygo2 Associates with MLL2 Histone Methyltransferase and GCN5 Histone Acetyltransferase Complexes To Augment Wnt Target Gene Expression and Breast Cancer Stem-Like Cell Expansion

2010 ◽  
Vol 30 (24) ◽  
pp. 5621-5635 ◽  
Author(s):  
Jiakun Chen ◽  
Qicong Luo ◽  
Yuanyang Yuan ◽  
Xiaoli Huang ◽  
Wangyu Cai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcriptional Activation ◽  
Cell Expansion ◽  
Breast Cancer Cells ◽  
Target Gene ◽  
Target Genes ◽  
Histone Acetyltransferase ◽  
Gene Activation ◽  
Histone Methyltransferase ◽  
T Cell Factor

ABSTRACT Resent studies have identified Pygopus as a core component of the β-catenin/T-cell factor (TCF)/lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in β-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with β-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wnt-dependent transcriptional program and cancer stem-like cell expansion.

scholarly journals Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

2018 ◽  
Author(s):  
Onur Tidin ◽  
Elias T. Friman ◽  
Felix Naef ◽  
David M. Suter
Keyword(s):  
Transcriptional Activation ◽  
Target Gene ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Tissue Growth ◽  
Live Cells ◽  
High Temporal Resolution ◽  
Transcriptional Responses

AbstractThe transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-β stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

scholarly journals Wnt/β-catenin Pathway-Mediated PPARδ Expression during Embryonic Development Differentiation and Disease

2021 ◽  
Vol 22 (4) ◽  
pp. 1854
Author(s):  
Tabinda Sidrat ◽  
Zia-Ur Rehman ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
Il-Keun Kong
Keyword(s):  
Embryonic Development ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Developmental Stages ◽  
Embryonic Stem ◽  
Hereditary Diseases ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Stem Cell Regulation ◽  
Early Developmental

The Wnt/β-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/β-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/β-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of β-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/β-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/β-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/β-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals Role for DNA replication in beta-globin gene activation.

1988 ◽  
Vol 8 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
T Enver ◽  
A C Brewer ◽  
R K Patient
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Covalent Modification ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Template Copy

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

Progesterone enhances target gene transcription by receptor free of heat shock proteins hsp90, hsp56, and hsp70

1991 ◽  
Vol 11 (10) ◽  
pp. 4998-5004
Author(s):  
M K Bagchi ◽  
S Y Tsai ◽  
M J Tsai ◽  
B W O'Malley
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Shock Protein ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Steroid Hormone ◽  
Receptor Activation ◽  
Target Cells ◽  
Dependent Manner ◽  
Shock Proteins

Steroid receptors regulate transcription of target genes in vivo and in vitro in a steroid hormone-dependent manner. Unoccupied progesterone receptor exists in the low-salt homogenates of target cells as a functionally inactive 8 to 10S complex with several nonreceptor components such as two molecules of 90-kDa heat shock protein (hsp90), a 70-kDa heat shock protein (hsp70), and a 56-kDa heat shock protein (hsp56). Ligand-induced dissociation of receptor-associated proteins such as hsp90 has been proposed as the mechanism of receptor activation. Nevertheless, it has not been established whether, beyond release of heat shock proteins, the steroidal ligand plays a role in modulating receptor activity. To examine whether the release of these nonreceptor proteins from receptor complex results in a constitutively active receptor, we isolated an unliganded receptor form essentially free of hsp90, hsp70, and hsp56. Using a recently developed steroid hormone-responsive cell-free transcription system, we demonstrate for the first time that the dissociation of heat shock proteins is not sufficient to generate a functionally active receptor. This purified receptor still requires hormone for high-affinity binding to a progesterone response element and for efficient transcriptional activation of a target gene. When an antiprogestin, Ru486, is bound to the receptor, it fails to promote efficient transcription. We propose that in the cell, in addition to the release of receptor-associated inhibitory proteins, a distinct hormone-mediated activation event must precede efficient gene activation.

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

scholarly journals HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity

2020 ◽  
Vol 117 (48) ◽  
pp. 30805-30815
Author(s):  
Mingzhe Shen ◽  
Chae Jin Lim ◽  
Junghoon Park ◽  
Jeong Eun Kim ◽  
Dongwon Baek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
Transcriptional Activation ◽  
Plant Immunity ◽  
Gene Activation ◽  
Transcriptional Corepressor ◽  
Defense Gene Expression ◽  
Dynamic Regulation ◽  
Defense Gene ◽  
Transcriptional Corepressors

Transcriptional regulation is a complex and pivotal process in living cells. HOS15 is a transcriptional corepressor. Although transcriptional repressors generally have been associated with inactive genes, increasing evidence indicates that, through poorly understood mechanisms, transcriptional corepressors also associate with actively transcribed genes. Here, we show that HOS15 is the substrate receptor for an SCF/CUL1 E3 ubiquitin ligase complex (SCFHOS15) that negatively regulates plant immunity by destabilizing transcriptional activation complexes containing NPR1 and associated transcriptional activators. In unchallenged conditions, HOS15 continuously eliminates NPR1 to prevent inappropriate defense gene expression. Upon defense activation, HOS15 preferentially associates with phosphorylated NPR1 to stimulate rapid degradation of transcriptionally active NPR1 and thus limit the extent of defense gene expression. Our findings indicate that HOS15-mediated ubiquitination and elimination of NPR1 produce effects contrary to those of CUL3-containing ubiquitin ligase that coactivate defense gene expression. Thus, HOS15 plays a key role in the dynamic regulation of pre- and postactivation host defense.

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