scholarly journals Orthogonal Control of Gene Expression in Plants Using Synthetic Promoters and CRISPR-Based Transcription Factors

2022 ◽  
Author(s):  
Shaunak Kar ◽  
Yogendra Bordiya ◽  
Nestor Rodriguez ◽  
Junghyun Kim ◽  
Elizabeth C Gardner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
System Level ◽  
Plant Signaling ◽  
Promoter Elements ◽  
Control Of Gene Expression ◽  
Form Complex ◽  
Pol Ii ◽  
Synthetic Promoters ◽  
Pol Iii

Abstract Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

scholarly journals Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors

2021 ◽  
Author(s):  
Shaunak Kar ◽  
Yogendra Bordiya ◽  
Nestor Rodriguez ◽  
Jungyun Kim ◽  
Elizabeth C Gardner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
System Level ◽  
Plant Signaling ◽  
Promoter Elements ◽  
Control Of Gene Expression ◽  
Form Complex ◽  
Pol Ii ◽  
Synthetic Promoters ◽  
Pol Iii

Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts

1994 ◽  
Vol 14 (9) ◽  
pp. 5910-5919
Author(s):  
S Connelly ◽  
C Marshallsay ◽  
D Leader ◽  
J W Brown ◽  
W Filipowicz
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Gene Promoters ◽  
Small Nuclear Rna ◽  
Promoter Elements ◽  
Pol Ii ◽  
Snrna Gene ◽  
Nuclear Rna ◽  
Pol Iii

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

scholarly journals Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

1994 ◽  
Vol 14 (9) ◽  
pp. 5910-5919 ◽  
Author(s):  
S Connelly ◽  
C Marshallsay ◽  
D Leader ◽  
J W Brown ◽  
W Filipowicz
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Gene Promoters ◽  
Small Nuclear Rna ◽  
Promoter Elements ◽  
Pol Ii ◽  
Snrna Gene ◽  
Nuclear Rna ◽  
Pol Iii

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

The heat shock response: A case study of chromatin dynamics in gene regulation

2013 ◽  
Vol 91 (1) ◽  
pp. 42-48 ◽  
Author(s):  
Sheila S. Teves ◽  
Steven Henikoff
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Control Of Gene Expression ◽  
Pol Ii ◽  
Chromatin Dynamics ◽  
Regulatory Processes ◽  
Shock Response ◽  
Rate Limiting

Recent studies in transcriptional regulation using the Drosophila heat shock response system have elucidated many of the dynamic regulatory processes that govern transcriptional activation and repression. The classic view that the control of gene expression occurs at the point of RNA polymerase II (Pol II) recruitment is now giving way to a more complex outlook of gene regulation. Promoter chromatin dynamics coordinate with transcription factor binding to maintain the promoters of active genes accessible. For a large number of genes, the rate-limiting step in Pol II progression occurs during its initial elongation, where Pol II transcribes 30–50 bp and pauses for further signals. These paused genes have unique genic chromatin architecture and dynamics compared with genes where Pol II recruitment is rate limiting for expression. Further elongation of Pol II along the gene causes nucleosome turnover, a continuous process of eviction and replacement, which suggests a potential mechanism for Pol II transit along a nucleosomal template. In this review, we highlight recent insights into transcription regulation of the heat shock response and discuss how the dynamic regulatory processes involved at each transcriptional stage help to generate faithful yet highly responsive gene expression.

Principles of Molecular Biology

2013 ◽  
pp. 76-87
Author(s):  
Steven E. Hyman ◽  
Eric J. Nestler
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Molecular Biology ◽  
Cell Surface ◽  
Environmental Context ◽  
Molecular Processes ◽  
Control Of Gene Expression ◽  
Extracellular Signals ◽  
Gene Environment

This chapter provides an overview of the fundamental molecular processes by which information is encoded in the genome and how this information is expressed within an environmental context. We describe what genes are, how they function, and how their expression into RNA and protein is regulated by signals from outside the cell. Particular attention is given to a series of stimulus-regulated transcription factors, which play important roles in transducing information from the cell surface to the nucleus. Work in this area has shown that the control of gene expression by extracellular signals is a critical arena for gene–environment interactions that are highly relevant to psychiatry.

scholarly journals De novo design of programmable inducible promoters

2019 ◽  
Vol 47 (19) ◽  
pp. 10452-10463 ◽  
Author(s):  
Xiangyang Liu ◽  
Sanjan T P Gupta ◽  
Devesh Bhimsaria ◽  
Jennifer L Reed ◽  
José A Rodríguez-Martínez ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput Screening ◽  
De Novo ◽  
Vital Role ◽  
Control Of Gene Expression ◽  
Core Motif ◽  
Expression Levels ◽  
Synthetic Promoters ◽  
The Core ◽  
Rich Diversity

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

The control of gene expression and cell proliferation by the epithelial apical junctional complex

2012 ◽  
Vol 53 ◽  
pp. 83-93 ◽  
Author(s):  
Domenica Spadaro ◽  
Rocio Tapia ◽  
Pamela Pulimeno ◽  
Sandra Citi
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Transcription Factors ◽  
Protein Complexes ◽  
Regulation Of Gene Expression ◽  
Small Gtpases ◽  
Junctional Complex ◽  
Nucleotide Exchange ◽  
Control Of Gene Expression ◽  
Apical Junctional Complex

The AJC (apical junctional complex) of vertebrate epithelial cells orchestrates cell–cell adhesion and tissue barrier function. In addition, it plays a pivotal role in signalling. Several protein components of the AJC, e.g. the cytoplasmic proteins β-catenin, p120-catenin and ZO (Zonula Occludens)-2, can shuttle to the nucleus, where they interact with transcription factors to regulate gene expression and cell proliferation. Other junctional proteins, e.g. angiomotin, α-catenin and cingulin, are believed to act by sequestering either transcription factors, such as YAP (Yes-associated protein), or regulators of small GTPases, such as GEF (guanine-nucleotide-exchange factor)-H1, at junctions. The signalling activities of AJC proteins are triggered by different extracellular and intracellular cues, including cell density, and physiological or pathological activation of developmentally regulated pathways, such as the Wnt pathway. The interplay between junctional protein complexes, the actin cytoskeleton and signalling pathways is of crucial importance in the regulation of gene expression and cell proliferation.

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