Transcriptional Interference in Convergent Promoters as a Means for Tunable Gene Expression

Bordoy, Antoni E.; Varanasi, Usha; Courtney, Colleen M.; Chatterjee, Anushree

doi:10.1021/acssynbio.5b00223

Cited by 30 publications

(57 citation statements)

References 49 publications

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“…Tunable transcriptional interference was first reported in bacteria (Bordoy et al 2016;Hao et al 2016), more recently in yeast (Chia et al 2017), and in human cells (Hollerer et al 2019). All of these studies highlight the notion that gene regulation by transcriptional interference is not binary with an on/off state, but rather can be utilized to tune the expression of regulated mRNAs during developmental gene expression programs.…”

Section: Discussionmentioning

confidence: 94%

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Jorgensen

Chen

Wende

et al. 2020

G3 Genes|Genomes|Genetics

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Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

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Section: Discussionmentioning

confidence: 94%

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Jorgensen

Chen

Wende

et al. 2020

G3 Genes|Genomes|Genetics

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“…To show this proof-of-concept in a more complex competitive environment, we next conducted three-strain matings between donor E. coli C600 (carrying RK2 in the control, with both RK2 and pET11a- opaL for the experimental group), targeted recipient E. coli BL21 (DE3) with GFP expressed from pHL662, and a non-targeted recipient E. coli DH5α with mCherry expressed from the plasmid pUV145 (22) (supplementary Fig. S2B).…”

Section: Resultsmentioning

confidence: 99%

“…E. coli NEB10-β was acquired from NEB and used as a host for propagating the pET11a-OriT vector. The pUV145 plasmid (22), was carried in an E. coli DH5a host. E. coli DH5α carrying pUV145 were employed in our three strain mating-toxicity assay.…”

Section: Methodsmentioning

confidence: 99%

Design of a de novo aggregating antimicrobial peptide and bacterial conjugation delivery system

Collins

Otoupal

Courtney

et al. 2018

Preprint

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26Traditional antibiotics are reaching obsolescence as a consequence of antibiotic 27 resistance; therefore novel antibiotic approaches are needed. A recent non-traditional approach 28 involves formation of protein aggregates as antimicrobials to disrupt bacterial homeostasis. 29 Previous work on protein aggregates has focused on genome mining for aggregation-prone 30 sequences in bacterial genomes rather than on rational design of aggregating antimicrobial 31 peptides. Here, we use a synthetic biology approach to design an artificial gene encoding the first 32 de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein 33 aggregator Lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic 34 peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent 35 aggregation and antimicrobial effect decreases. Further, to deliver this artificial gene, we 36 developed a probiotic approach using RK2, a broad host range conjugative plasmid, to transfer 37 opaL from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying the 38 opaL gene by adding the RK2 origin of transfer. We show that opaL is non-toxic to the donor, 39 allowing for maintenance and transfer since its expression is under control of a promoter with a 40 recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we 41 observe selective growth repression in T7 polymerase expressing recipients. This technique 42 could be used to target desired pathogens by selecting pathogen-specific promoters to control 43 opaL expression. This system provides a basis for the design and delivery of novel antimicrobial 44 peptides. 45 46 47 3 48 Importance 49The growing threat of antibiotic resistance necessitates new treatment options for 50 bacterial infections that are recalcitrant to traditional antimicrobials. Existing methods usually 51 involve small-molecule compounds which interfere with essential processes in bacterial cells. By 52 contrast, protein aggregates operate by causing widespread disruption of bacterial homeostasis 53 and may provide a new method for combating infections. We used rational design to create and 54 test an aggregating de novo antimicrobial peptide, OpaL. In addition, we employed bacterial 55 conjugation to deliver the opaL gene from donor bacteria to recipient bacteria while using a 56 strain-specific promoter to ensure that OpaL was only expressed in targeted recipients. To the 57 best of our knowledge, this represents the first design for a de novo peptide with aggregation-58 mediated antimicrobial activity. We envision that OpaL's design parameters could be used in 59 developing a new class of antimicrobial peptides to help treat antibiotic resistant infections. 60 61 1 Introduction 62 Traditional antibiotic treatments are losing efficacy as bacteria gain resistance to 63 available antimicrobial compounds. This resistance arises through a variety of mechanisms such 64 as altered target sites, excl...

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“…Direct contact of bacterial RNAPs has not been observed during head-on RNAP collisions (15), and it is generally understood that interference of one RNAP on another may be mediated through DNA supercoiling (16,17) rather than due to direct collisions of transcriptional machinery. Consequently, the act of cis-antisense transcription has been shown to reliably down-regulate gene expression (9,13,(18)(19)(20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

Engineering Transcriptional Interference through RNA Polymerase Processivity Control

O'Connor

Bordoy

Chatterjee

2020

Preprint

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Antisense transcription is widespread in all kingdoms of life and has been shown to influence gene expression through transcriptional interference (TI), a phenomenon in which one transcriptional process negatively influences another in cis. The processivity, or uninterrupted transcription, of an RNA Polymerase (RNAP) is closely tied to levels of antisense transcription in bacterial genomes, but its influence on TI, while likely important, is not well-characterized. Here we show that TI can be tuned through processivity control via three distinct antitermination strategies: the antibiotic bicyclomycin, phage protein Psu, and ribosome-RNAP coupling. We apply these methods toward TI and tune ribosome-RNAP coupling to produce 38-fold gene repression due to RNAP collisions. We then couple protein roadblock and RNAP collisions to design minimal genetic NAND and NOR logic gates. Together these results show the importance of processivity control for strong TI and demonstrate the potential for TI to create sophisticated switching responses.

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Transcriptional Interference in Convergent Promoters as a Means for Tunable Gene Expression

Cited by 30 publications

References 49 publications

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Design of a de novo aggregating antimicrobial peptide and bacterial conjugation delivery system

Engineering Transcriptional Interference through RNA Polymerase Processivity Control

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