A zinc-finger fusion protein refines Gal4-defined neural circuits

Raghu, Shamprasad Varija; Mohammad, Farhan; Chua, Jia Yi; Barros, Claudia S.; Lam, Joanne; Loberas, Mavis; Sahani, Sadhna; Claridge‐Chang, Adam

doi:10.1101/228718

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…[6,7] Fusion proteins can also be used as transgene-specific regulators to boost expression in mammalian cells. [8][9][10][11] Both approaches, enhancer elements and fusion protein regulators, require the addition of DNA target sequences into the pDNA, which may not always be possible or desirable.…”

Section: Introductionmentioning

confidence: 99%

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

Enwerem

Warga

Dugoni

et al. 2021

Preprint

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Purpose: Targeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. Results: We validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA.

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Section: Introductionmentioning

confidence: 99%

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

Enwerem

Warga

Dugoni

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…We are attempting to engineer chimeric proteins that can activate as well as deactivate at specific temperatures allowing us to alter neuronal activity with short stimuli. Another potential is to use temperature as a driving factor to activate these proteins and induce cellular changes while simultaneously altering the expression of target genes [187][188][189][190]. A first step towards designing chimeric proteins is to determine the biophysical properties if these proteins and understand their structure function relationship.…”

Section: Conclusion and Summarymentioning

confidence: 99%

Identification, characterization and use of novel thermogenetic tools in drosophilia melanogaster

Mishra¹

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Extrinsic control of neural activity is necessary to decipher the neural mechanisms underlying behavior. Molecular tools that employ light (optogenetics) or temperature (thermogenetics) are primarily used for extrinsic manipulation of neurons. While the available tools offer precise temporal and spatial resolution, their caveats lie in the limited number of tools that can be used simultaneously to alter neuronal activity. The overlapping spectrum of activation of optogenetic tools prevents their simultaneous use in preparations. Similarly, the lack of thermogenetic tools that can function in the physiological range of organisms has restricted their use. The use of thermogenetic tools is limited to two members from the Transient receptor family of proteins, TrpA1 and TrpM8 to activate neurons, and one protein that reduces synaptic output, Shibirets. A major drawback to the Trp channels is their response to both temperature and voltage changes. Hence, the discovery of a new temperature sensitive Gustatory Receptor protein provided an opportunity to mine for other temperature sensitive proteins and develop novel thermogenetic tools. In this thesis we report the identification of several thermosensitive proteins, their characterization, and use in studying the learning and memory of freely moving flies. In the first chapter, we probed several Gustatory receptors for their temperature sensitivity using the heat box. The heat box is a high throughput system that enables us to test and track the behavior of single flies in response to temperature. The top and bottom of heat box chamber has Peltier elements that allow for control of temperature with a resolution of 1[degrees]C. We overexpressed several Gustatory receptors one at a time pan neuronally in Drosophila melanogaster and exposed them to various assays. Our initials results imply that at least 2 Drosophila melanogaster Gustatory receptors other than Gr28bD are temperature sensitive. To increase the repertoire of thermosensitive proteins, we assayed for temperature response properties of Gr28bD orthologs from 5 other Drosophila species that occupy different habitats in the world. We rationalized that flies in different habitats will have Gr28bD orthologs with unique temperature response properties designed to sustain in that habitat. Of the 5 proteins we tested, we found that 4 proteins are temperature sensitive at different temperatures. While pan-neuronal overexpression is a robust method to determine the temperature responsiveness of a protein, it does not recapitulate the natural environment the protein is present in. In D. melanogaster, Gr28bD is present in specialized heat sensing cells in the antenna, called Hot Cells. There are 3 Hot cells on each of the two antennae. There is however no physiological information on the where the orthologs are expressed. Since Gr28bD is used for rapid heat avoidance in flies, we rationalized that its orthologs too sever a similar function in their host species and are expressed in the peripheral regions. Hence, in the second chapter, we tested for the avoidance behavior of flies using two choice assays. We made mutant flies that lacked Gr28b proteins, including Gr28bD in the antennae. We then examined the ability of the orthologs to rescue the heat avoidance behavior in these mutants. We found that all the orthologs respond to temperature differences albeit, at different temperatures. Above a threshold temperature, flies rescued with some orthologs could not differentiate between small temperature differences, suggesting that the activity of the orthologs might saturate beyond certain temperatures. Some homologs responded to temperature only when expressed in Hot Cells, thus leading us to examine the presence of accessory proteins it the hot cells that might be enhancing the thermosensitive properties of these homologs. We found several candidate proteins that can studied further to determine their role in the temperature sensing in the hot cells. When used as thermogenetic tools, thermosensitive proteins are in localized environments in small cluster of cells. In the third chapter, we expressed Gr28bD in small clusters of dopaminergic neurons in the fly brain with an aim to understand the role of activation of dopaminergic neurons in operant place learning and memory paradigm. In addition to examining their learning scores at different temperatures, we investigated other behaviors of the flies during the training. Contrary to previous results from our lab that showed that dopaminergic neurons are not important for place learning and memory, we found that activation of a specific subset of dopaminergic neurons does alter place learning and memory. Our findings new laid the groundwork for more experiments to investigate dopaminergic modulation of place learning and memory.

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A zinc-finger fusion protein refines Gal4-defined neural circuits

Cited by 2 publications

References 26 publications

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

Identification, characterization and use of novel thermogenetic tools in drosophilia melanogaster

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