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microPublication / Biology / A single amino acid change...
A single amino acid change in the EGL-46 transcription factor causes defects in BAG neuron specification
Rasoul Godini1, Kasper Langebeck-Jensen2 and Roger Pocock1
1Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria 3800, Australia
2Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen, Denmark
Correspondence to: Roger Pocock (roger.pocock@monash.edu)
These authors contributed equally.
Figure 1: (A) Schematic of the egl-46 genomic locus showing the rp4 genetic lesion (TGC>TAC). (B) Schematic of the EGL-46 protein showing the amino acid change (C185Y) caused by rp4. (C) Quantification of Pgcy-33::gfp expression defects in egl-46(rp4) animals. Transgenic expression of a fosmid (WRM0636bB06) containing the entire egl-46 genomic locus rescues the loss of Pgcy-33::gfp expression in the BAG neurons observed in egl-46(rp4) mutant animals. Circles indicate gfp expression level in the pair of left and right BAG neurons.

Description

The BAG neurons control multiple aspects of Caenorhabditis elegans behavior, such as sensing environmental gases (oxygen and carbon dioxide), regulation of systemic fat levels and egg laying (Brandt et al. 2012; Guillermin et al. 2011; Juozaityte et al. 2017; Zimmer et al. 2009). To identify factors that control BAG specification, we performed a forward genetic mutagenesis screen using the Pgcy-33::gfp reporter, which is exclusively expressed in the BAG neurons. We isolated a new allele (rp4) that exhibits a loss of Pgcy-33::gfp expression. Using the one-step whole-genome sequencing and SNP mapping strategy (Doitsidou et al. 2010) we mapped the genetic lesion to the egl-46 gene, encoding a zinc finger transcription factor homologous to mammalian INSM1/2, which we had previously shown to be important for BAG specification (Rojo Romanos et al. 2015). The new lesion we identified egl-46(rp4) (TGC>TAC) causes a single amino acid change in a highly conserved cysteine residue (C185Y) that lies in the first zinc finger domain of EGL-46, which would be predicted to affect DNA binding. Analysis of Pgcy-33::gfp expression in the rp4 allele reveals that it exhibits the same phenotype as the previously published rp13 deletion allele, which is an out-of-frame deletion that removes the zinc finger domains (Rojo Romanos et al. 2015). Therefore, rp4 acts as a strong loss-of-function/null allele and may be of use to those researchers interested in elucidating additional functions of EGL-46.

Methods

Request a detailed protocol

In the forward genetic screen, the BAG reporter strain Pgcy-33::gfp; Pdop-3::rfp was mutagenized using ethyl methanesulfonate. Mutants with decreased GFP expression in the BAG neurons were isolated using the automated COPAS biosorter platform. The one-step whole-genome sequencing and SNP mapping strategy (Doitsidou et al. 2010) was used to identify the genetic lesion of the isolated rp4 allele. Phenotypic analysis of Pgcy-33::gfp BAG expression was performed as described previously (Rojo Romanos et al. 2015).

Reagents

RJP22 rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp)

RJP56 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp)

RJP4585 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp); rpEx2046 (WRM0636bB06) 1ng/µl + Punc-122::gfp 30ng/µl Line 1

RJP4586 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp); rpEx2047 (WRM0636bB06) 1ng/µl + Punc-122::gfp 30ng/µl Line 2

Strains will be available at the CGC.

References

Brandt, J.P., Aziz-Zaman, S., Juozaityte, V., Martinez-Velazquez, L.A., Petersen, J.G., Pocock, R., and Ringstad, N. (2012). A single gene target of an ETS-family transcription factor determines neuronal CO2-chemosensitivity. PLoS One 7, e34014.
PubMed
Doitsidou, M., Poole, R.J., Sarin, S., Bigelow, H., and Hobert, O. (2010). C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS One 5, e15435.
PubMed
Guillermin, M.L., Castelletto, M.L., and Hallem, E.A. (2011). Differentiation of Carbon Dioxide-Sensing Neurons in Caenorhabditis elegans Requires the ETS-5 Transcription Factor. Genetics 189, 1327-39.
PubMed
Juozaityte, V., Pladevall-Morera, D., Podolska, A., Norgaard, S., Neumann, B., and Pocock, R. (2017). The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety. Proc Natl Acad Sci U S A 114, E1651-E1658.
PubMed
Rojo Romanos, T., Petersen, J.G., Riveiro, A.R., and Pocock, R. (2015). A novel role for the zinc-finger transcription factor EGL-46 in the differentiation of gas-sensing neurons in Caenorhabditis elegans. Genetics 199, 157-163.
PubMed
Zimmer, M., Gray, J.M., Pokala, N., Chang, A.J., Karow, D.S., Marletta, M.A., Hudson, M.L., Morton, D.B., Chronis, N., and Bargmann, C.I. (2009). Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron 61, 865-879.
PubMed

Funding

NHMRC - GNT1105374 and GNT1137645 to R.P.

Author Contributions

Rasoul Godini: Formal analysis, Investigation, Methodology, Validation, Visualization, Data curation, Writing - review and editing
Kasper Langebeck-Jensen: Formal analysis, Investigation, Data curation, Methodology, Visualization
Roger Pocock: Formal analysis, Methodology, Investigation, Data curation, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing.

Reviewed By

David Miller

History

Received: February 11, 2020
Accepted: February 25, 2020
Published: February 25, 2020

Copyright

© 2020 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Godini, R; Langebeck-Jensen, K; Pocock, R (2020). A single amino acid change in the EGL-46 transcription factor causes defects in BAG neuron specification. microPublication Biology. 10.17912/micropub.biology.000224.
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