Enhancer-driven random gene overexpression (ERGO): a method to study gene function in Chlamydomonas
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Abstract
Gene overexpression can be used to study gene function and is more suitable to characterize essential and redundant genes than gene knockout. A forward genetic approach based on random gene overexpression, also known as activation tagging , was previously used to study gene function in angiosperms. However, such an approach has never been applied to algae. Here, we present enhancer-driven random gene overexpression (ERGO), a forward genetic screen that we utilized to study genes involved in carotenoid metabolism in the green alga Chlamydomonas reinhardtii . We generated a library of over 33,000 insertional mutants in a yellow-in-the-dark background strain, which is incapable of producing chlorophyll in the dark. Each mutant contained a randomly inserted enhancer, E hist cons , capable of activating gene expression in the C. reinhardtii nuclear genome. After visually screening the mutant colonies for a color change from yellow to orange, we isolated a mutant with increased carotenoid content and remarkable resistance to high-light stress. RNA-seq data analysis revealed substantial upregulation of a gene, that we name CMRP1 , encoding a putative F-box protein. CRISPR-mediated knockout of this gene resulted in decreased carotenoid concentrations, confirming that CMRP1 is involved in the regulation of carotenoid metabolism. Our study shows that a gene overexpression screen can be successfully adapted to C. reinhardtii and potentially other plants and algae, thereby expanding the palette of genetic tools to study gene function.
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Enhancer-driven random gene overexpression (ERGO): a method to study gene function in Chlamydomonas
Your enhancer-insertion library is a useful tool for probing carotenoid regulation, and the CMRP1 follow-up is both convincing and compelling. The long-range activity of the enhancer in your top hit is intriguing and raises a few questions about how ERGO should be interpreted.
If an insertion can influence genes across ~2 Mb, then many nearby loci are plausible targets. How confident are you that CMRP1 is the primary driver rather than one member of a broader set of co-activated genes? More generally, because NHEJ insertions favor open and insertion-tolerant regions, regulators positioned in less permissive chromatin may never be sampled. Insertions that disrupt essential genes, or essential neighboring genes, would also eliminate the …
Enhancer-driven random gene overexpression (ERGO): a method to study gene function in Chlamydomonas
Your enhancer-insertion library is a useful tool for probing carotenoid regulation, and the CMRP1 follow-up is both convincing and compelling. The long-range activity of the enhancer in your top hit is intriguing and raises a few questions about how ERGO should be interpreted.
If an insertion can influence genes across ~2 Mb, then many nearby loci are plausible targets. How confident are you that CMRP1 is the primary driver rather than one member of a broader set of co-activated genes? More generally, because NHEJ insertions favor open and insertion-tolerant regions, regulators positioned in less permissive chromatin may never be sampled. Insertions that disrupt essential genes, or essential neighboring genes, would also eliminate the corresponding clones before screening, also impacting sampling. Do you have a sense of how much of the genome is protected in this way? Along those lines, have you looked at whether enhancer effectiveness varies with chromatin context, and whether some genomic regions tend to dampen or block enhancer activity?
Did you characterize the expression of neighboring genes at all to distinguish between CMRP1-driven changes and insertion-related ones? Given that many insertions are tandem or structurally complex, did you assess whether enhancer copy number, truncation, or orientation contributed to the expression patterns or phenotypes you observed?
Finally, the use of ERGO here implements a pigment phenotype in the yellow-in-the-dark background. Do you envision pairing the enhancer library with non-colorimetric reporters or selectable screens to expand beyond carotenoid metabolism in the future?
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