Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation
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eLife assessment
Powers and colleagues reveal that commonly used "genetic markers" (selectable cassettes that allow for genome modification) may lead to unintended consequences and unanticipated phenotypes. These consequences arise from cryptic expression directed from within the cassettes into adjacent genomic regions. In this work, they identify a particularly strong example of marker interference with a neighboring gene's expression and develop and test next-generation tools that circumvent the problem. The work will be primarily of interest to yeast biologists using these types of tools and interpreting these types of data.
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Abstract
Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51 , despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1 . This behavior resembled the ‘neighboring gene effect’ (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.
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Author Response
Public Evaluation Summary:
Powers and colleagues reveal that commonly used "genetic markers" (selectable cassettes that allow for genome modification) may lead to unintended consequences and unanticipated phenotypes. These consequences arise from cryptic expression directed from within the cassettes into adjacent genomic regions. In this work, they identify a particularly strong example of marker interference with a neighboring gene's expression and develop and test next-generation tools that circumvent the problem. The work will be primarily of interest to yeast biologists using these types of tools and interpreting these types of data.
Thank you for your time and thoughtfulness in assessing our manuscript. We agree the immediate and most direct importance of our findings is to those using cassette-based genome …
Author Response
Public Evaluation Summary:
Powers and colleagues reveal that commonly used "genetic markers" (selectable cassettes that allow for genome modification) may lead to unintended consequences and unanticipated phenotypes. These consequences arise from cryptic expression directed from within the cassettes into adjacent genomic regions. In this work, they identify a particularly strong example of marker interference with a neighboring gene's expression and develop and test next-generation tools that circumvent the problem. The work will be primarily of interest to yeast biologists using these types of tools and interpreting these types of data.
Thank you for your time and thoughtfulness in assessing our manuscript. We agree the immediate and most direct importance of our findings is to those using cassette-based genome editing in yeast or interpreting data that comes from these experiments. However, the relevance of our findings is not limited to yeast researchers, as yeast deletion phenotypes and synthetic phenotypes are often used to guide studies in other organisms. For example, just one popular synthetic genetic interaction study from yeast (Costanzo et al, Science 2010) has been cited over 1100 times since 2010, and a large subset of these citations are not from studies focused on budding yeast.
The central finding of our work (which we regret was not sufficiently highlighted in the original manuscript), is important to an even broader scientific community: because eukaryotic promoters are inherently bidirectional, divergent promoter activity from genome-inserted expression cassettes can drive off-target gene neighboring gene repression.
Although instances of cassette induced off-target effects have been described previously, the mechanism behind these effects was previously unknown. Our study leveraged a strong case of selection cassette-driven off-target effects to identify the mechanism by which these confounding phenotypes occur. Our finding that cassettes of disparate sequence composition and expression level are competent to drive disruption of neighboring gene expression helped us determine that bidirectional promoter activity, inherent to most eukaryotic promoters, drives this effect. Thus, our data suggests a much wider pool of overlooked mutants are potentially affected by effects like the “neighboring gene effect” (NGE, Ben-shtrit et al. Nature Methods 2012) than previously considered. We find that bidirectional promoter activity from expression cassettes occurs at all cassette-inserted loci analyzed, but the resultant divergent transcripts are often terminated before disrupting neighboring genes, apparently through the mechanisms terminating most endogenous divergent transcripts (eg. CUTs; Xu et al. Nature 2009; Schultz et al. Cell 2013). These data help explain why some loci are sensitive to disruption of neighboring gene expression while others are immune. Based on identification of this mechanism of action, we find that a simply “insulating” the promoter internal to the inserted cassette with transcription termination sequences prevents this type of off-target effect. We share these updated editing tools with the community to decrease confounding off-target effects in future studies.
Because the mechanisms driving these off-target effects are fundamental, they are likely occurring in other eukaryotes. Considering the specific cassette induced LUTI-based mis-regulation reported here, this off-target mis-regulation could be seen, regardless of organism, if the following conditions are met:
- Insertion of a cassette housing a bidirectional promoter
• Most, if not all, promoters have bidirectional activity (Teodorovic, Walls, and Elmendorf, NAR 2007; Xu et al., Nature 2009, Neil et al, Nature 2009, Trinklein et al. Genome Research 2004, Seila et al., Science 2008, Core and Lis Science 2008; Preker et al Science 2008), including commonly used mammalian promoters (CMV and EF1alpha; Curtin et al. Gene Therapy 2008; SV40: Gidoni et al. Science 1985). Insulator use is rare in construct design and has been primarily used in cases in which the concern is protecting expression of the expression cassette from the local chromatin environment. Although not the dominant mode of gene deletion in mammalian cells, expression cassettes are commonly inserted for knock-in experiments, for example, in the form of antibiotic resistance genes or fluorescent protein-encoding genes.
• It is interesting that in their native context in both yeast and mammals, most promoters do not produce a stable divergent transcript. In yeast, this results from mechanisms including the NNS termination pathway coupled to Rrp6/exosome-mediated RNA degradation (Schultz et al. Cell 2013). The TEF1 promoter is a prime example, with evidence for a divergent transcript that is visible only when RRP6 is deleted (Xu et al., Nature 2009) or when nascent transcripts are analyzed (Churchman and Weissman, Nature 2011). In mammals, the NNS pathway does not serve this role, but rather the production of stable divergent transcripts is limited by early polyA signals that prevent transcriptional interference from naturally occurring more pervasively and the instability of the resultant short transcripts (Ntini et al, NSMB 2013; Almada et al, Nature 2013). Note that persistence of a stable (detectable) transcript is not needed for neighboring gene disruption to occur, but the production of a transcript that extends into the regulatory sequences for a neighboring gene’s transcript is.
- A neighboring gene within a distance that allows transcription interference without intervening transcription termination
• This is hard to assess systematically, but natural transcription interference and LUTI occur in both human and yeast cells (Chen et al., eLife 2017; Chia et al. eLife 2017; Hollerer et al., G3 2019; Otto and Cheng et al., Cell 2018; Van Dalfsen et al. Dev Cell 2018). Data from our lab suggests this regulation can even be effective up to spans of ~2KB (Vander Wende et al, bioRxiv is an interesting example), so it seems that the artificial regulation described here could have similar range.
• Although yeast genes are more closely spaced than those in human or mice, there are many gene dense regions in these organisms cases and it has been shown that roughly ¼ of head-to-head oriented genes are within 2KB in human (Gherman, Wang, and Avramopolous, Human Genomics, 2009)
A neighboring gene in the divergent orientation to the cassette (ie. Head-to-head orientation; should be present in half of cassette insertions)
Competitive uORF sequences in the extended 5’ transcript region
• This is, again, hard to systematically assess, but our studies indicate that approximately half of AUG uORFs are effective at competing with main ORF translation. Because almost every intergenic region houses at least one AUG this may not be a major limiting factor. As in yeast, AUG uORF translation has been seen to be pervasive in naturally 5’ extended human transcripts (Floor and Doudna, eLife, 2016 as just one example).
While these conditions must be met to match the exact LUTI-based repression that we report at the DBP1/MRP51 locus, even situations in which only conditions 1 and 2 are met could drive potent transcriptional interference impacting neighboring gene expression.
Our findings offer a new perspective important for designing or interpreting genome engineering experiments in any organism, and identification of a mechanism for neighboring gene effects of expression cassette insertion allow it to be prevented in future studies.
We regret the narrow framing of our study in the initial manuscript, but hope that our revised manuscript better demonstrates how our findings fit into existing literature regarding neighboring gene effects from cassette insertion, and that their broad relevance is now clear.
Reviewer #1 (Public Review):
This manuscript presents information that will be of great interest to yeast geneticists - standard gene deletions can lead to misleading phenotypes due to effects on adjacent genes. The experiments carefully document this in one case, for the DBP1 gene, and present additional evidence that it can occur at additional genes. An improved version of the standard gene replacement cassette is described, with evidence that it functions in an improved fashion, insulated from affecting adjacent genes.
We appreciate the reviewer’s enthusiasm for the data in our study, and their perspective that this will be of great interest to the yeast community. We hope that we have improved the writing in the revised manuscript to emphasize our finding that a conserved feature of eukaryotic gene regulation drives this effect suggests it likely to be occurring in other organisms.
Reviewer #2 (Public Review):
The impact of the work will be for yeast researchers in the clear and careful presentation of a case study wherein phenotypes might be ascribed to the knockout of a particular gene but instead derive from effects on a neighboring gene. In this case, a transcript expressed from within or adjacent to a knockout of DBP1 by a selectable marker towards the adjacent gene MRP51 interferes with the adjacent gene's normal transcription start sites. Furthermore, although neighboring MRP51 ORF is present on the longer mRNA isoform that is generated, it is not efficiently translated. The authors expand on this phenotypic observation to demonstrate that a substantial fraction of selectable marker insertions can generate transcription adjacent to or within and going away from, selectable markers.
The strengths of the work are that the derivation of the observed phenotypes for the dpb1∆ alleles is clearly and carefully elucidated and the creation of new selectable marker cassettes that overcome the potential for cryptic transcript emanation from or near to the selectable markers. This is valuable for the community as a clear demonstration of how only the exact right experiments might detect underlying mechanisms for potentially misattributed phenotypes and that many times these experiments may not be performed.
Thanks very much to the reviewer for their thoughtful assessment of our manuscript. We are thrilled that they find the work to be valuable for yeast researchers, and more broadly, to those interested in avoiding misinterpretations of mutant phenotypes. We propose this to be a mechanism that is likely to be important beyond yeast studies and hope that we have made this clearer in the revised manuscript.
While understandable in terms of how the experiments likely played out, the manuscript seems in between biology and tool development, as the biology in question was related to a gene that is not the focus of this lab. The tool development is likely to be useful but potentially non-optimal.
We agree with the reviewer’s point that this is a good opportunity to improve the standard yeast cassettes further and have now done so. We now include a further improved pair of cassettes that minimize shared sequences (Figure 3H). These and the previously described constructs (Figure 3F) will all be deposited at Addgene and we hope that they will be of value to the yeast community.
The reviewer’s comment also made us realize that our previous presentation of the work was not ideal. We have adjusted the order of data in the revised manuscript, including swapping the data in Figures 3 and 4 and adding a Figure 5 to further emphasize the mechanism that we identify to drive this off-target effect, rooted in bidirectional promoter activity. While we hope the new cassettes are useful to others, they also serve a specific biological role in this manuscript, which is to show that bidirectional transcription driven from existing cassettes is the cause of the off-target effect that we report.
The mechanism for interference identified in this example case (via a long undecoded transcript isoform (LUTI) has already been described for other loci and in a number of species, including in work from the Brar lab. The concept of marker interference with neighboring genes has also been increasingly appreciated by a number of other studies.
Indeed, because of our recent research interests, we were aware that natural LUTI-based regulation was widespread prior to this study, but even we were surprised to see it occurring in this artificial context. The idea that constitutive LUTI-based repression can be easily driven at loci that are not otherwise LUTI-regulated is an interesting point to consider in designing gene editing approaches. We agree with the reviewer that a greater discussion of previously published work regarding marker interference is necessary to understand the novelty of our findings, including the discussion of some work that should have been cited and discussed in the original manuscript (Ben-Shitrit et al. Nature Methods 2012 and Egorov et al. NAR 2021, in particular). In the reframing of our revised manuscript, we aimed to emphasize the novel aspects of our work, and how they relate to previous reports of the “neighboring gene effect” (NGE). Although the phenomenon of the NGE had been reported, it was not previously clear what caused it to occur, which made it impossible to prevent in planning new approaches or to diagnose in existing data. In revealing this unexpected mechanism driven by bidirectional promoter activity that is general to expression cassette-based editing, rather than resulting from any particular cassette sequence, we were able to design constructs to prevent this from occurring in future studies. Moreover, because bidirectional promoter activity is a highly conserved feature of eukaryotic gene expression, this finding suggests that the type of off-target effect that we describe here is likely to occur with expression cassette insertion in more complex eukaryotes, as well. To our knowledge, this has not been widely considered as a possibility.
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eLife assessment
Powers and colleagues reveal that commonly used "genetic markers" (selectable cassettes that allow for genome modification) may lead to unintended consequences and unanticipated phenotypes. These consequences arise from cryptic expression directed from within the cassettes into adjacent genomic regions. In this work, they identify a particularly strong example of marker interference with a neighboring gene's expression and develop and test next-generation tools that circumvent the problem. The work will be primarily of interest to yeast biologists using these types of tools and interpreting these types of data.
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Reviewer #1 (Public Review):
This manuscript presents information that will be of great interest to yeast geneticists - standard gene deletions can lead to misleading phenotypes due to effects on adjacent genes. The experiments carefully document this in one case, for the DBP1 gene, and present additional evidence that it can occur at additional genes. An improved version of the standard gene replacement cassette is described, with evidence that it functions in an improved fashion, insulated from affecting adjacent genes.
-
Reviewer #2 (Public Review):
The impact of the work will be for yeast researchers in the clear and careful presentation of a case study wherein phenotypes might be ascribed to the knockout of a particular gene but instead derive from effects on a neighboring gene. In this case, a transcript expressed from within or adjacent to a knockout of DBP1 by a selectable marker towards the adjacent gene MRP51 interferes with the adjacent gene's normal transcription start sites. Furthermore, although neighboring MRP51 ORF is present on the longer mRNA isoform that is generated, it is not efficiently translated. The authors expand on this phenotypic observation to demonstrate that a substantial fraction of selectable marker insertions can generate transcription adjacent to or within and going away from, selectable markers.
The strengths of the work …
Reviewer #2 (Public Review):
The impact of the work will be for yeast researchers in the clear and careful presentation of a case study wherein phenotypes might be ascribed to the knockout of a particular gene but instead derive from effects on a neighboring gene. In this case, a transcript expressed from within or adjacent to a knockout of DBP1 by a selectable marker towards the adjacent gene MRP51 interferes with the adjacent gene's normal transcription start sites. Furthermore, although neighboring MRP51 ORF is present on the longer mRNA isoform that is generated, it is not efficiently translated. The authors expand on this phenotypic observation to demonstrate that a substantial fraction of selectable marker insertions can generate transcription adjacent to or within and going away from, selectable markers.
The strengths of the work are that the derivation of the observed phenotypes for the dpb1∆ alleles is clearly and carefully elucidated and the creation of new selectable marker cassettes that overcome the potential for cryptic transcript emanation from or near to the selectable markers. This is valuable for the community as a clear demonstration of how only the exact right experiments might detect underlying mechanisms for potentially misattributed phenotypes and that many times these experiments may not be performed. While understandable in terms of how the experiments likely played out, the manuscript seems in between biology and tool development, as the biology in question was related to a gene that is not the focus of this lab. The tool development is likely to be useful but potentially non-optimal. The mechanism for interference identified in this example case (via a long undecoded transcript isoform (LUTI) has already been described for other loci and in a number of species, including in work from the Brar lab. The concept of marker interference with neighboring genes has also been increasingly appreciated by a number of other studies.
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