Updated March 1, 2016
- Genetic interactions involving two or more mutations that result in suppression or enhancement of a given phenotype, also synergistic (synthetic) interactions
- Co-transfection experiments in which two or more genes are expressed in a heterologous system to assess functional interaction
- Expression of one gene alters the phenotypic outcome of a mutation in another gene; the two genes may or may not be from the same species. In the literature, these types of experiments are variably referred to as: functional complementation, rescue experiments, or suppression
The IGI evidence code is used for annotations based on experiments reporting the effects of perturbations in the sequence or expression of one or more genes or gene products. IGI is also used for experiments that interrogate functional interactions between two or more genes or gene products when co-expressed, for example, in a cell line. Additional uses of IGI include experiments in which the expression of one gene affects the phenotypic outcome of a mutation in another gene.
Key to deciding whether or not to use the IGI or IMP (Inferred from Mutant Phenotype) evidence code is consideration of the point of reference (i.e., what is being compared) to determine a possible interaction. If experiments interrogate the effects of multiple mutations or differences from the control, then use IGI. If experiments interrogate the effects of a single mutation or difference from the control, then use IMP.
The IGI evidence code requires curators enter a stable database identifier for the interacting entity in the With/From field of the Gene Association File (GAF). Independent interactors may be captured in the With/From field by separating each entry with a pipe. If the interaction experiment involves multiple perturbations simultaneously, e.g. triply mutant strains, then the respective interactors are separated with a comma.
Examples where the IGI evidence code should be used:
Genetic interactions such as suppression, enhancement, synergistic (synthetic) interactions, etc.
This use of the IGI evidence code refers to the more “traditional” genetic interaction experiments performed in model organisms, such as Saccharomyces cerevisiae, as well as more recent approaches adopted in a number of different systems such as RNA-mediated knockdown or genome editing techniques. Note that genetic interaction experiments may be performed with both loss-and gain-of-function mutations. Consequently, curators will need to use their expertise to determine whether interaction phenotypes resulting from gain-of-function mutations are informative about the normal, wild type role of a gene or gene product.
- Example 1: Double loss-of-function mutations resulting in enhancement of a mutant phenotype
- Localized cell wall degradation is essential for proper cell fusion in the fission yeast, Schizosaccharomyces pombe. This process is accomplished by the localized action of degradative enzymes including several distinct glucanases that act on differentpolysaccharides. Deletion of multiple glucanases in S. pombe results in decreasing efficiency of cell fusion indicating thateach enzyme contributes additively to this process.
- exg3 fungal-type cell wall disassembly involved in conjugation with cellular fusion (GO:1904541) PMID:25825517 IGI agn2
- agn2 fungal-type cell wall disassembly involved in conjugation with cellular fusion (GO:1904541) PMID:25825517 IGI exg3
- Example 2: Gain-of-function mutation
- The response to axonal injury requires the activities of MAP kinase and cAMP signaling pathways that are required, for example, for signaling growth cone formation. In C. elegans, the activity of the upstream-most kinase in one of the MAPK signaling pathways, DLK-1, is stimulated by Ca2+ influx mediated by the EGL-19 voltage-gated calcium channel. EGL-19’s regulatory role in the MAPK-mediated axon regeneration pathway was determined, in part, through doubly mutant animals containing an egl-19 hypermorphic mutation that results in occasional action potentials with significantly prolonged plateau phases and a dlk-1 loss-of-function mutation that showed a reduced axon regenerative response when compared to egl-19 alone.
- EGL-19 positive regulation of MAPK cascade involved in axon regeneration (GO:1904922) PMID:20203177 IGI DLK-1
- Note that in this example, reciprocal IGI annotations are not made, as the GO term selected for EGL-19 does not make sense for DLK-1.
- Example 3: Synergistic (synthetic) interactions
- Disruption of the MSB2 gene in S. cerevisiae has no appreciable effects on the cell's ability to activate the High-Osmolarity Glycerol (HOG) pathway upon osmotic stress, or on cellular growth on high-osmolarity media. To identify potential osmosensors in the SHO1 branch of the HOG pathway, the authors screened for a mutant that is osmosensitive only in an msb2Δ background and recovered mutations in the HKR1 gene. Like MSB2, mutations in HRK1 alone confer no osmosensitivity to the cells.
- MSB2 hyperosmotic response (GO:0006972) PMID:17627274 IGI HKR1
- HKR1 hyperosmotic response (GO:0006972) PMID:17627274 IGI MSB2
Co-transfection experiments include those experiments where two or more gene products are expressed in a heterologous system, such as a cell line, for the purposes of interrogating a functional interaction between them.
- Example 1: Co-transfection of G protein-coupled receptors (GPCRs)
- In C. elegans, the response to dauer pheromone, a mixture of small molecules, is mediated by G protein-coupled receptors (GPCRs). Genetic analysis has implicated two GPCRs, SRBC-64 and SRBC-66, in a signaling pathway that responds to specific components of dauer pheromone. To assess the biochemical role of SRBC-64 and SRBC-66, the gene products were expressed singly or in combination in HEK293 cells. Only when expressed in combination were the GPCRs able to enhance forskolin-stimulated cAMP production.
- SRBC-64 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-66
- SRBC-66 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-64
Expression of one gene affects the phenotype of a mutation in another gene
These types of experiments are described in various ways in the published literature, but generally involve expressing a wild-type copy of one gene in the background of a mutation in a second, different gene to determine if the expressed gene can mask the phenotype of the mutated gene. The two genes may or may not be from the same species. When genes from different species are analyzed it is often with the intent of demonstrating functional conservation between species.
- Example 1: Genes from different species
- C. elegans contains two genes, lgg-1 and lgg-2, with sequence similarity to the Saccharomyces cerevisiae ubiquitin-like protein Atg8 that is required for autophagosome biogenesis. Transformation of lgg-1, but not lgg-2, into atg8 deletion mutants in nitrogen starvation medium results in increased survival compared to atg8 mutants alone, indicating that lgg-1 can functionally complement budding yeast atg8.
- lgg-1 (C. elegans) macroautophagy (GO:0016236) PMID:20523114 IGI atg8 (S. cerevisiae)
- For these annotations, the With/From column should list the identifier for the endogenous gene that is complemented by the heterologously expressed gene being annotated. In annotations from cross-species functional complementation experiments, the gene referred to in the With/From column will thus be from a different species than the gene being annotated.
- Example 2: Different genes from the same species
- The planar cell polarity pathway is critical for a number of biological processes including epidermal wound repair. Activity of the GRHL3 transcription factor is essential for efficient wound repair in mice and human cell lines. Wound repair requires activation of the RhoA small GTPase to effect the cellular polarization, actin polymerization and epidermal migration critical to wound closure. The gene encoding the RhoGEF RhoGEF119, a RhoA GTPase activator, is a transcriptional target of GRHL3, and RHOGEF119 activity is also required for wound repair. Expression of human RhoGEF119 in human Grhl3-kd cell lines rescues the actin polymerization defects resulting from loss of Grhl13, indicating a role for RhoGEF119 in regulation of actin cytoskeletal organization during wound repair.
- ARHGEF19 positive regulation of actin cytoskeleton organization (GO:0032956) PMID:20643356 IGI GRHL3
- GRHL3 positive regulation of actin cytoskeleton organization (GO:0032956) PMID:20643356 IGI ARHGEF119
- Note that rescue experiments may be used to help determine the order in which gene products act within a biological pathway or process.
- Example 3: Different genes from the same species
- Localized assembly of a filamentous actin (F-actin) network at the leading edge of D. discoideum cells is required for proper chemotaxis towards the cAMP chemoattractant. The organization of actin filaments is regulated by intracellular pH; an increase in pH is necessary for chemotaxis and required the Na+/H+ exchanger Ddnhe1. Expression of DdAip1, the D. discoideum ortholog of Actin-interacting protein 1, suppresses the chemotaxis defect of Ddnhe1 mutants by restoring the F-actin network, thus illustrating DdAip1's role in actin filament polymerization.
- aip1 actin filament polymerization (GO:0030041) PMID:20668166 IGI nhe1
When NOT to use IGI
A mutation in one gene affects some property of another gene
Some experiments assess a functional interaction between one or more gene products by examining the effects that mutations in one gene have on the properties of another. These types of experiments are annotated using the IMP (Inferred from Mutant Phenotype) evidence code and the target, or affected gene product, may be captured as an Annotation Extension. The key here is that the genetic perturbation is directed at only one of the gene products in the experiment.
- For example, treatment of cells with GSK3B antagonists results in nuclear accumulation of the GATA6 transcription factor. This experiment indicates that GSK3B negatively regulates GATA6 localization.
- GSK3B negative regulation of protein localization to nucleus (GO:1900181) PMID:23624080 transports_or_maintains_localization_of GATA6
Expression of a gene is used to restore the normal function of the same gene
Evidence for a gene's role in a given biological process can be evaluated by expression of a wild-type copy of the gene to "rescue" the phenotype of a mutation in that gene. These experiments, since they involve the same gene, are not considered genetic interactions and may instead be used to support an IMP annotation.
- For example, loss-of-function mutations in the C. elegans phosphoinositol-5-phosphatase inpp-1 exhibit defective Ca2+ signaling in the AWA chemosensory neuron in response to odorant stimulus. Expression of inpp-1 from a genomic fosmid clone or from an AWA-specific promoter restores the wild-type AWA-mediated odorant response.
- inpp-1 response to odorant (GO:1990834) IMP
Expression of a miRNA affects expression of a target gene as determined via a reporter assay
A reporter assay is a common way to determine the target(s) of a miRNA. The 3'UTR of an mRNA containing specific miRNA binding sites fused to a reporter gene is transfected into cells together with the miRNA. If the mRNA is a bona fide target the miRNA binds to and reduces the expression of the reporter gene. The assay is assessing the action of the miRNA on the target, not how the two entities work together to affect some process, therefore the evidence code should not be IGI. Since the effect of the miRNA can be determined without any perturbation, the evidence code used is IDA. Often the authors will perform a perturbation experiment as well, but this is not required to see the effect of the miRNA on the target.
- For example, in luciferase reporter assays with a construct containing a full-length Snai1 3′UTR sequence, miR-133 transfection strongly repressed the luciferase activity by 60%. Mutations of either predicted miR-133-binding site in the Snai1 3′UTR reduced the responsiveness to miR-133, which was almost absent with mutations of both sites, suggesting direct binding of miR-133 to both sites (Fig.4C).
- Human miR-133a mRNA binding involved in posttranscriptional gene silencing (GO:1903231) PMID:24920580 IDA has_direct_input SNAI1
- Human miR-133a gene silencing by miRNA (GO:0035195) PMID:24920580 IDA regulates_expression_of SNAI1