Agenda
Bluejeans Conference Line
Review New IGI Documentation
IGI: Inferred from Genetic Interaction
• Genetic interactions such as suppression, enhancement, synergistic (synthetic) interaction, etc.
• Co-transfection experiments in which more than one gene is expressed in a heterologous system to assess functional interaction
• Functional complementation in which a gene from one species is used to complement a mutation in a different species
• Rescue experiments in which expression of one gene rescues the phenotype of a mutation in another gene
• Inference about one gene drawn from the phenotype of a mutation in a different gene??
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
functional complementation experiments, phenotypic rescue, and inferences made about one gene drawn from the phenotype of mutations
in a different 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.
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 different
polysaccharides. Deletion of multiple glucanases in S. pombe results in decreasing efficiency of cell fusion indicating that
each 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 gain-of-
function mutation and a dlk-1 loss-of-function mutation that showed a reduced axon regenerative response when compared to
egl-19(gf) 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
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.
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
Functional complementation
Functional complementation refers to experiments in which a gene from one species complements a mutation in another. 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
Rescue experiments
One way in which functional interactions between two or more genes is assessed is through phenotypic rescue experiments. In these
experiments, the expression of one gene is used to complement, or rescue, the mutant phenotype resulting from mutations in a second
gene. Rescue experiments may be used to help determine the order in which gene products act within a biological pathway or process.
Example
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
When NOT to use IGI
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
Minutes