Genetic Techniques for Biological Research
Corinne A. Michels
Copyright q 2002 John Wiley & Sons, Ltd
ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
6 Epistasis Analysis
OVERVIEW
The Random House Dictionary of the EnglishLanguage-Unabridged Edition (1966)
defines epistasis asa genetic term describing the‘interaction between nonallelic
genes in which one combination of such genes has a dominant effect over other
combinations’.The key word to remember is ‘nonallelic’, i.e. different genes.
Epistasis (from the Greek meaning ‘stand above’) is the masking of the phenotype
of a mutation in one gene by the phenotype of a mutation in another gene (Huang
& Sternberg, 1995). One geneis said to be epistatic to another when the double
mutant strain exhibits the phenotype of that mutantgene. This is in clear contrast to
the terms dominant and recessive, which describe the relationship between different
alleles of the same gene. It is very important not to confuse these concepts.
Epistasis analysis is used to determine if genes with related mutant phenotypes act
in the same or different pathways, and, if in the same pathway, to place them in a
linear order relative to one another based on the step in the pathway controlled by
that gene. In otherwords,one
uses epistasis analysis to construct an order-offunction map that reflects the sequence of events in a pathway controlled by several
genes.
The use of epistasis analysis for the study of complex pathways was suggested
moreor less simultaneously by twoindependent
research groups working in
different fields. Jarvik & Botstein (1973) reportedthe isolation of temperaturesensitive and cold-sensitive mutationsthat block phage P22 morphogenesis, i.e.
assembly of the phage particle. They used a combination of double mutant studies
and reciprocal temperature shifts (made possible by their use on conditional ts and
CS mutants)todeterminetheorder
of events in phage assembly. Theirwork
demonstrated that head and tail assembly were independent processes but that both
were dependent on phage DNA replication. Hereford & Hartwell (1974) used
epistasis analysis to order events in the Saccharomyces cell division cycle. They used
temperature-sensitive mutants that blocked the cellcycle at morphologically distinguishable points. The execution points of these genes were ordered relative to the
block produced by the cell cycle inhibitor a-factor by temperature-shift experiments
and relative to each other by double mutant studies.
To determine the epistatic relationship
between two genes, mutations in these genes
must have distinguishable phenotypes. Epistasis analysis is undertaken only after the
initial steps of genetic analysis. Mutationsare isolated and placed into complementation groups. Then, representative alleles are selected for a detailed characterization of phenotype so as to reveal subtle differences in phenotype not obvious
from initial characterization. This could be a complete morphological analysis, such
as in secretory pathway mutants or cell division cycle mutants, or intensive biochemical characterization, such as for DNA replication mutants or mutantsaffecting
metabolicpathways.
Occasionally, different alleles exhibit somewhat different
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phenotypes. The researcher can now capitalize on these phenotypic differences in the
epistasis analysis.
For the purposes of epistasis analysis, there are two types of pathway in living
systems, substrate-dependentpathways and switchregulatorypathways (Huang &
Sternberg, 1995). A substrate-dependent pathway consists of an obligate series of
steps or reactions that are required to produce a final outcome. The outcome can be
as simple as the synthesis of a nutrient such as an amino acid or a macromolecule,
or can be as complex as the formation of aribosome.Thesubstrate-dependent
pathway can be thought of as a progression of events or even as a river flowing
downstream with separate tributaries joining at different points and finally flowing
into the lake. Another view is as a series of positive reactions each dependent on a
source of substrate and a functional gene product for the successful completion of
each step in the pathway. Moreover, the product of the more upstream step is used
as the substrate of the downstream step. If there is no substrate available or if any
one of the enzymes is missing or inactive, then the pathway will be blocked at that
step. A production line at a factory would be considered to be this type of pathway.
It is dependent on the input of parts (substrates) and workers (gene products) to
assemble these parts.
A switch regulatory pathway consists of a series of genes or gene products that
alternate between two states, ‘on’ and ‘off’. The components of this pathway are
usually acting directly on each other as opposed to
on substrates, as occurs in a
substrate-dependent pathway. The activity of a switch regulatory pathway is regulated by an upstream signal that stimulates the pathway and produces the downstream response. Environmental changes, cell-cell interactions, zygote formation,
and mitogenic signals are only a few of the signals that can act as initiators of a
switch regulatory pathway. The downstreamresponse can be altered gene expression,
cell division, or the initiation of a developmental process such as pattern formation.
Mutations in the genes encoding components of a switch regulatory pathway can
lock the component into a permanently ‘on’ or permanently ‘off’ state. This has the
effect of separating the downstream response from the initiating signal. Mutations
that allow the response to be produced even in the absence of a stimulatory signal or
despite the presence of an inhibitory signal are referred to as constitutive mutations.
The isolation of constitutive mutations is a strong indicator that one is dealing with
a switch regulatory pathway.
In a switch regulatory pathway the actionof a particular component,or regulatory
factor, can be either positive or negative. The function of a positive regulatory factoris
to activate the next component in the pathway (its downstream component) when it
is in its active form. A recessive (loss of function) mutation in a gene encoding a
positive regulatory factor blocks the pathway. A dominant (gain of function) mutation in a gene encoding a positive regulatory factor produces a protein capable of
functioning constitutively even in the absence of upstream activation of the pathway.
A negative regulatory factor in the activated state inactivates the next downstream
component in the pathway. Therefore, arecessive, loss of function mutation in a gene
encoding a negative regulatory factor allows the next step in the pathway to be
constitutively active. A dominant, gain of function mutation in a gene encoding a
negative regulatory factor produces a gene product capable of constitutively inhibiting thepathway evenin the absence of the signal. By determining whether the
ANALYSIS
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mutation is dominant or recessive, constitutive or blocks the response, the geneticist
can decide whether the gene product is a negative or positive regulatory factor.
Determining whether the pathway under investigation is a substrate-dependent or
switch regulatory pathway is not a simple task. Nonetheless, as will be seen below, it
is important because the interpretation of the results of double mutant analysis
depends on the type of pathway. The characterization of mutant alleles of pathway
genes canprovide some clues. As discussed above, if constitutivemutantsare
obtained in any of the pathway genes, then one can conclude that the pathway is a
switch regulatory pathway.Moreoften
thannot, a switch regulatorypathway
controls more than one downstream response. As a result mutations in a switch
regulatory pathway affect a number of phenotypic traits, such as the expression of
several genes, andare said to be pleiotropic. The identification of pleiotropic
mutants is suggestive of a switch regulatory pathway. If no constitutive mutations in
pathwaycomponents
have been identified, onecan proceed with an epistasis
analysis undertheassumption
that one is dealing with asubstrate-dependent
pathway. For complex processes this is unlikely to be the case. More likely, the
initial characterization of mutations has not uncovered all the genes in the pathway
or isolated a sufficiently varied array of mutant alleles. As the genetic analysis of the
pathway proceeds newgenes and/or newalleles willbe identified and thetrue
character of the pathway will be revealed in full detail. This will become clearer as
examples from the literature are discussed.
EPISTASIS ANALYSIS OF A SUBSTRATE-DEPENDENT
PATHWAY
Let us say that GENl and GEN2 are related because mutations in both genes
decrease the production of Z. Mutations in GENl give phenotype A, and mutations
in GEN2 give phenotype B. Only recessive loss of function allelesof GENl and
GEN2 have been isolated. No constitutive allelesof GENl or GEN2 have been
identified and mutations in these genes alter Z production but appear not to affect
otherphenotypes. We assumethat this is asubstrate-dependentpathway
and
proceed with the epistasis analysis.
The four mechanisms of genetic interaction between GENl and GEN2 are given
in Table 6.1 (based on Hereford & Hartwell, 1974) and the resulting phenotype of
the single or double mutation with regard to production of Z is indicated.
Models 1 and2:Proteins Genlp andGen2p participate in different steps of the same
pathway and protein Genlp acts in a step that is upstream (Model 1) or downstream (Model 2) of the step catalyzed by protein Gen2p.
Model 3: Proteins Genlp andGen2parecomponents
parallel pathways for Z production.
of two independent and
Model 4: Proteins Genlp and Gen2p act at the same step and in conjunction with
one another.
To determine the epistatic relationship between these two genes, one constructs a
strain that is mutant at bothgenes and observes the phenotype of the double mutant
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Table 6.1 Epistasisanalysis of asubstrate-dependentpathway
Phenotype of
gen2
single
Phentotype of
genl gen2
double
A
B
A
A
B
B
A
B
Unique
A
B
Phenotype of
genl
single
mutationmutationmutation
~~
Model 1
GEN2
GENI
Model 2
GENIGEN2
Model 3
~
A
d
-
GENI
-----W
GEN2
Model 4
GENI, GEN2
P
=B =A
or
unique
strain. If the double mutant phenotype is A, then GENI is epistatic to GEN2 and
GENI encodes the upstream component in the pathway. Alternately, if the double
mutant phenotype is B, then GEN2 is epistatic to GENI and GEN2 encodes the
upstream component. In summary, in a substrate-dependent pathway, if the double
mutant exhibits a phenotype identical to one or the other mutant genes, then that
gene is epistatic and encodes the more upstream component in the pathway.
If Model 3 or 4 describes the relationship, the results can be more difficult to
interpret. The term ‘unique’ used in Table 6.1 indicates that thephenotype is
different from either phenotype A or B. It can be qualitatively related to phenotypes
A and B but quantitatively more extreme. For example, mutations in two different
RAD genes might partially reduce the rate of recombination but to different extents,
while thedoublemutant
completely blocks all recombination.This
type of
interaction is called enhancement and will be discussed in detail in Chapter 9.
Another classic example of two parallel pathways affecting a single trait comes from
Drosophila. The reddish brown eye color results from a mixture of two pigments
synthesized by parallel pathways. Mutations in one of these pathways that blocks
red pigment productionproducesbrown
eyeswhile mutations in thealternate
pathway that blocks brown pigment production produces red eyes. Flies defective
for the production of both pigments, that is double mutants, have white eyes. White
eyes is a unique phenotype and could not have been predicted from observing the
phenotypes of the single mutants. The double mutant in Model 4 might exhibit a
unique phenotype or could be phenotype A or B. What will distinguish Model 4
from Models 1 and 2 is that the phenotype of the double mutant is likely to vary
with the alleles. Thus, epistasis analysis has limitations. The researcher will have to
proceed to other methods, such as suppressor and enhancer analysis or coimmunoprecipitation, to support a proposed model.
EPISTASIS ANALYSIS OF A SWITCH REGULATORY
PATHWAY
In a switch regulatory pathway, the epistatic
gene encodes the downstream component. Because this
type
of pathway involves both negative and positive
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EPISTASIS ANALYSIS
components, it is not possible to come up with a simple table describing all possible
results. Instead, several examples of hypothetical switch regulatory pathways will be
presented for the reader to ponder. Then some sample results will be described and
the reader can practice analytical skills. In the pathways given below, an arrowhead
indicates thatthe action of theprotein or signal is positive anda vertical line
indicates that theaction of theprotein or signal is negative. For each pathway
the reader should determine the phenotype of mutants in each protein component.
The classes of mutationstoconsiderare
alleles thatput the component in the
permanently ‘on’ state and thosethatput
it in the permanently ‘off’ state.The
possible phenotypes are constitutive or no response produced in the presence of
signal.
- - - -
Regulatory pathway 1
Signal
Protein A
Regulatory pathway 2
Signal
Protein X
Protein C
Response
+Protein Y +Protein Z
Response
Protein B
The experiments listed below present the results of epistasis analysis of mutations
in genes GENl, GEN2, GEN3,and GEN4. Mutations in these genes affect the same
process and may be in a common pathway.A number of alleles of each are available
including constitutive alleles. Experiments 1-5 provide an example of an epistasis
analysis of a switch regulatory pathway. Use these results as a practice exercise.
Determine whether each gene encodes a positive or negative regulator and the
order-of-function of the genes in thepathway. As a guide, the results of each
experiment are followed by their interpretation. Regulatory pathway 3 synthesizes
these conclusions into an order-of-function map of the pathway.
Experiment 1: A recessive mutation in GENl does not respond to the signal. A
recessive mutation in GEN2 is constitutive. A strain carrying both mutations (genl
gen2) is constitutive. (Conclusions: Genl protein is a positive regulator.Gen2
protein is a negative regulator. GEN2 is epistatic to GENI and the product of GEN2
acts downstream of the product of GENI.)
Experiment 2: A recessive mutation in GEN2 is constitutive. A recessive mutation in
GEN4 blocks the response to the signal. A strain carrying both mutations
(gen2
gen4) doesnotrespond
to the signal. (Conclusions: Gen4protein is a positive
regulator. GEN4 is epistatic to GEN2 and the product of GEN4 acts downstream of
the product of GEN2.)
Experiment 3: A dominantconstitutive alleleof GENl is identified. A recessive
mutation in GEN3 blocks the response to the signal. A strain carrying both mutations (GENI-c gen3) does not respond to the signal. (Conclusions: Genl-c protein is
an activated form of the protein that is locked in the ‘on’ state. Gen3 protein is a
positive regulator. GEN3 is epistatic to GENl indicating that the product of GEN3
acts downstream of the product of GENI.)
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Experiment 4: A dominantconstitutive allele of GEN4 is isolated. A recessive
mutation in GEN3 blocks the response to the signal. A strain carrying both mutations (gen3GEN4-c) is unable to respond tothe signal. (Conclusion:GEN3 is
epistatic to GEN4 and the product of GEN3 acts downstream of the product of
GEN4.)
Experiment 5: A dominantconstitutive allele of GEN3 is isolated. A recessive
mutation in GEN4 blocks the response to the signal. A strain carrying both mutations (GEN3-c gen4) does not respond to the signal. (Conclusion: GEN4 is epistatic
to GEN3. This result taken together with the results of Experiment 4 suggests that
the products of GEN3 and GEN4 could act at the same step.
- - -
Regulatory pathway 3
Signal
GENl
GEN2
GEN3, GEN4
-
Response
EPISTASIS GROUP
Genes encoding proteins that function in the same pathway or process will exhibit
epistasis, as defined in the discussion presented above. Such genes are said to be
members of an epistasis group. A well-studied example of an epistasis group is the
RAD52 epistasis group (Paques & Haber, 1999). Mutations ingenes
such as
RADSO, RAD51, RAD52, RAD54, RAD.55, RAD57, RAD59, SPO11, and M R E l l
exhibit defects in recombination and double-strand break repair. Construction
of
doublemutant
strainsdemonstratesepistaticrelationshipsamongthevarious
members of this group of genes. Mutations in RAD3 or RAD6, which like RAD52
were originally isolated because of their increased sensitivity to X-ray radiation, do
not exhibit epistasis with the RAD52 epistasis group or with each other. When
paired with RAD52 or other members of the RAD52 epistasis group, the results are
consistent with Model 3 in Table 6.1 and clearly indicate that RAD3 and RAD6 are
in distinct pathways. Thus, despite certain similarities in phenotype, RAD52, RADS,
and RAD6 are members of different epistasis groups.
REFERENCES AND FURTHER READING
Avery, L. & S. Wasserman (1992) Ordering gene functions: the interpretation of epistasis in
regulatory hierarchies. Trends Genet. 8: 3 12-316.
Botstein, D. & R.Maurer
(1982) Genetic approaches to the analysis of microbial
development. Ann. Rev. Genet. 16: 61-83.
Hereford, L.M. & L.H. Hartwell (1974) Sequential gene function in theinitiation
of
Succhuromyces cerevisiae DNA synthesis. J. Mol. Biol. 84: 445-461.
Huang, L.S. & R.W. Sternberg (1995) Genetic dissection of developmental pathways.
Methods Cell Biol. 4 8 : 97-122.
Jarvik, J. & D. Botstein (1973) A genetic method for determining the order of events in a
biological pathway. Proc. Nut1 Acad.Sci. USA 70: 2046-2050.
Paques, F. & J.E. Haber (1999) Multiple pathways of recombination induced by doublestranded breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63: 349-404.