Exploring the Molecular Etiology of Dominant-Negative Mutations

Herskowitz (1987) provided the classical definition of dominant-negative (DN) mutations as those leading to mutant polypeptides that disrupt the activity of the wild-type gene when overexpressed. Although Herskowitz' definition referred basically to intralocus interactions, it is now recognized that interlocus (e.g., trans-acting) interactions can also lead to dominance (Omholt et al., 2000) and to DN effects (DNEs; Veitia 2002). DNEs are thought to have an impact on plant genome evolution. Indeed, Gibson and Spring (1998) have suggested that paralogous genes encoding multidomain proteins might be overretained in polyploids because mutations in such genes could produce DN phenotypes. From a practical viewpoint, DN mutations have been widely used to study gene function in plants, for example, in situations where null mutations of functionally redundant genes result in no phenotype. Here, I explore the mechanistic foundations for DNEs and some of their quantitative aspects to describe the general features of their effects at the molecular level, improved understanding of which should help in the design of DN proteins. Students are accustomed to simplified concepts of dominance and recessivity. These extreme terms stem from the analysis of qualitative characters for which only a few states are scored. More quantitative studies of the phenotypic effects of a mutation have led to proposals for a more exhaustive classification (Müller, 1932). The terms amorph, hypomorph, and hypermorph have been applied to alleles (and gene products) displaying no activity, lower, or higher activity than the wild-type allele, respectively. As noted above, an important source of dominant phenotypes is the production of a mutant protein that interferes with the action of the wild-type one, and alleles producing these DNEs are called antimorphs. The effects of strong antimorphs, such as those reviewed by Herskowitz (1987), can be easily understood. However, the quantitative dimensions of more complex DNEs require further examination in the era of high-throughput genetics and genomics. The simplest examples of DN mutations come from factors that oligomerize, wherein the mutant polypeptide can poison the complex. For instance, in the context of a dimer AA, if the mutant protein a renders dimers Aa and aa inactive (which is easily conceivable, as we will see below), in the heterozygote there will be production of 25% AA, 50% Aa, and 25% aa, leading to only 25% of activity with respect to the wild type. This is to be contrasted with the case of a heterozygous A/- organism (carrying one wild-type and one loss-of-function allele), which is expected to have 50% activity relative to the wild type. This kind of reasoning has led to the proposal that heterozygous DN mutations cause more severe effects than simple null alleles of the same gene (Strachan and Read, 2004). Moreover, if a is overexpressed, the proportion of dimer containing only normal subunits will decrease. Thus, the current shorthand definition of a DN implies that (1) the phenotype of the heterozygote A/a is worse than that of A/- and (2) overexpression of a leads to a stronger phenotype in the heterozygote than when [A] = [a]. A Typical Example of a DN Mutation. Signal transduction system involving a dimeric receptor whose cytoplasmic portion contains a PK domain. Upon interaction with an extracellular ligand, the PK activity of one monomer phosphorylates the cytoplasmic side of the other monomer. If a mutant monomer lacks the cytoplasmic portion, only 25% of the dimeric receptor will work properly, which in most cases is not enough to elicit a normal response. Other examples involve mutations in structural proteins that form polymers (see a general quantitative treatment for multimeric proteins in the Supplemental Materials online). Mutations in such proteins are frequently dominant, and heterozygous individuals carry a mixture of normal and abnormal protein molecules that copolymerize. For example, a missense mutation in the actin 2 gene in Arabidopsis (act2-2D) leads to defects in the initiation and elongation of root hairs, the elongation of root epidermal cells, and growth in aerial portions. Not surprisingly, in transgenic plants overexpressing the mutant gene, actin filament bundles in root epidermal cells are shorter than in the wild type or in the null mutants (Nishimura et al., 2003). DNEs can also arise in newly formed polyploids and hybrids. During allopolyploid and hybrid formation, the parental genomes do not contribute equally to the transcriptome and the proteome of the resulting individual. The alleles contributed by each parent can be expressed at different levels and often encode different isoforms. Thus, a portion of the changes reported in newly formed polyploids and hybrids, either transcriptional or at another phenotypic level (Adams et al., 2003), could be due to DNEs in the context of multimeric complexes. If we consider a protein working as a homodimer, the orthologous proteins in the two parental lineages might have accumulated mutations that change their primary sequences but not the quaternary structure and the activity of the dimer. However, if the parental lines contribute incompatible forms (i.e., A and a), a tetraploid will have 50% of functionally impaired Aa dimers (i.e., here AA and aa are functional), which might have a phenotypic impact depending on the particular metabolic or signaling system and the environmental conditions. DNE Mutations Can Also Appear in Homodimeric Ligands. (A) Wild-type case. The dimer AA interacts with the receptor with two times more energy than Aa. This translates into a huge difference in terms of affinities (see Supplemental Materials online). (B) Coexpression (equimolar amounts) of a wild-type and a DN subunit unable to interact with the receptor. (C) Sequential interaction between a ligand and its receptor (here, a piece of DNA or RNA with two binding sites). The dimer is formed during DNA/RNA binding. The first monomer interacts with DNA (RNA) with an energy E1, and the newcomer monomer is attracted by both the first monomer sitting on its binding site (E2) and by the nearing free binding site (E1). In sum, the newcomer monomer is attracted more strongly than the first one (with E1+E2). This is a source of cooperativity. The triangle represents a DN protein. The curve representing Y versus protein concentration is a hyperbola ranging from 0 (no occupation of RR) and 1 (saturation) (formal treatment in the Supplemental Materials online). An analysis of this system allows us to explore some quantitative subtleties of DN mutations. We will consider two possibilities: (1) when a has completely lost its binding capacity and (2) when it has retained residual binding. Binding Curves for a Dimer AA (Aa or aa) Interacting with a Target RR. To display the responses of different genotypes on the same graph, Y is represented as a function of protein concentration (A or a) relative to an arbitrary maximal output for each genotype (range between 0 and 1). Of course, the concentrations of A (or a) and of dimers is a function of the strength or duration of a signal. In absolute terms, for any value of x (which depends directly on the intensity of the stimulus), A/- will have two times less monomers and dimers than A/A. As expected, the curve for A/- (light blue) is always lower than that of A/A (dark blue). The position of the curve for A/ a, where a exerts a DNE, depends on the residual activity of a. (A) The monomer a has kept only 1/100 of the intrinsic binding activity of a normal monomer (i.e., almost no binding of aa). It behaves as a canonical DN protein (red curve) and overexpression of a (2× and 5×) with respect to A leads to a consistent decrease in total binding activity (gray and black curves, respectively). When a has lost its activity, binding of Aa or aa is virtually negligible. (B) The monomer a has kept approximately one-third of the intrinsic binding activity of A. The hypomorphic character of a is obvious from the inspection of the curve a/a (i.e., there is binding). However, the curves of A/- and A/a almost coincide, which means that in the heterozygote, a behaves apparently as a null allele. This is explained by a subtle DNE. Overexpression of a leads to paradoxical results. This simple system is also instructive for understanding the interplay between hypomorphy and negative dominance. Let us consider that a has kept residual intrinsic binding activity (i.e., by definition, a is hypomorphic). This means that aa is able to bind the receptor, although more weakly than the wild-type dimer. One interesting hypomorphic allele encodes a monomer a, which has retained a binding capacity of ∼1/3 of the wild-type activity (according to the model underlying the results presented in Figure 3). In this case, the curves for A/- and A/a are virtually superimposable. In other words, the mutant a, which is clearly hypomorphic, behaves as an apparently null allele in the presence of A. This might be explained by a molecular DNE in which part of the binding impairment of Aa is compensated by the residual binding of aa, so that total binding is similar to that of the heterozygote A/-. Of course, alleles encoding proteins with lower intrinsinc (i.e., per monomer) binding capacity will behave more like the traditional DN mutations. DNEs are frequently studied by overexpressing the relevant allele, with the expectation that its overexpression will strongly affect the activity of the wild-type protein. This is always so for the extreme DN alleles (i.e., a is completely inactive). However, when a retains residual activity (i.e., slightly below one-third normal according to the model in Figure 3), a typical DNE is obtained when there is expression of A and a at similar concentrations. Interestingly, depending on the extent of the defect, when a is overexpressed, there may be conditions (two- or fivefold overexpression) that lead to a situation where there is higher binding activity than in A/- or in A/a when [A] = [a] (Figure 3B). Some DN mutations lead to even more complex interactions, as observed in the case of a DN mutation of the PITX2a homeodomain protein that leads to a dominant eye disorder in human (Axenfeld-Rieger syndrome). PITX2a can form homodimers in the absence of DNA, but in its presence, binding is cooperative. Cooperativity in this context refers to the facilitating effect that one monomer bound to DNA (i.e., a form of the receptor RR) exerts on the binding of another monomer. The observation of cooperativity for PITX2a excludes the possibility that all the protein binds DNA as preassembled dimers. PITX2a monomers have a well-documented tendency to form dimers in solution, but when their concentration is low, most of them will recognize DNA as monomers. Then, an incoming monomer can be attracted concertedly by the bound monomer and by a nearby free DNA binding site (Figure 2C). This effect is pronounced at low protein concentrations and generates cooperativity, while at higher concentrations, preassembled dimer will bind DNA preferentially (by definition, in a noncooperative fashion). The DN mutation K88E, which occurs in the DNA binding homeodomain of PITX2a, hinders the interaction of the protein with DNA but not its dimerization. Not surprisingly, the coexistence of the K88E variant (a) with the wild type (A) greatly reduces cooperativity. Indeed, at low protein concentration, only the normal monomer A will recognize DNA and attract preferentially, in concert with DNA, another normal monomer (diluted by the presence of the mutant). The attraction of a monomer a is much weaker as only the monomer A bound to DNA can interact with it (i.e., a cannot see DNA). At higher equimolar concentrations, abnormal dimers (Aa and aa) appear in solution leaving in the end 25% of normal dimers that strongly bind to DNA (Saadi et al., 2003). This example will be discussed further below to see how a defect of one monomer affects not only DNA binding but transcription itself. Trans-DNEs Due to Overexpression. (A) Irreversible assembly of trimer A-B-A allowing intermediate dimers AB and BA (represented). (B) An excess of the molecular bridge B (1.5×) leads to a dramatic decrease in yield of ABA due to the production of intermediates that cannot be completed because of the lack of enough A monomers. More classical trans-DN mutants have been described for Gα subunits of heterotrimeric G-proteins involved in signal transduction. Such mutants have been used extensively to study G protein–coupled receptor (GPCR)–dependent signal transduction. Mutations in different regions of Gα subunits can give rise to trans-DNEs by three different mechanisms: sequestration of the Gβγ subunits, sequestration of the activated GPCR by the heterotrimeric Gαβγ complex, and sequestration of the activated GPCR by nucleotide-free Gα (Barren and Artemyev, 2007). Other Trans-DNEs. (A) Monomers A and B can form homo- and heterodimers. (B) Overexpression of A alters the ratio between the different dimers. (C) Introduction of a strong DN mutant a leads to the production of a substantial amount of nonfunctional dimers (Aa, aa, aB, etc.). DNEs also can arise in the context of transcriptional regulation. For example, a DNE can be obtained by removing the transactivation of a leaving only the DNA binding domain. This can behave as a of This is to in For example, the protein in is expressed as three The polypeptides an transcriptional while the form lacks As the and into homo- and the behaves as a DN et al., This is also the case for and in which are able to in to leads to the of the and them into that signal transduction et al., 2003). In plants, a of proteins function as transcriptional In proteins containing a DNA binding but the transactivation are involved in aspects of epidermal These proteins interact with other and due to the absence of a behave as DN and by inactive and of the DNA binding can also lead to This in As noted above, the gene encodes a DN of this of proteins (with DNA binding and can be expressed However, the expression of containing only the on protein activity A similar is also expected in For instance, the genome of Arabidopsis encodes proteins to bind DNA and proteins with a less than for binding et al., 2003). These binding may function like as negative of proteins the of unable to bind DNA et al., 2003). effects are expected in to the which a DNA binding a and for Arabidopsis encodes all of which are to function as homo- et al., 2004). Some of these are and may lack In a example in plants, et the function of the in signaling the of a DN form of which a transcriptional and to the function of the wild-type protein when expressed in transgenic Moreover, in with has been in the with by transcription are also to gene et al., 2002). of this by or expression of one relative to involved in the same complex can abnormal phenotypes. DNEs in (A) with two binding (gray recognized in a by the A or its form a, which behaves as a (B) Cooperativity may be due to the attraction of an incoming monomer by A sitting on the DNA and a DNA (C) two monomers sitting on their DNA binding will attract the much more strongly than only one monomer bound to is when a monomer lacks the domain. a containing two binding for A. The same binding are also recognized by the variant a, which might as a We that there may be cooperativity during interaction between A molecules with the This might also be so for the interactions between a, and the One source of cooperativity (i.e., A to form dimers in solution, but this is during DNA binding). possibility is that monomers are unable to interact in solution and that interaction of one monomer with DNA leads to an change that the of bound A for an incoming monomer. It is also less that there are no interactions and that binding of one monomer to DNA leads to a change in the site that its for a newcomer monomer. the case, cooperativity means that the A = occurs more than A = Due to the of the molecular that the most to transcription is the by two molecules of This also means that if the for the between the complex and the is the for the of and the will be much higher than the of see To transactivation activity in this we will the and 2 these an for the transcriptional as a function of the concentration of A (and a) can be as described by Veitia and Veitia and (see Supplemental Materials online). a simple in conditions can be (1) the wild-type situation (2) when there is a allele and when there is of A and a a the transactivation domain. In the case, we can two different when the mutation of a cooperativity or when A and a interact we can also explore a situation where the transactivation capacity of a is normal and cooperativity and when cooperativity is normal but transactivation capacity is of a (with to the A or with DN a. The represents as a function of the per allele production of A (a) relative to a maximal The output in terms of concentrations of A (a) depends directly on the strength of a signal that the production of A In the particular case of the heterozygote for any value of it will have two times less A protein than A/A. Thus, the of for the heterozygote A/- at any (light blue) are lower than in the normal A/A (dark but for of is In A/a when a lacks the transactivation capacity in absence of cooperativity between A and a, there is a tendency to with concentrations of A and a, as the to the by other molecules of A When cooperativity between A and a is the of the curve for A/ a is at = as the represents only 25% of the When there is residual transactivation and cooperativity is the in A/a is not at = 1 but at a lower level the curve for A/a that of A/-. This allele a is hypomorphic if the system at low levels of A and a, while it is DN for higher concentrations. are in the Supplemental Materials in A/a when a lacks the transactivation in the absence of to the classical DN definition, the curve is lower at any than that of A/-. However, there is a tendency to with concentrations of A and a. In A to preferentially the as it interactions with incoming A monomers. However, it is that at low protein concentration occurs less in A/a than in the wild-type In a monomer unable to interactions will lead to a DNE. The situation is completely different at the other when cooperativity between A and a is Indeed, the of the curve for A/a is at = This is expected because which transcription (i.e., the of and are represents only 25% of the at A example is provided by an mutation in the This the of the human gene, which contains binding A of containing the DNA binding but the is of a DNE that transcriptional However, this effect is obtained only when the DN is much more strongly expressed and than the wild-type protein et al., 2004). As above, this is may be due to absence of interactions between molecules on this of One or Binding for PITX2a and DN activity in the presence of the wild-type of the wild type and its DN how at low of DNA, the of the with two is times stronger (i.e., than the of the with only one site due to cooperativity and As at of the of the with two binding is of of the wild type The in as expected, less dramatic for the with only one binding and with of the and from and and the for (Saadi et al., 2003). As expected when the transactivation capacity of a is normal and cooperativity a DNE that leads to a to a null allele in a heterozygous of this kind of mutant is an described by and A mutation can affect the levels of cooperativity less An interesting case when cooperativity to approximately of the normal level (according to the underlying the results presented in Figure and transactivation capacity is In these the variant a behaves as a hypomorphic allele in the a/a and as a null allele in A/a (i.e., A/a = not This the lack of between and null When there is residual transactivation (i.e., and cooperativity is the in A/a is not at = 1 but at a lower with capacity can be easily in some The is provided the which contains two regions and involved in the of the transcriptional in the lead to a decrease in transactivation capacity and 2007). A of such an allele with the wild-type should behave as As in Figure the curve for A/a that of A/-. Thus, the same allele can be hypomorphic if the system at low levels the curves each and can be DN in molecular terms at higher protein concentrations. An can be for instance, a protein a that interacts with the of wild-type For a of concentrations the heterozygote A/a will to behave like A/A. However, at only 25% of the will be which interacts with the with in a heterozygote at low protein concentrations it is more to the at of the will be Thus, the curves for A/a and A/- each other at some a are not equally to a DN When there is strong cooperativity and the of a to a DN protein should on the of binding on the The simplest case to is when a lacks a transactivation but interacts with A. If the that transcription is the one with wild-type as above, the maximal can be the the in the Supplemental Materials For a with two binding will be 25% with respect to the wild-type for three binding and for binding A and a are expressed at equimolar For more complex the is not and the analysis of not with The and examples described how DNEs can arise from both and The how the between and other of mutants are not For instance, two different how DN effects can lead a hypomorphic allele to behave as an loss-of-function allele in a Moreover, overexpression of a normal allele can trans-DNEs if the gene interacts with other an of DN mutants in the context of transcriptional the of cooperativity to elicit strong negative effects and that the of to a DN depends on the structure of the A understanding of the mechanistic foundations for DNEs should help in the design of DN proteins to study gene The is in the of this Supplemental of the in I A. and two for their on this and for and for some interesting