| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online December 14, 2007; 10.1105/tpc.107.055053 The Plant Cell 19:3843-3851 (2007) © 2007 American Society of Plant Biologists
Exploring the Molecular Etiology of Dominant-Negative Mutations[W]Université Denis Diderot/Paris VII (Unité de Formation et de Recherche/Department of Biology), 75005 Paris, France 1 Address correspondence to veitia{at}cochin.inserm.fr.
Herskowitz (1987)
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 MOLECULAR BASIS OF CANONICAL DN MUTATIONS
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
A textbook example of a DNE at the level of signal transduction involves a membrane receptor whose cytoplasmic portion contains a protein kinase (PK) domain. These receptors often dimerize upon interaction with an extracellular ligand, and the PK activity of one monomer phosphorylates the cytoplasmic side of the other monomer (i.e., cross-phosphorylation), which triggers signal transduction (Figure 1
). In a heterozygous individual expressing one normal allele and one mutant allele encoding a protein lacking the cytoplasmic portion, only 25% of the dimeric receptor will work properly. Often 25% receptor activity is not enough to effectively transduce the signal, and so the actual impact of a DN allele may be greater than that of a knockout allele, where 50% activity might elicit a response (depending on the settings of the particular system and probably on the environmental conditions). The Leu-rich repeat receptor-like Ser/Thr kinase (RLK) ERECTA represents such an example in Arabidopsis. This protein is involved in the regulation of organ shape and inflorescence architecture. A truncated ERECTA protein lacking the cytoplasmic PK domain (
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 AN INTERPLAY BETWEEN HYPOMORPHY AND NEGATIVE DOMINANCE DN mutations also can appear in homodimeric ligands (Figures 2A and 2B ). Indeed, additional quantitative insights into DNEs can be obtained by examining the case of a ligand AA that interacts with a homodimeric protein receptor or a bipartite DNA or RNA sequence (RR). For simplicity, we will assume that dimers AA (and also Aa and aa in a heterozygote) are irreversibly formed and recognize the target RR with the affinity constants KAA, KAa, and Kaa, respectively. Based on this assumption, the activity of AA and its variants, Aa and aa, can be studied quantitatively by assessing the degree of saturation (Y) of their target: Y = [occupied RR]/[total RR]. For simplicity, we will further assume that the biological response is proportional to Y.
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.
When there is coexpression of A and a and the latter has completely lost its binding capacity, according to the example of the dimer given in the introduction, intuition suggests that Y, at any point, will be
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 TRANS-DNEs
Stoichiometric imbalances within a protein complex can further blur the distinction between DN and other alleles such as hypermorphic alleles (Veitia, 2002
More classical trans-DN mutants have been described for G  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 G subunits can give rise to trans-DNEs by three different mechanisms: sequestration of the Gβ subunits, sequestration of the activated GPCR by the heterotrimeric Gβ complex, and sequestration of the activated GPCR by nucleotide-free G (Barren and Artemyev, 2007).
Negative transdominance may also arise in oligomers sharing common subunits. For example, if AA, AB, and BB have different functions, overexpression of A may shift the balance toward a molar excess of AA and AB, with negative consequences for the function of BB (Figures 5A and 5B
). This happens for the mammalian Id helix-loop-helix (HLH) transcription factors (TFs), which lack a DNA binding domain. Id proteins interact with other basic helix-loop-helix (bHLH) proteins that contain both DNA binding and dimerization domains. Not surprisingly, constitutive expression of Id-1, containing only the dimerization domain, strongly deregulates bHLH function (Sun, 1994
DNEs IN TRANSCRIPTIONAL REGULATION
DNEs also can arise in the context of transcriptional regulation. For example, a DNE can be obtained by removing the transactivation domain of a modular TF, leaving only the DNA binding domain. This truncated factor can behave as a competitive inhibitor of transcription. This is known to occur in nature. For example, the C/EBP protein in mammals is expressed as three alternative polypeptides. The longer polypeptides contain an N-terminal transcriptional activation domain, while the short form lacks it. As the long and short isoforms assemble into homo- and heterodimers, the latter behaves as a natural DN (Zahnow et al., 1997
Removal of the DNA binding domain can also lead to DNEs. This happens in bHLH TFs. As noted above, the Id-1 gene encodes a naturally occurring DN inhibitor of this family of TFs. Complete bHLH proteins (with DNA binding and dimerization domains) can be expressed constitutively. However, the regulated expression of Id-1, containing only the dimerization domain, imposes regulation on bHLH protein activity (Sun, 1994
A simple model of transcriptional activation can be used to explore some quantitative subtleties of DN mutations in this context. Studies of viral systems and Drosophila have shown that transcription often displays a sigmoidal relationship with respect to the concentration of a TF. In the case of a system responding to a single type of activator (A), this sigmoidal response can be dissected into two main components: cooperative binding of A to the promoter (p) of a target gene and synergy (Figure 6
). Synergy is the result of the concerted interactions between the molecules of A already bound to the promoter and the transcriptional machinery (Carey, 1998
Consider a promoter, p, containing two binding sites for A. The same binding sites are also recognized by the variant a, which might act as a competitive inhibitor. We assume that there may be cooperativity during interaction between A molecules with the promoter. This might also be so for the interactions between A, a, and the promoter. One possible source of cooperativity was mentioned above (i.e., A tends to form dimers in solution, but this is enhanced during DNA binding). Another possibility is that monomers are unable to interact in solution and that interaction of one monomer with DNA leads to an allosteric change that increases the affinity of bound A for an incoming monomer. It is also possible, though less likely, that there are no A–A interactions and that binding of one monomer to DNA leads to a change in the neighboring site that increases its affinity for a newcomer monomer. Whatever the case, cooperativity means that the reaction pA + A = pAA occurs more readily than p + A = pA.
Due to the existence of synergy, the molecular species that contributes the most to transcription is the promoter occupied by two molecules of activator: pAA. This also means that if the affinity constant for the association between the complex pA and the polymerase is KpolA, the K for the association of pAA and the polymerase will be much higher than 2K (of the order of K2polA; see Zlotnick, 1994 With a rather simple equation in hand, several conditions can be explored: (1) the wild-type situation A/A, (2) when there is a missing allele (A/-), and (3) when there is coexpression of A and a truncated version a lacking the transactivation domain. In the latter case, we can distinguish two different situations: (3a) when the mutation of a abolishes cooperativity or (3b) when A and a interact cooperatively. Finally, we can also explore a situation (4) where the transactivation capacity of a is normal and cooperativity absent, and (5) when cooperativity is normal but transactivation capacity is partial.
Figure 7
shows that a TR, relative to the maximum output of the promoter versus [A] exhibits a sigmoidal relationship, ranging between 0 and 1. Saturation reflects the maximum response of the system, but this does not imply that the promoter functions only at saturation. According to the figure, and in general, the values on the curve for the heterozygote A/- at any point are lower than in A/A (for each value of relative [A], the heterozygote A/- has two times less [A] in absolute terms than the wild type). Interestingly, at low relative concentrations of A, the shift between the curves is very pronounced Y(A/-) is
What happens in A/a when a lacks the transactivation domain in the absence of cooperativity? According to the classical DN definition, the curve is lower at any point than that of A/-. However, there is a tendency to reach saturation with increasing concentrations of A and a. In fact, A tends to occupy preferentially the promoter as it ensures cooperative interactions with incoming A monomers. However, it is apparent that promoter recognition at low protein concentration occurs less readily in A/a than in the wild-type condition. In practice, a truncated monomer unable to ensure cooperative interactions will lead to a weak DNE. The situation is completely different at the other extreme, when cooperativity between A and a is fully maintained. Indeed, the plateau of the curve for A/a is reached at TR = 0.25. This is expected because pAA, which drives transcription (i.e., the contributions of pAa and paa are negligible), represents only 25% of the occupied promoter species at saturation.
A potential example is provided by an artificial mutation in the TF FOXL2. This TF represses the promoter of the human steroidogenic acute regulatory gene, which contains multiple putative binding sites. A version of FOXL2 containing the DNA binding domain but lacking the C-terminal domain is capable of inducing a DNE that impairs transcriptional repression. However, this effect is obtained only when the DN version is much more strongly expressed (5x and 10x) than the wild-type protein (Pisarska et al., 2004
A more telling example is provided in Figure 8
, which represents the response of two different promoters containing one or two binding sites for the TF PTX2a and its DN version, as described earlier (Saadi et al., 2003
As expected intuitively, when the transactivation capacity of a is normal and cooperativity absent, a very mild DNE appears that leads to a behavior close to a null allele in a heterozygous state. Isolation of this kind of mutant is possible using an elegant yeast genetic screen described by Burz and Hanes (2001) . A mutation can affect the levels of cooperativity less dramatically. An interesting case arises when cooperativity drops to approximately one-tenth of the normal level (according to the parameters underlying the results presented in Figure 7) and transactivation capacity is normal. In these conditions, the variant a behaves as a hypomorphic allele in the homozygote a/a and as a null allele in A/a (i.e., A/a = A/-; data not shown). This highlights again the lack of distinct boundaries between hypomophic, DN, and null alleles.
When there is residual transactivation (i.e., 1<Kpola<KpolA) and cooperativity is normal, the plateau in A/a is not reached at TR = 1 but at a lower level. Alleles with partial activation capacity can be easily produced in some cases. The paradigm is provided the yeast TF Gal4, which contains two activating regions (ARI and ARII) involved in the recruitment of the transcriptional machinery. Deletions in the acidic region ARII lead to a decrease in transactivation capacity (Ptashne and Gann, 2002 All target promoters within a cell are not equally sensitive to a DN TF. When there is strong cooperativity and synergy, the sensitivity of a promoter to a DN protein should depend on the number of binding sites on the DNA. The simplest case to visualize is when a lacks a transactivation domain but interacts cooperatively with A. If the promoter species that drives transcription is the one fully loaded with wild-type protein, as assumed above, the maximal TR can be calculated using the formula (of the binomial probabilities) given in the Supplemental Materials online. For a promoter with two identical binding sites, maximum TR will be 25% with respect to the wild-type condition output: for three binding sites, 12.5%, and for four binding sites, 6.25% (when A and a are expressed at equimolar concentrations). For more complex situations, the answer is not intuitive and requires the analysis of models not dealt with here. CONCLUSION The various theoretical and experimental examples described above illustrate how DNEs can arise from both intra- and interloci interactions. The exploration shows how the boundaries between DNs and other classes of mutants are not clear. For instance, two different models demonstrate how DN effects can lead a hypomorphic allele to behave as an apparent loss-of-function allele in a heterozygote. Moreover, overexpression of a normal allele can induce trans-DNEs if the gene product interacts with other (normal) subunits. Finally, an exploration of DN mutants in the context of transcriptional control shows the importance of cooperativity to elicit strong negative effects and that the response of target promoters to a DN TF critically depends on the structure of the formers. A better understanding of the mechanistic foundations for DNEs should help in the design of DN proteins to study gene function.
Supplemental Data
Acknowledgments I thank A. Russo, I. Saadi, and two anonymous referees for their helpful comments on this article and N. Eckardt for comments, editing, and for providing some interesting references. Footnotes
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.055053 REFERENCES
Adams, K.L., Cronn, R., Percifield, R., and Wendel, J.F. (2003). Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proc. Natl. Acad. Sci. USA 100: 4649–4654. Barren, B., and Artemyev, N.O. (2007). Mechanisms of dominant negative G-protein alpha subunits. J. Neurosci. Res. 85: 3505–3514.[CrossRef][Medline] Birchler, J.A., Bhadra, U., Bhadra, M.P., and Auger, D.L. (2001). Dosage-dependent gene regulation in multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev. Biol. 234: 275–288.[CrossRef][Web of Science][Medline] Burz, D.S., and Hanes, S.D. (2001). Isolation of mutations that disrupt cooperative DNA binding by the Drosophila bicoid protein. J. Mol. Biol. 305: 219–230.[CrossRef][Web of Science][Medline] Carey, M. (1998). The enhanceosome and transcriptional synergy. Cell 92: 5–8.[CrossRef][Web of Science][Medline] Deppmann, C.D., Acharya, A., Rishi, V., Wobbes, B., Smeekens, S., Taparowsky, E.J., and Vinson, C. (2004). Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: A comparison to Homo sapiens B-ZIP motifs. Nucleic Acids Res. 32: 3435–3445. Fukazawa, J., Sakai, T., Ishida, S., Yamaguchi, I., Kamiya, Y., and Takahashi, Y. (2000). Repression of shoot growth, a bzip transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12: 901–915. Gibson, T.J., and Spring, J. (1998). Genetic redundancy in vertebrates: Polyploidy and persistence of genes encoding multidomain proteins. Trends Genet. 14: 46–49.[CrossRef][Web of Science][Medline] Herskowitz, I. (1987). Functional inactivation of genes by dominant negative mutations. Nature 329: 219–222.[CrossRef][Medline] Müller, H.J. (1932). Further studies on the nature and causes of gene mutations. In Proceedings of the Sixth International Congress on Genetics, D.F. Jones, ed (Menasha, WI: Banta Publishing Company), pp. 215–255. Nakajima, H., Suzuki, K., and Iwamoto, I. (2003). Lineage-specific negative regulation of STAT-mediated signaling by proteolytic processing. Cytokine Growth Factor Rev. 14: 375–380.[CrossRef][Web of Science][Medline] Nishimura, T., Yokota, E., Wada, T., Shimmen, T., and Okada, K. (2003). An Arabidopsis ACT2 dominant-negative mutation, which disturbs F-actin polymerization, reveals its distinctive function in root development. Plant Cell Physiol. 44: 1131–1140. Omholt, S.W., Plahte, E., Oyehaug, L., and Xiang, K. (2000). Gene regulatory networks generating the phenomena of additivity, dominance and epistasis. Genetics 155: 969–980. Papp, B., Pal, C., and Hurst, L.D. (2003). Dosage sensitivity and the evolution of gene families in yeast. Nature 424: 194–197.[CrossRef][Medline] Pisarska, M.D., Bae, J., Klein, C., and Hsueh, A.J. (2004). Forkhead l2 is expressed in the ovary and represses the promoter activity of the steroidogenic acute regulatory gene. Endocrinology 145: 3424–3433. Ptashne, M. (2007). Words. Curr. Biol. 17: R533–R535.[CrossRef][Web of Science][Medline] Ptashne, M., and Gann, A. (2002). Genes and Signals. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Ramsay, N.A., and Glover, B.J. (2005). MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci. 10: 63–70.[CrossRef][Web of Science][Medline] Saadi, I., Kuburas, A., Engle, J.J., and Russo, A.F. (2003). Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome. Mol. Cell. Biol. 23: 1968–1982. Short, T.W. (1999). Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose. Plant Physiol. 119: 1497–1506. Shpak, E.D., Lakeman, M.B., and Torii, K.U. (2003). Dominant-negative receptor uncovers redundancy in the Arabidopsis ERECTA Leucine-rich repeat receptor-like kinase signaling pathway that regulates organ shape. Plant Cell 15: 1095–1110. Stevens, R.C., and Davis, T.N. (1998). Mlc1p is a light chain for the unconventional myosin Myo2p in Saccharomyces cerevisiae. J. Cell Biol. 142: 711–722. Strachan, T., and Read, A. (2004). Human Molecular Genetics. (Oxford, UK: Oxford University Press). Sun, X.-H. (1994). Constitutive expression of the Id1 gene impairs mouse B cell development. Cell 79: 893–900.[CrossRef][Web of Science][Medline] Toledo-Ortiz, G., Huq, E., and Quail, P.H. (2003). The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15: 1749–1770. Veitia, R.A. (2002). Exploring the etiology of haploinsufficiency. Bioessays 24: 175–184.[CrossRef][Web of Science][Medline] Veitia, R.A. (2003). A sigmoidal transcriptional response: cooperativity, synergy and dosage effects. Biol. Rev. Camb. Philos. Soc. 78: 149–170.[Medline] Veitia, R.A., and Nijhout, H.F. (2006). The robustness of the transcriptional response to alterations in morphogenetic gradients. Bioessays 28: 282–289.[CrossRef][Web of Science][Medline] Yan, C., Wang, P., DeMayo, J., DeMayo, F.J., Elvin, J.A., Carino, C., Prasad, S.V., Skinner, S.S., Dunbar, B.S., Dube, J.L., Celeste, A.J., and Matzuk, M.M. (2001). Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol. Endocrinol. 15: 854–866. Zahnow, C.A., Younes, P., Laucirica, R., and Rosen, J.M. (1997). Overexpression of C/EBPbeta-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer. J. Natl. Cancer Inst. 89: 1887–1891. Zlotnick, A. (1994). To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes. J. Mol. Biol. 241: 59–67.[CrossRef][Web of Science][Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | THE PLANT CELL | PLANT PHYSIOLOGY | |
|---|---|---|---|
-kinase) produces a DNE when expressed under the control of the native ERECTA promoter and terminator. Transgenic plants expressing the mutant 
