transposable elements belong to the Activator-Dissociation and .... the Dissociation locus (Ds) for its propensity ... work on Ac and Ds revealed that both could.
AMER. ZOOL., 29:549-555 (1989)
Maize Transposable Elements in Development and Evolution1 NINA V. FEDOROFF Carnegie Institution ofWashingtOJi, Department of Embryology, 115 West University Parkway, Baltimore, Maryland 21210
SYNOPSIS. Transposable elements were first discovered in maize by Barbara McClintock more than 40 years ago. Today it is apparent that transposable elements are a common component of the genetic material in virtually all organisms. The best studied maize transposable elements belong to the Activator-Dissociation and Suppressor-mutator families. They are short DNA sequences that consist of genes required for mobility and regulation. Both the expression and the mobility of transposable elements are regulated in development by a mechanism that relies on the methylation of element sequences critical for expression. Elements can be stably inactivated by the same mechanism, persisting in the genome in a cryptic form for long periods. The ability of the host organism to regulate the highly mutagenic transposable elements may be critical to their survival, as well as their utility as agents of genomic change. INTRODUCTION
Transposable elements are segments of genetic material capable of moving from place to place in an organism's chromosomes. They are organized for mobility: the genes of a transposable element code for enzymes that cleave and resect cut DNA molecules. Such enzymes mobilize the genes that encode them because they cleave DNA at special recognition sequences that bracket the genes, freeing them from their original chromosomal position and promoting their attachment to a newly cut site somewhere else in the genome. The genes and the bracketing recognition sequences define the transposable element, a self-contained mobile genetic unit. Mobile genes are exceptional. Most of the genes that define an organism have fixed chromosomal positions. Indeed, the fundamental task of genetics throughout the first part of the 20th century, and one that remains central even in an era dominated by molecular biology, is identifying and determining the chromosomal location of genes. Mobile genes are generally inessential to an organism's immediate survival. They serve solely to assure their own mobility. Moreover, the movement of transposable elements often results in the production of additional copies of the ele-
1 From the Symposium on Science as a Way of Knowing—Cell and Molecular Biology presented at the Annual
Meeting of the American Society of Zoologists, 2730 December 1988, at San Francisco, California.
ment. This means that a transposable element can outreplicate the genome of the organism in which it resides, accumulating multiple copies of itself in the organism's chromosomes. Transposable elements are ubiquitous. Although they were first identified in maize plants by Barbara McClintock in the 1940s, it is now apparent that they are present in virtually all organisms in which they have been carefully sought. They can comprise a rather substantial fraction, as much or more than 10%, of an organism's DNA. Moreover, transposable elements are responsible for many, perhaps even a majority, of spontaneous mutations in some types of organisms. They cause a variety of mutations, ranging from insertion mutations and major chromosomal rearrangements to small sequence changes. In sum, transposable elements are common and abundant constituents of the genome, yet have no obvious survival value and are highly mutagenic. As a consequence, it is almost impossible to avoid questions about why there are transposable elements, how organisms survive their deleterious mutagenic activities, and how and why they persist in evolution. But in taking on the question of transposable elements' evolutionary significance, one immediately shoulders the baggage of two extreme and perhaps extremely naive views of biological evolution. At one end of the spectrum is the "phenotypic paradigm," the notion that an organism represents a refined bit of engineering, opti-
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mized inside and out, phenotype to genetic markers located distal to it on the genotype, by natural selection (Doolittle short arm of maize chromosome 9. and Sapienza, 1980). This conviction, McClintock soon understood that an addioverstated here almost to parody, under- tional genetic locus was required to actilies attempts to attribute phenotypically vate the process of chromosome breakage selectable function to all of an organism's at the Ds locus and named it the Activator DNA. At the other end of the spectrum is locus (Ac). In 1948, McClintock reported the "selfish DNA" paradigm, which reaches that the Ds locus could change its chroa limit interpretation where the organism mosomal location or transpose from one is viewed as the genes' way of perpetuating chromosomal position to another themselves. One need not approach this (McClintock, 1948). This was the first extreme to construct a sensible argument report that a genetic locus could move. for transposable elements as quintessential Over the next few years, McClintock's "selfish DNA" (Orgel and Crick, 1980). work on Ac and Ds revealed that both could In the following narrative, I will sum- transpose into genes, causing a familiar kind marize what has been learned about the of unstable mutation, known in many by-now classical transposable element fam- organisms for its ability to cause variegailies identified and studied genetically by tion. But it was the results of McClintock's McClintock. I will discuss what has been careful genetic experiments that explained understood through both genetic experi- the variegation. McClintock showed that mentation and, more recently, through the when Ds moved away from its initial posistructural and functional analysis of trans- tion, its movement was occasionally assoposable element DNA sequences cloned ciated with the origin of a mutation that from the maize genome using recombinant prevented pigmentation from developing DNA techniques. I will avoid the quagmire in the maize plant and kernel. The mutaof evolutionary arguments to the best of tion was unstable. It reverted during plant my ability without side-stepping the central and kernel development to give a varieissue. This I perceive to be not how trans- gation pattern, with pigmented (wildtype) posable elements came to be, but how tissue sectors arising in an unpigmented organisms and elements co-exist, in the (mutant) background. The unstable mutashort and long run, in a developmental and tion and the newly inserted Ds element, as in an evolutionary time frame. That they judged by the new position of chromosome co-exist (and, therefore, co-evolve) is a fact. breakage, coincided with the c locus, which How they co-exist is decipherable and was known to be a gene whose correct functherefore describable. But I suspect that to tion was necessary for pigment production. ascribe purpose or evolutionary motive in Moreover, the instability of the mutation the sense inherent in either the "pheno- was conditional. It was unstable only in the typic" or the "selfish DNA" paradigm (or presence of Ac. any mixture or modification thereof) may McClintock identified and then analysed fulfill a human need for purpose more than the occasional progeny plants in which the it explains. unstable mutation had reverted in the germline, heritably restoring normal gene function and pigmentation. Such progeny T H E STRUCTURE AND REGULATION OF plants no longer showed chromosome MAIZE TRANSPOSABLE ELEMENTS breakage at the c locus in the presence of The Activator-Dissociation element family Ac. This implied that reversion of the McClintock identified the first transpos- mutation was associated with the transpoable element by its ability to cause chro- sition of Ds to yet another new location. mosome breakage at its site of insertion McClintock therefore concluded that the (for reviews, see McClintock, 1951; Fedo- mutation arose by transposition of Ds into roff, 1983, 1989a). She initially named it the c locus and that its somatic instability the Dissociation locus (Ds) for its propensity in the presence of Ac could be explained to promote the dissociation and loss of by the transposition of the element away
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from the locus during development of the plant. McClintock subsequently established that both Ac and Ds could cause insertion mutations, although the mutations had slightly different properties. Mutations caused by Ac, unlike those caused by Ds, were inherently unstable, suggesting the Ac could promote its own transposition. Moreover, an Ac mutation could occasionally change into a Ds-like mutation directly, suggesting a close relationship between the two kinds of transposable elements. McClintock further showed that there were several different kinds of Ds elements, some of which could and some of which could not break chromosomes. The DNA sequences corresponding to Ac and several different Ds elements have been cloned during the past few years (reviewed in Doring and Starlinger, 1986; Fedoroff, 1989a). The two Ac elements whose nucleotide sequence has been determined are identical. Ac is a DNA sequence of 4,563 nucleotides, whose ends comprise an inverted repetition (IR) of the 11 base pair (bp) sequence CAGGGATGAAA. The element appears to consist of a single gene. The gene codes for an enzyme, called a transposase, required for transposition of the element. The element-encoded transposase also promotes transposition of Ds elements. Ds elements were denned genetically by their ability to transpose and break chromosomes in the presence of an Ac element. The Ds elements that have been cloned and analysed comprise a structurally heterogeneous group of DNA sequences. Some Ds elements are clearly derived from Ac elements by internal deletions of parts of the element sequence coding for the transposase. Such elements cannot move because they no longer code for a functional transposase. But they remain capable of moving when transposase is supplied by a structurally intact Ac element located somewhere else in the genome. The Ds elements that break chromosomes have a unique structure. They are double elements consisting of one short, internally deleted Ac element inserted in inverted orientation directly into the middle of a second copy of the
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same element. In addition to transposing and causing chromosome breakage, such double Ds elements have been implicated in a variety of major chromosomal rearrangments, including duplications, deletions and translocations. Additional Ds elements have been identified whose resemblance to the Ac element is much more limited. Indeed, there are elements that transpose in the presence of an Ac element whose sequence similarity to Ac does not extend far beyond the IRs (Doring and Starlinger, 1986). The known functions of the Ac element are all connected with its ability to transpose and appear to be attributable to the activity of the element-encoded transposase. Ac's ability to promote transposition of Ds elements, as well as break chromosomes at Ds insertion sites and promote chromosomal rearrangements, is disrupted by mutations that simultaneously eliminate the element's ability to move itself. However, there is evidence that an element-encoded gene product is involved in negative regulation of transposition frequency. That such a regulatory mechanism exists has been inferred from the observation that Ac elements transpose less frequently and later in plant development as the number of elements in a plant increases. Because mutations that eliminate the element's ability to transpose also eliminate its ability to contribute to this so-called "dosage effect," it may be that the same gene product is involved in both transposing and regulating transposition frequency. An additional negative genetic regulatory mechanism affects the ability of the Ac element to move and promote the transposition of Ds elements. McClintock observed that the Ac element could undergo a reversible inactivation which simultaneously eliminated all of its genetically detectable activities. An inactive Ac element is genetically indistinguishable from a Ds element, but is structurally unaltered and can return to an active form. Active and inactive Ac elements differ in the extent of methylation of certain cytosine residues near the end of the element containing the beginning of the transposase gene. As
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described in greater detail for the maize Suppressor-mutator transposable element below, methylation of specific element sequences appears to be the molecular mechanism by which the plant regulates the element's activity. The Suppressor-mutator element family The second family of transposable elements identified and studied in detail by McClintock is designated Suppressor-mutator (Spm; for reviews, see McClintock, 1965; Fedoroff, 1983, 1989a). The Spm element family, like the Ac-Ds element family, comprises elements that can transpose autonomously, as well as elements that are mobile only in the presence of a fully functional Spm. The Spm element is about twice as large as the Ac element (8.3 kilobases) and is bracketed by the 13 bp IR sequence CACTACAAGAAAA. The Spm element is known to have at least two genetic functions, one of which is a transposase. The correspondence between the element's genetically defined functions and its genes is not yet clear. There is some reason to suspect that the element has two partially overlapping genes. Unlike what is observed in the Ac-Ds element family, most transposition-defective Spm (dSpm) elements are simple derivatives of the complete element with internal deletions of various sizes. The Spm element is automutagenic, hence an element-encoded protein is involved in generating the internal deletions that give rise to dSpm elements. Intraelement deletions commonly reduce or abolish the element's ability to both transpose itself and promote the transposition of dSpm elements located at other chromosomal positions. This observation provides the evidence that an element-encoded gene product is necessary for transposition of the element. dSpm element insertions into genes affect gene expression in different ways. In some cases, expression of the gene is either affected only slightly or reduced by the insertion mutation, while in other cases it is completely abolished. Expression of a mutant gene with a dSpm insertion is often different in the presence of a fully functional Spm element elsewhere in the
genome than it is in its absence. The expression of some mutant genes is inhibited by the Spm element, while expression of others is enhanced. Studies on the interactions between a functional Spm element and mutant genes with dSpm insertions led to the formulation of the hypothesis that expression of such a mutant gene can come under the control of the molecular system that regulates expression of the element itself (Masson et al., 1987). The properties of certain mutant genes suggested that the Spm element carries a gene for a regulatory molecule, probably a protein, that is necessary for the element to remain in an active form. As described above for the Ac element, Spm elements undergo reversible changes between the active and inactive forms. Genetic and molecular studies on active and inactive elements have provided further insight into the regulation of element activity (reviewed in Fedoroff, 19896). Genetic studies on Spm elements undergoing inactivation have revealed that the responsible genetic mechanism is incremental, heritable, and capable of imposing a variety of differential patterns of element expression in the plant's developmental cycle. The Spm element can exist in one of three genetically distinguishable forms: 1) a stably and heritably active form, 2) a programmable form in which it shows differential expression during development, and 3) a stably and heritably inactive form, termed cryptic (Banks et al., 1988). Elements in the programmable form can exhibit one of many heritable programs or patterns of differential expression within the plant. Two distinct components have been discerned in the programming mechanism: 1) the phase setting and 2) the phase program. The phase setting determines whether the element is active or inactive, while the phase program governs the heritability of the phase setting, as well as its pattern of reversal during development. Molecular studies have revealed that active, programmable and cryptic elements can be distinguished by the level of methylation of cytosine residues in the sequence at the beginning of an element-
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encoded gene. Cryptic Spm elements are almost fully methylated both in the vicinity of the beginning of the gene and throughout the element. Elements in the stably and heritably active form are methylated at cytosine residues throughout most of the element's sequence, but not near the beginning of the gene. Programmable Spm elements are methylated at a subset of possible sites near the beginning of the gene and fully methylated throughout the rest of the element. Thus it appears that element methylation is closely correlated with the element's expression pattern. Partially methylated, programmable elements undergo changes in their expression potential during development. Such elements, for example, are much more likely to be expressed in ears produced on tillers than they are in ears produced on the plant's main stalk. Moreover, an element is more likely to be transmitted in an active form through female, than through male, gametes. These differences are reflected in the methylation levels of elements in different plant parts. Although a given expression program is relatively heritable, it can change. There are both stochastic and directed changes in an element's program. Stochastic changes are evidenced by plant-to-plant differences in the developmental expression pattern which can, in turn, be heritable. The two sources of directed change reside in the plant itself and in the interaction between active and inactive elements. Spm elements in the programmable form can not only show a differential pattern of expression in development, but can also be re-programmed during development without undergoing a change in phase setting. It has been observed, for example, that an inactive element is much more likely to be reactivated in the next plant generation if it is transmitted through tiller gametes than if it is transmitted through the gametes produced on the main stalk of the same plant. An active Spin element in the same genome with an inactive programmable Spm element activates the latter. This supports the hypothesis articulated earlier that the Spm element encodes a positive regulatory
gene product. The interaction between the inactive element and the positive regulatory gene product provided by the active element also promotes re-programming of the element. For example, an inactive programmable Spm has a higher probability of being transmitted to progeny in an active form from a plant that contains a second active element than from a plant that does not. Once again, such changes in programming are correlated with the extent to which sequences at the beginning of the element's gene sequence are methylated. The same genetic mechanism appears to be responsible both for the different developmental programs of gene expression and for enforcing the inactivity of the cryptic Spm element. A cryptic Spm element is not active in the presence of a second element, but the presence of the active element increases its likelihood of undergoing spontaneous activation. A cryptic Spm element undergoes spontaneous reactivation in about 1 in 100,000 plants. By contrast, activation of a cryptic Spm is observed in up to 1 in 100 plants containing an active element. Moreover, partly activated cryptic Spm elements show patterns of differential activity in development. That is, they become programmable Spm elements. DISCUSSION
It is becoming increasingly apparent that the activities of the transposable elements Ac and Spm are subject to a striking form of regulation. Both elements appear to be susceptible to an inactivation mechanism associated with, and probably mediated by, methylation of element sequences critical to expression. Although little is understood about how the Ac element maintains its ability to be expressed, the results of recent studies on the Spm element have established that a gene product encoded by the element itself is required to keep the element active. The emerging role of this positive regulator is double. It appears able to activate an inactive element, so long as the element is not in the very deeply inactive cryptic form. In addition, the Spm's regulatory gene product serves to promote the heritable reactivation of the element. The genetic properties of the Spm ele-
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ment's inactivation mechanism imply that the underlying molecular events are both heritable and reversible. They also suggest that multiple events contribute incrementally to establishing and maintaining the inactive state. The different element forms have been found to be distinguishable on the basis of the extent of methylation near the beginning of an element gene sequence. Moreover, the genetic properties of the inactivation mechanism are just those anticipated if multiple independent methylation events can occur and if the stability of the inactive state is proportional to the extent of methylation of a region containing multiple methylation-sensitive sites. It appears likely, therefore, that differential methylation of a sequence containing multiple methylatable sites determines the element's pattern of expression. The important observations that an Spm element-encoded gene product both activates expression of a partially methylated inactive element and promotes its demethylation and heritable activation explain how the element maintains itself in a heritably active form. Once activated, an element produces the gene product that is required to maintain it in an active form. That is, the element encodes a positive autoregulatory gene product which activates the element and maintains it in a heritably active form. The observed regular differential patterns of element expression in development bear the implication that different plant parts differ in their ability to impose and maintain element-inactivating methylation. Differences in element activity during development correlate well with the position on the plant of the meristem producing the reproductive structure that transmits the element to progeny. This observation, in turn, suggests that the element has come under the control of a basic genetic mechanism involved in determination of the developmental fate of a given meristem. Simply stated, then, it appears that the Spm element is negatively regulated by a genetically stable, but reversible epigenetic mechanism mediated by methylation of
multiple cytosine residues within an element sequence critical for its expression. Countering the plant's ability to inactivate it is an S/wi-encoded positive autoregulatory mechanism which not only activates element expression, but serves to reverse the heritable, methylation-associated, inactivation of the element. The importance of this mechanism in a developmental time frame is that it makes it possible to program an element in such a way that it is expressed in many cells of many tissues, but that its activity in one or more than one germline is minimized to avoid heritable genetic damage to either the element itself or the plant genome. Minor changes in the element's expression pattern appear to arise continuously. Some affect the timing and tissue-specificity of element expression. Thus there exists the constant possibility of reprogramming the element to permit earlier element expression and transposition. It is perhaps equally significant that transposable elements can exist in a cryptic form. In view of the propensity of active elements to mutate by self-inflicted intraelement deletions, their long-term survival in a genetically competent form may be enhanced by or depend on the plant's ability to stably inactivate the element. Both the early observations of McClintock and more recent experiments carried out by other investigators suggest that cryptic transposable elements become active under conditions that favor chromosome damage. This may be attributable to altered patterns of DNA synthesis and methylation associated with the repair of chromosomal damage. The fact that transposable elements promote extensive genetic change in an organism and become active under conditions that stress the genome may provide the key to their retention, especially in rapidly evolving organisms such as maize. Indeed, the properties of the elements and of the positive and negative genetic mechanisms that govern their activity suggest that transposable elements are a stress-inducible, self-limiting system for rapid genome change.
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Fedoroff, N. 19896. About maize transposable elements and development. Cell 56:181-191. Banks, J., P. Masson, and N. Fedoroff. 1988. Molec- Masson, P., R. Surosky, J. Kingsbury, and N. V. Fedoular mechanisms in the developmental regulation roff. 1987. Genetic and molecular analysis of the of the maize Suppressor-mutator transposable eleSpm-dependent a-m2 alleles of the maize a locus. ment. Genes Dev. 2:1364-1380. Genetics 177:117-137. Doolittle, W. and C. Sapienza. 1980. Selfish genes, McClintock, B. 1948. Mutable loci in maize. Carthe phenotype paradigm and genome evolution. negie Inst. Wash Yrbk. 48:155-169. Nature 184:601-603. McClintock, B. 1951. Chromosome organization and Doring, H.-P. and P. Starlinger. 1986. Molecular genie expression. Cold Spring Harbor Symp. genetics of transposable elements in plants. Ann. Quant. Biol. 16:13-47. Rev. Genet. 20:175-200. McClintock, B. 1965. The control of gene action in Fedoroff, N. 1983. Controlling elements in maize. maize. Brookhaven Symp. Biol. 18:162-184. In J. Shapiro (ed.), Mobile genetic elements, pp. 1- Orgel, L. E. and F. H. C. Crick. 1980. Selfish DNA: 63. Academic Press, New York. The ultimate parasite. Nature 184:604-607. Fedoroff, N. 1989a. Maize transposable elements. In M. Howe and D. Berg (eds.), Mobile DNA. ASM Press, Washington. (In press) REFERENCES
