How maize transposable elements escape negative selection

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How maize transposable elements escape negative selection

T h e concept of transposable genetic elements was developed by Barbara McClintock four decades ago, through genetic studies in maize. Soon after the molecular isolation of transposons from E. coil, it became apparent that transposable elements are natural components of most genomes, found in representatives of virtually all organisms. Only then did the importance of ALFONS GIERL McClintock's discoveries become widely recognized. The molecular analysis of maize transposable ele- The transposable element systems En/Spm atul Ac affect ments began less than ten years ago. Representatives geuc structure and control the expression of geuss. In of many types of mobile elements have been found, some cases, the deleterious consequences of tnserffonal and can be grouped into two classes. The first class mutagetws/s are reduced because certain members of comprises elements that transpose by reverse tran- thesefamilies of elements mimic introns. Thepotential scription of an RNA intermediate. This category can be he.fits of such imterac~ns, and a multilevel control of divided into retrovirus-like elements, which are transposititm activity, might explain 'survival' of these flanked by long direct terminal repeats, and nonviral eleme~ during evolutio~ retrotransposons, which terminate in a poly(A) stretch of variable length. In maize, the Bsl element typifies the first subdivision, whereas the Cin4 element Structuraland functionalcharacteristicsof D~/Spm belongs to the nonviral retrotransposons. The coding andAc The genetic and molecular characteristics of En/Spm regions of Bsl and Cin4 share homology with viral and Ac elements have recently been reviewed in dereverse transcriptases. The second class comprises elements with terminal tail1-3. In summary, the En/Spm and Ac families each ininverted repeats; these elements probably transpose clude autonomous and nonautonomous elements. The directly from DNA to DNA by excision and re- autonomous elements, En/Spm and Ac, encode funcintegration (this has been demonstrated convincingly tions required for transposition. The nonautonomous only for the Ac system). The majority of maize elements elements (receptor or defective elements) usually reprethat have been studied molecularly fall into this class sent internal deletion derivatives of the autonomous elements, but have retained the ability to react in trans (Table 1). Most of the molecular and genetic data that have to functions expressed by the autonomous elements of been accumulated concern two families of elements: the same family. The defective En/Spm elements have the enhancer (En) or suppressor-mutator (Spm) and been termed I or dSpm elements, and Ds designates the activator (Ac) families. Using these two systems as defective elements of the Ac family. Transposition of En/Spm and Ac seems to be a examples, this article describes the regulation of transposition and the subtle interactions between the ele- nonreplicative process ('cut and paste'), although it ments and the 'host'. Most of the features described might be linked to chromosomal replication. Upon appear to be unique to plant transposable elements insertion, En/Spm and Ac cause a duplication of target sequences of typically 3 bp and 8 bp, respectively. and particularly to those of maize. T,~mm 1. Maize t t m t s p o s a b ~ ~ t s

Elements TIRb

TSDc

Size 8287

En/Spm

CACTACAAGAAAA

3

MpIl Ac

CACTACCGGAATr C/TAGGGATGAAA

3 8

Bg Mu

CAGGGA/a*V~/TACTr/cTATCG 8 200-500 bp 9

rDt rmrh

CAG/ATGTr/ATFAAATC 80 bp

8 9

w i t h t e r m i n a l inverted repeats

En and Spm am virtually identical; Spm is 8283 bp long and differs from En at six single nucleotide positions. ErdSpm encode at least two products necessary for transposition. Elements with similar TIPs and internal homology have been found in Antirrhfnum majus (Taml) and Glycine max (Tgm) TIR similar to En/Spm, sequence not yet complete 9 kb Ac encodes one product necessary for transposition 4563 and shares with Tam3 of A. majus a similar TIR and internal sequence homology. TIR is similar to Ac, sequence not yet complete 5 kb 1.2-2 kb A diverse family of elements that share the 200 bp TIPs. Because of the relatively long TIPs, they are reminiscent of foldback elements of Drosophila. The autonomous element has not yet been identified A nonautonomous memi~erof the Dt family 704 A nonautonomous member of the Mrh family 246

aAutonomous elements are in bold type. bTerminalinverted repeat, eratget site duplication. XlGMAY1990 VOL 6 NO. 5 l l l l l

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EVIEWS abundance of the Ac message Both elements belong to the Tfll~ in poly(A)+ mRNA is about class of elements that have •• :.c}~ c} G..' 10-8, far less than one transhort terminal inverted script per ce1112. The En/Spm repeats (TIRs). The TIRs and promoter is somewhat subterminai regions are stronger, but because of difrequired as substrates for ferential splicing of the pretransposition of En/Spm 2.3 cursor transcript, tnpA mRNA and Ac't. occurs at a frequency of 10-6 En/Spm encodes at least and tnpB mRNA at a frequentwo products, which are decy of 10-a. Therefore tnpB is rived by alternative splicing rate limiting for transposition from a precursor transcript s. FIG n in the En/Spm system. The most abundant product Regulation of the En/Spm promoter. The En/Spm proAnother type of elementhas been termed tnpA (Ref. moter is at the left (5') end of the element. The tnpA pro- specific regulation was de6). The 69 kDa tnpA protein tein, which is encoded by the En/Spm element, binds to tected when the inhibitory has been shown to bind to a the 12 bp motifs(open arrows) in the promoter upstream 12 bp sequence motif that region. This region is thus sheltered against methylation effect of the En-I102 element is reiterated several times and the promoter remains active. Overexpression could on En/Spm transposition was within the subterminal be avoided by binding of tnpA to the motif overlapping analysed13. This internal deregions6. The tnpA product the TATAbox; this would imply a lower binding affinity letion derivative of En exto this motifthan to the other motifs. presses an aberrant polycorresponds to the supprespeptide (tnpR) that shares sor function, which was defined genetically for the En/Spm system (see below). homology with both tnpA and tnpB. The tnpR polyAnother function, which has been termed tnpB (Ref. peptide probably represses transposition by competitive 3), may be represented by one (or all) of the three inhibition of tnpA and/or tnpB function. Since there are other alternative splice products that have been detect- about 50-100 En/Spn-i-homologous elements (mainly edS, all of which contain sequences derived from the deletion derivatives) distributed throughout the maize two open reading frames present in the first intron of genome, it is feasible that products encoded by some of tnpA. It has been speculated, from a comparison with these elements could modulate ErdSpm transposition. En/Spm-related elements from other plant species, that In the Ac system, an inverse correlation between the product encoded by the open reading frames inter- the number of active Ac elements and transposition acts with the 13 bp TIRs and in fact may accomplish frequency has been observed. This negative dosage endonucleolytic cleavage at the element's end#. effect is only observed in maize and not when Ac is The single transcription unit of Ac probably transformed into other plant species 14. The molecular encodes only one product, which is required to pro- basis of negative dosage is not yet understood. mote transposition 4. Surprisingly, this product does not A positive regulator has been postulated for interact with the 11 bp TIRs of Ac, but rather binds to En/Spm is on the basis of the genetic observation that a hexamer motif that is reiterated several times in the inactive elements can be reactivated by an active elesubterminal regions of AcT. The exact mechanism of menfl 6, There is some evidence that this role is played Ac transposition is thus also unknown. by tnpA, which binds in the upstream promoter region of En/Spm and could therefore shelter this region from Regulation of translm, ition methylation (Fig. 1). This implies that the presence of Since transposition is tightly linked to mutation, it tnpA is essential to maintain its own expression. Overmight be expected to be regulated to a level that is not expression of element-encoded functions could be deleterious to the cell. This is illustrated by the phe- prevented by the fact that the innermost tnpA-binding nomenon of hybrid dysgenesis in Drosophila 8, or by site at the 5'-end of En/Spm overlaps with the TATA the high mutability that is associated with the mutator box of the promoter. Binding to this site might result (Mu) transposable element system of Zea mays. In in repression of the promoter; in this sense tnpA active Mu lines, the mutation frequency is 30-50 times would be an autoregulator. the spontaneous mutation rateg. However, the Mu system changes frequently to an inactive state, and the DNA r~luence alterations after excision deleterious consequences of the high mutation rate are Soon after the first excision products (empty donor thereby avoided. sites) of plant transposable elements were analysed, it Transposition of Ac and En/Spm is regulated by became apparent that the wild-type gene sequence is element-specific mechanisms and by cellular functions. usually not precisely re-established after excision. ThereBoth the Ac and the En/Spm systems can reversibly fore gene function may be restored to varying degrees, change from an active to an inactive state. Inactivity is ranging from zero to full activity. In a systematic analycorrelated with an increased level of methylation of C sis of En/Spm excisions from the waxy gene, Schwarzresidue#0,11, while reactivated elements are hypo- Sommer et aL~7 found that only one in ten excision methylated; this indicates that the elements are subject events was precise. The analysis of these altered to negative control by cellular functions. sequences, 'footprints', led to the formulation of models Relatively low gene expression is also a conse- for plant transposable element excision and integration. quence of element-specific features. The promoters of According to the model of Saedler and Neversm, the transposable elements are rather weak, and the transposition takes place by a 'cut and paste' . . . . . .

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~EVIEWS mechanism. The transpo,~ase the insertion site23. Wessler20 recognizes the ends of tb~: elehas pointed out that these ment and introduces staggered 'introns' have arisen within the nicks at the ends of the target past 25 years. Of course such site duplication, in the same 'introns' are lost in transfashion as postulated for reposition of the defective insertion. Footprints result element, but they could turn from the action of DNA repair into stable introns if c/s-acting enzymes, which act on sequences necessary for transthe protruding single-stranded position were deleted. In this fringes at the excision site and respect, it is interesting to note at the element in the 'transthat in the En/Spm system, position complex' (Fig. 2). The such deletions are promoted formation of footprints has also by the presence of the autonbeen explained by Coen et omous element, perhaps by al. ]9, but in their model the prematurely aborted attempts :) ~CAITGCAA; _enzymatic cutting activity for at excision. Raboy and his colexcision and re-insertion is difleagues 2't have reported a seferent: a staggered nick, the quence of events that led to size of which corresponds to formation of a stable intron the target site duplication, is from a dSpm element; in this only formed at the recipient case, two terminal nucleotides = [ OTACOTT J ¢ site, whereas the element at of the element that are essen't (C~A A TT~G, fC ~ AAAA j.' the donor site is released by tial for transposition were curing more precisely at the deleted. FIGM Splicing does not precisely ends of the element. Excision Saedler and Nevem model for excision of a plant remove all sequences of the generates two hairpin structransposable elementTM. Wavy lines represent the tures at the site of excision, termini of the element. Staggered nicks have been element from pre-mRNA. As a and footprints are generated introduced by transtxxsase at the ends of the target result the protein sequence is by resolution of these hairpins site duplication (shown in boxes). Exonuclease usually altered, with conseand ligation of the products. degradation (dashed half circles) and DNArepair syn- quences similar to those of thesis occur (thick dashed lines) at the single-stranded element excision. NevertheIf the element is integrated into exon sequences, and if fringes. The products are f'mallyligatedto yielda par- less, the ability to function as ticular footprint. introns probably mitigates the the number of nucleotides impact of transposable eleadded or deleted is a multiple of three, then the consequence will be an allele ment insertion on gene expression 20. coding for an altered gene product. If the insertion is in regulatory sequences of a gene, new patterns or En/Spm can control the function of genes in opposite ways levels of gene expression may result. The functions encoded by the autonomous element Since the germinal excision frequency of elements such as En/Spm or Ac is in the range of a few per cent may control the activity of genes into which defective (and can occasionally reach as high as 50o,4), this elements have integrated. Negative control has been mechanism is a potent generator of mutations. In ad- observed for the ErdSpm system with the so-called suppressible alleles. As described above, in certain dition, if, for example, transposable elements were inserted in a particular set of genes in only part of a alleles most of the inserted sequences of a defective element are removed by splicing from pre-mRNA, given population, mutant alleles of these genes would arise in this part of the population with a relatively resulting in an intermediate level of gene expression. high frequency. Thus the generation of new variation However, this only happens in the absence of the upon which selection could act, perhaps even leading autonomous EndSpm element. In its presence, the forto speciation, could be accelerated by the action of marion of the pre-mRNA is blocked by the suppressor function of En/Spm and expression of the gene transposable elements. containing the defective element is thus abolished. The suppressor function resembles a negative regulatory Defective elements can function as intmns In several alleles, the insertion of defective circuit in which an En/Spm-encoded protein acts as a elements like Ds or dSpm reduces but does not repressor. The repressor recognizes and binds a abolish gene expression. The insertions in these alleles defined c/s-element located within the defective element. The bound protein probably stericaUy hinders are found within the transcribed regions of the genes involved. These alleles produce transcripts virtually progress of RNA polymerase through the gene, resultthe same size as wild-type ones because most of ing in prematurely terminated transcripts at the site of the element sequences are removed by splicing insertion. There are two mutant alleles of En/Spm that are almost defective fn transposition but still encode (reviewed in Ref. 20). In several cases this has been analysed in detai12L22. In summary, the elemen~ carry the suppressor function. Because both these alleles splice donor and acceptor sites close to their termini, express intact tnpA, this protein is a likely candidate or they 'activate' cryptic splice sites that are close to for the suppressor function. It binds to the 12 bp

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TIG MAY1990 VOL.6 NO. 5

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FIGI1

acLivaLion

UGR I

I ~ suppres

,s i on

T

al-ml J

sequence motifs in the ends of the defective element, and could thereby block transcription readthrough as and formation of the primary transcript (Fig. 3). Binding of tnpA can probably also have an opposite effect on gene expression; for example, the expression of the o~1-m2 allele of the A1 gene is activated in the presence of En/Spm (Fig. 3). In the t x l - m 2 allele, insertion of a dSpm element disrupts the promoter region of the A1 gene z6, thereby inhibiting gene expression. However, in the presence of En/Spm-encoded functions, A1 expression is partially restored. It has been suggested that binding of U l l t ~..... uons i,J ~t~, dPfective _ .~ , l~,t,° element in the A1 promoter activates this promote# 6. These control systems formally resemble the classical operon model, in which a c/s regulatory unit (defective element) responds in t r a m to signals (tnpA) emitted by the regulator (that is, the autonomous element; Fig. 3).

En/~pm-encode~

to

Concluding remarks The generation of DNA sequence diversity and the ability to create novel regulatory circuits may have given maize transposable elements an important role in evolution. There are also other indications that transposable elements make a significant contribution to biological processes. For example, when the sequences of different alleles are compared, sequence alterations that are most easily explained as footprints of excised elements are as frequently found as point mutationst7. This strongly suggests that transposable elements contribute to genetic variation in nature. A completely different argument to support the notion that transposable elements might provide the flexibility for populations to adapt successfully to environmental changes, comes from the observation that transposable elements are persistent in maize breeding populations (reviewed in Ref. 3). In these populations, the number of copies of the Uq transposable element increased under moderate selection pressure, or when selection for a particular trait was reversed. However, Uq was lost in the process of homogenization of the genome by inbreeding, which is a selection against variability. Elucidation of the extent to which transposable elements contribute to the genetic flexibility responsible for population adaptation is an interesting area of future research.

Control of A1 gene expression by the En/Spm system. The two alleles of the A1 gene contain dSpm elements integrated at different sites; tnpA protein, which is expressed in trans by" the autonomous En/Spm element, controls A1 expression in opposite ways. The ~xl-ml allele is expressed in the absence of tnpA protein (suppressor function) because the inserted element is removed by splicing from the precursor transcript. In the presence of tnpA protein, formation of the al-ml primary transcript is blocked, because tnpA protein binds to the dSpm element that is inserted within the transcribed region of A1. The al-m2 allele is not expressed in the absence of tnpA, presumably because the insertion has disrupted essential promoter elements. In the presence of tnpA protein, A1 expression is partly restored, probably because tnpA protein binds to the dSpm element that is inserted in the promoter region, and thereby activates the A1 promoter.

References 1 Peterson, EA. (1987) CRCCrit. Rev. PlantSci. 6, 105-208 2 Fedoroff, N. (1989) in Mobile DNA (Howe, M and Berg, D., eds), pp. 375-411, ASM Press 3 Gierl, A., Saedler, H. and Peterson, P.A. (i989) Annu. Rev. Genet. 23, 71-85 4 Coupland, G., Baker, B., Schell, J. and Starlinger, P. (1988) EMBOJ. 7, 3653-3659 5 Masson, P., Rutherford, G., Banks, J.A. and Fedoroff, N. (1989) Cell 58, 755-765 6 Gierl, A., Ltitticke, S. and Saedler, H. (1988) EMBOJ. 7, 4045-4053 7 Kunze, R. and Starlinger, P. (1989) EMBOJ. 8, 3177-~185 8 Engels, W.R. (1983) Annu. Rev. Genet. 17, 315-344 9 Chandler, V. and Walbot, V. (1986) Proc. NatlAc~d. Sci. b3A 83, 1767-1771 10 Banks, J.A., Masson, E and Fedoroff, N. (1988) Genes Dev. 2, 1364-1380 I1 Schwartz, D. and Dennis, E. (1986) Mol. Gt,~. Genet. ?05, 476--482 12 Kunze, R., Stochaj, U., Lauf,J. and Starlinger, P. (1987) EMBOJ. 6, 1555-1563 13 Cuypers, H. et al. (1988) EMBOJ. 7, 295~¢--2960 14 Jones, J.D.G., Carland, E M., Maliga, P. and D0oner, H.K. (1989) Science 244, 204-207 15 Nevers, P. and Saedler, H. (1977) Nature268 109-115 16 McClintock, B. (1971) Carnegie lnst. Wash#~gton Yearb. 70, 5-17 17 Schwarz-Sommer, Zs. etal. (1985)FJfBOJ. 4, 591-597 18 Saedler, H. and Nevers, P. (1985) EMBOJ. 4, 585-590 19 Coen, E.H., Carpenter, R. and Martin, C. (1986) Cell47, 285-296 20 Wessler, S.R. (1989) Gene82, 127-133 2/ Wessler, S.R., Baran, G. and Varagona, M. (1987) Science 237, 916--918 22 Schiefelbein, J.W., Raboy, V., Kim, H.Y. and Nelson, O.E. (1988) in Proc. Int. Syrup. Plant Transposable Elements (Nelson, O., ed.), pp. 261-278, Plenum 23 Simon, R. and Starlinger, P. (1987) Mol. Gen. Genet. 209, 198--199 24 Raboy, V., Kim, H-Y., Schiefelbein, J.W. and Nelson, O. (1989) Genetics 122, 695-703 25 Gierl, A., Schwarz-Sommer, Zs. and Saedler, H. (1985) F_dVIBOJ. 4, 579-583 26 Schwarz-Sommer, Zs. et al. (1987) EMBOJ. 6, 287-294

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"riG MAY1990 VOL 6 NO. 5