J. Pathol. 185: 342–344 (1998)

EDITORIAL

REGULATION OF THE WILMS’ TUMOUR SUPPRESSOR(WT1) GENE BY AN ANTISENSE RNA: A LINK WITH

GENOMIC IMPRINTING?

* .

University of Bath, Department of Biology and Biochemistry, South Building, Bath BA2 7AY, U.K.

SUMMARY

Antisense transcripts are typically associated with the down-regulation of gene expression. In this issue, Moorwood et al. presentevidence that an antisense RNA can enhance expression of the Wilms’ tumour suppressor locus WT1. We suggest that the unusualfunction of the WT1 antisense RNA might relate to the recent discovery of an antisense transcript that is involved in regulating imprintedexpression of the murine Igf2r gene, particularly since there is some evidence that the WT1 gene is regulated by genomic imprinting inhumans. ? 1998 John Wiley & Sons, Ltd.

KEY WORDS—gene expression; genomic imprinting; IGF2R; transcription; tumour suppressor; WT1

INTRODUCTION

The identification of the Wilms’ tumour suppressorgene, WT1, was reported in 1990.1,2 Cytogenetic analy-sis of heterozygous constitutional deletions encompass-ing chromosome 11p13 had indicated the involvement ofat least two genes in patients with WAGR syndrome.These genes, WT1 and Pax-6, were subsequently char-acterized and can be held to account for most, if not all,of the defects that constitute WAGR syndrome, namelyWilms’ tumour, aniridia, genito-urinary anomalies, andmental retardation. Pax-6 is mutated in human aniridiapatients as well as in small-eye (sey) mice.3 WT1 hasbeen confirmed as a tumour suppressor, being mutatedin around 10 per cent of all Wilms’ tumours as well as insome tumours of non-kidney origin. Also, it is almostalways mutated in Denys–Drash syndrome, in whichpatients display a range of genito-urinary anomalies,together with nephropathy and a Wilms’ tumour pre-disposition.4 The importance of WT1 for the correctdevelopment of the genito-urinary tract was underlinedby gene targeting at the murine wt1 locus. Micehomozygous for a null mutation had kidney andgonadal agenesis.5

Like many other genes with pivotal roles in develop-ment, both WT1 and Pax-6 encode transcription fac-tors; these fall into different classes, distinguished by theDNA-binding motifs that they possess. Pax-6 containsregions encoding a paired-box type DNA-binding motif,while WT1 includes four zinc-finger motifs located at itscarboxyl-terminus. It is notable that in the characterized

*Correspondence to: Andrew Ward, University of Bath, Depart-ment of Biology and Biochemistry, South Building, Bath BA2 7AY,U.K.

CCC 0022–3417/98/040342–03 $17.50? 1998 John Wiley & Sons, Ltd.

mutant WT1 alleles carrying nonsense or missense muta-tions, many of the mutations affect the zinc fingers, suchthat binding to the normal target DNA sequences islikely to be abolished.4 These types of mutation are themost prevalent in Denys–Drash syndrome patients(present in over 90 per cent of characterized cases),where they are almost invariably constitutionally hetero-zygous point mutations and are therefore thought to beacting in a dominant negative fashion. These mutantWT1 proteins either are truncated, such that they lackzinc fingers, or contain amino-acid substitutions thateither disrupt finger folding or replace residues known tobe critical for the recognition of target DNA sequences.The dominant negative mode of action has beenascribed to self-association of the WT1 protein, which isknown to occur through two N-terminal domains,and can result in an inactive complex when wild-typeand Denys–Drash syndrome mutant proteins areco-expressed.6

THE LEVELS OF WT1 EXPRESSION MAY BECRITICAL FOR NORMAL DEVELOPMENT OF

THE GENITO-URINARY TRACT

A great deal of effort has gone into the search forclinical mutations of WT1, and also into the characteri-zation of the DNA sequences and target genes to whichWT1 can bind. Relatively little is known about theregulation of its levels of expression. This is clearlyimportant if changes in the level of wild-type WT1protein, per se, contribute to the pathological pheno-types of WAGR and Denys–Drash syndrome, andindeed it has been argued that this could be the case(reviewed in ref. 4). The nephropathy, which is charac-

Received 2 February 1998Accepted 3 April 1998

343EDITORIAL

teristic of Denys–Drash syndrome but absent in WAGRpatients, might be a function of dominant negatively-acting WT1 complex, whereas the genito-urinaryanomalies, which are a common feature of both syn-dromes, might result from WT1 haplo-insufficiency. Atleast two key pieces of evidence appear to be at oddswith this idea. First, there is the lack of phenotype inmice heterozygous for a wt1 null mutation.5 Second,there is a sub-set of Denys–Drash syndrome patients(with nephropathy) with a mutation that causes animbalance in the ratio of naturally occurring WT1isoforms. Several WT1 isoforms are normally expressedand some of these result from an alternative splicingevent such that three amino acids are either present(WT1+KTS) or absent (WT1"KTS) from the linkerregion between the third and fourth zinc fingers. ThemRNAs encoding WT1+KTS are usually about four-fold more abundant than those encoding WT1"KTS,7but this ratio can be disturbed by an intronic mutation,effecting a single base substitution, that disrupts WT1mRNA splicing. The same heterozygous constitutionalmutation has been found in 3 of 48 reported Denys–Drash syndrome cases and the normal ratio of WT1isoforms is disrupted because the mutant allele no longerexpresses WT1+KTS mRNA (reviewed in ref. 4). It ispossible that the altered WT1+KTS:WT1"KTS ratiois responsible for the nephropathy in these patients andit remains plausible, at least in humans, that the deple-tion of wild-type WT1 protein (perhaps specificallyWT1+KTS) underlies the aberrant development of thegenito-urinary tract.

Just as too little wild-type protein has been implicatedas a clinically relevant factor, there is a growing body ofevidence to suggest that an excess of normal WT1 canhave pathological consequences. In contrast withthe intragenic mutations found in Wilms’ tumours,mesotheliomas, and leukaemias (reviewed in ref. 4),overexpression of wild-type WT1 has been reportedin several tumours and tumour cell lines, includingovarian tumours,8 leukaemias,9–12 and malignantmelanocytes.13

ANTISENSE RNAs AND GENOMIC IMPRINTING

One intriguing discovery that is likely to have animportant bearing on the regulation of WT1 expressionlevels is the presence of an antisense mRNA whichoverlaps the first exon of the WT1 gene14,15 (Fig. 1a).The antisense transcripts arise from a promoter withinintron 1 of WT1 and they are up-regulated by WT1itself.16 Consequently, it may not seem surprising thatthe paper by Moorwood et al. in this issue,17 demon-strates that the WT1 sense and antisense messages areco-localized to the same structures in developing humankidneys—except that the experiments also show that thehighest levels of expression for both transcripts occur inthe same places. Since antisense RNAs are generallyassociated with down-regulation of their cognate sensepartners,18 the expectation would be for high levels ofantisense RNA to correlate with low levels of senseRNA (and vice versa). Moreover, the authors went on to

? 1998 John Wiley & Sons, Ltd.

demonstrate that in a cultured cell line (T5A1), expres-sion of WT1 from a stably integrated transgeneincreased following the introduction of a constructexpressing the antisense RNA. Elevated expression ofWT1 protein was greatest in the cells with lower expres-sion of the introduced antisense RNA and it was notedthat the lower levels of antisense RNA better reflectedthe antisense:sense RNA ratio observed in vivo.Moorwood et al. quite reasonably conclude that theantisense RNA might increase WT1 transcription (pre-sumably this would not involve an interaction of theantisense RNA with the sense promoter, since the con-struct used to drive WT1 expression in the T5A1 cell lineuses a heterologous promoter), transcript stability, orthe rate of translation. Perhaps the inverse correlationbetween antisense RNA levels and elevation of WT1protein could be due to the antisense RNA having apositive action on one of these processes, when itis expressed at lower levels, and a negative effecton one of the other processes, when it is expressedat supra-physiological levels.

Although both the in vivo role of the antisense RNAand its mechanism of action remain obscure, it isinteresting to make a comparison with another unusualantisense RNA that has recently been discovered at themouse Igf2r locus.19 There are obvious parallels withWT1 in that the antisense transcripts arise from apromoter within an intron towards the 5* end of theIgf2r gene, such that sense and antisense transcriptsoverlap only within their 5* extremities (Fig. 1). It is alsonoteworthy that the equivalent human gene, IGF2R, canbe classified a tumour suppressor, since intragenic muta-tions have been found in a number of liver and breastcancers.20,21 However, the unusual and exciting findingrelates to the fact that Igf2r is an imprinted gene, withonly the maternally inherited allele normally beingexpressed22 (Fig. 1b). It is clear from the study by Wutzet al.19 that the Igf2r antisense transcripts are usuallyexpressed from the paternally inherited allele and that

Fig. 1—Schematic representations of the 5* regions of WT1 (a) andIgf2r (b). Sense RNAs are depicted by solid arrows and antisenseRNAs by broken arrows

J. Pathol. 185: 342–344 (1998)

344 EDITORIAL

loss of antisense transcription (following deletion of theantisense promoter region) resulted in expression of thenormally silent paternal sense Igf2r gene. Thus, althoughthe Igf2r antisense RNA does act to down-regulate thesense gene, this does not necessarily require a directinteraction of the two transcript species. Instead, expres-sion of one transcript species excludes expression of theother species from the same chromosome, a situationreminiscent of at least one other pair of imprinted genes,Igf2 and H19. These genes, which are not physicallylinked to Igf2r, are located about 80 kb apart, with Igf2being expressed only from the paternal chromosome andH19 from the maternal chromosome. It is largely on thebasis of these two examples that a more general modelof intrachromosome competition has been proposedto form part of the mechanism regulating imprintedgenes.23

The link between WT1 and Igf2r/IGF2R can beextended, since evidence for imprinting of the WT1 genealso exists. Although WT1 expression in fetal kidneyand Wilms’ tumours is non-imprinted24 (or biallelic), itsexclusive expression from one allele has, in some indi-viduals, been observed in the placenta and fetal brain(maternal),25 as well as in fibroblasts and peripherallymphocytes (paternal).26 This means that imprinting ofWT1 is both tissue-specific within individuals and poly-morphic in the population. While this makes it difficultto assess the significance of WT1 imprinting, it should benoted that tissue restriction of imprinted gene regulationhas been found in several of the imprinted genes thathave been characterized.27 Perhaps more pertinently,imprinting of IGF2R also appears to be polymorphic inhumans, with biallelic expression occurring in mostindividuals.28–30

In the context of a discussion on the potential clinicalrelevance of altered levels of WT1 expression, its regu-lation by genomic imprinting is tantalizing. Changes inexpression associated with loss of imprinting mutationsat other imprinted loci have been linked with severaldevelopmental disorders (e.g., Igf231) and, at the veryleast, the presence of imprinting represents anotherindication that tight regulation of WT1 levels is criticallyimportant. The discovery of the oppositely imprintedsense and antisense Igf2r transcripts raises the questionthat a similar intrachromosome competition couldoperate at the WT1 locus in those tissues where it isimprinted. If this is the case, the competition mechanismmust be overridden in tissues that exhibit biallelicexpression, to allow sense and antisense transcriptionfrom the same chromosome. Alternatively, perhaps theantisense WT1 transcript is a vestige of the imprintingmechanism that has acquired a novel role in maintainingappropriate levels of expression that is still unlike thefunction of most known antisense RNAs.

One thing is clear. Future estimates of the frequencyof WT1 involvement in developmental disorders andcancer will need to take account of mutations outsidethe structural gene that could affect its levels of expres-sion. A search through all the potential regulatorysequences around WT1 is a daunting task, but theantisense transcriptional unit would be an interestingplace to begin looking.

? 1998 John Wiley & Sons, Ltd.

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