Impressions of imprints

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Impressions of imprints ROLFOHLSSON,DENISEBARLOW*AND AZIMSURANfl" DEPARTMENTOFANIMALDE~XLOPMENTANDGENETICS,UPPSALAUNIVERSm',NORBYVAGEN18A,~752 36 UPPSALA,SWEDEN. *RESEARCHINSTITUTEOFMOLECULARPATHOLOGY,DRBOHR-GASSE7, A-1030WIEN,AUSTRIA.

tWmCOMECRCINSTITLrIE,TENNISCOURTROAD,CAMBPdDGE,UK0321QR. Parental (genomic) imprinting means that a subset of autosomal loci are expressed in a parent-of-origindependent manner. Importandy, such patterns of gene expression do not conform to classical Mendelian inheritance and have potentially farreaching implications for genetics, evolution, developmental biology and pathology. With the aim of collating the current data and rationalizing the different aspects of imprinting, a Nobel conference on 'Parental imprinting: causes and consequences' was recently held in Stockholm, Sweden (12-14 June 1994). The first and last presentations of the meeting both addressed a central question in the imprinting field: are the monoallelic expression patterns established by repression or activation/derepression mechanisms? Interestingly, both the /gt2 and Igt2r genes were reported to be expressed biallelically during preimplantation development, suggesting that monoallelic expression may eventually be achieved by repression. However, it appears that a complex series of events may be involved postzygotically to achieve monoailelic expression. In the case of l~2r at least, the imprinting status may involve an activation of the maternal allele via a methylated intronic CpG island inherited from the maternal gamete, which is silenced when inherited (unmethylated) from the paternal germ line (D. Baflow, Vienna, Austria; R. Jaenisch, Cambridge, USA). To explain this situation, both D. Solter (Freiburg, Germany) and D. Barlow invoked the presence of stage- and cell-typespecific 'readers' of imprints. Such readers may operate to both suppress and activate gene transcription. The period of preimplantation development appears to be a watershed for the process of X-chromosome inactivation. As reviewed by M. Lyon (Didcot, UK), Xist is a strong candidate for a gene with a key role in this process and its expression precedes X inactivation. In females, Xist

is expressed only from the inactive X chromosome while it is silent on the active X chromosome. While Xist is imprinted in the mouse placenta such that only the patemally derived copy is transcriptionally active, it is randomly inactivated in the embryo proper where X inactivation is also random. A. Surani (Cambridge, UK) offered explanations for this complex pattern of allele usage. R appears that Xist is expressed only from the patemaUy derived allele up to the eight-cell stage. At this point, the parental imprints are erased, which may account for the expression of Xist in gynogenotes as well as for random expression of Xist at around gastrulation. The H19 and Igf2genes are close neighbours and yet are expressed from opposite parental genomes. These and other considerations have prompted suggestions that the two loci share a common enhancer that is controlled by the epigenetic status at the 1119 locus (S. Tilghman, Princeton, USA). Importantly, in this model, H19 must dominate lgf2, such that the transcriptional activity of H19 inhibits lgf2 transcription in cts. To provide firm evidence in favour of this model, Tiighman is currently attempting to knock out the two enhancers downstream of H19. If the model is correct, the loss of function of the t119 enhancer would be expected to perturb not only transcription of t119, but also transcription of Igt'2. Two exceptions to this model were presented. First, the leptomeninges of the mouse, which express Ig/'2 biallelicaUy, nevertheless express only the maternally derived 1119 allele (R. Ohlsson, Uppsala, Sweden). Second, while the Pl promoter of the human IGF2 gene directed biallelic expression during both pre- and postnatal development, the P2-P4 promoters had a complex allele usage. In one example, transcription was directed from the P2 and P4 promoters on opposite parental alleles, despite monoallelic H19 transcription. It is possible, TIG DECEMI~ER1994 VOL. 10 No. 12

therefore, that one promoter can be imprinted, but that a neighbouring promoter can override the effects of imprinting. This plasticity of imprinting was suggested to reflect a situation in which the repertoire of transcription factors may reinterpret or bypass epigenetically controlled regulatory elements (which is potentially applicable in tissue-specific imprinting) (R. Ohlsson). It is possible, therefore, that the model of Igf2/H19 regulation proposed by Tilgl-anan may need to be modified, a possibility that is emphasized by recent studies of the Ins2 locus, which is contiguous with IGF2 (Ref. 1). The current models of IGF2 and 1-119 imprinting mechanisms have a bearing on the human BeckwithWiedemann syndrome (BWS). This disease is characterized by organomegaly, involves genetic imprinting and has been linked to chromosome 11p15.5 (B. Beckwith, Loma Linda, USA). IGF2 is an attractive candidate gene since it maps to this region, is expressed primarily from the paternal allele and produces a growthpromoting activity. A double dose of IGF2 activity could be generated, either by duplication of the paternal IGF2 allele or by loss of imprinting, such that two IGF2 alleles are transcriptionally active (R. Weksberg, Toronto, Canada). Alternatively, the hemihypertrophy that is characteristic of BWS could be generated by specific deletion of the growthretarding function of an imprinted gene. Candidates include the HI9 gene, ectopic expression of which represses the neoplastic phenotype of rhabdomyosarcoma cells (B. Tycko, New York, USAL and a novel gene located near IGF2that encodes a zinc-finger protein (M. Mannens, Amsterdam, The Netherlands). BWS individuals have a predisposition to tumorigenesis, and this is also likely to involve imprinting. A. Reeve (Otago, New Zealand) discussed the preferential retention of the paternal 11p15.5 region in Wiims' turnout, which is common in

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BWS patients. The genetics of such in PWS. U. Francke showed that one processes and their consequences imprinted gene, SNRPN, which has were the theme of O. Core's (Sudbury, been proposed to encode a comCanada) presentation. He suggested ponent of the RNAsplicing nmohinery that mitotic crossing-over involving in the CNS, maps to the locus involved imprinted loci might be a more com- in PWS. SNRPN is expressed only mon process in human diseases than from the paternally derived allele, as has hitherto been realized. In this might be predicted. However, there context, the Wilms' tumours that are more candidate genes in this showed loss of heterozygosity ful- region of chromosome 15. One of filled all the criteria of his model them is ZNF127, an imprinted locus system. To explain involvement of that encodes a zinc-finger protein IGF2 in the Wilms' turnouts without (R. Nicholls). ZNF127 belongs to a loss of heterozygosity, A. Feinberg growing list of genes that are func(Baltimore, USA) and A. Reeve both tionally imprinted in a tissue-specific invoked loss of imprinting of IGF2; manner (B. Cattanach, Didcot, UK; that is, biallelic expression of the R. Nicholls). Both the human gene locus. It was intriguing to learn, then, ZNF127 and its mouse homologue that 1-119is not expressed at an ap- are monoallelicaUy expressed in the preciable level in such tumours. The brain and the kidney, but biallelically loss of transcriptional activity of 1t19 expressed in the liver. could also be correlated with the It is not yet clear whether CpG acquisition of paternal-like methyl- met_hy!ation actually constitutes the ation in the maternal 1-119 allele gametic imprint. Although there are (B. Tycko; A. Feinberg). These results exceptions, as exemplified by the fit Tflghman's model very nicely: ff intronic CpG island of /pO°2r, the maternal 1-119 is subdued, then methylation patterns laid down durmaternal IGF2 is allowed to be ing gametogenesis are generally activated. erased during preimplantation develThis was not the only example of opment. Although studies of mice in an 'imprinting mutation'. B. Horst- which the gene encoding DNA hemke (Essen, Germany) discussed methyltransferase has been knocked the effects of CpG methylation in the out (R, Jaenisch) provide direct Prader--Wiili (PWS) and Angelman evidence that the CpG methylation (AS) syndromes. These human dis- patterns are a manifestation of the eases have been mapped to chromo- functional imprint, these might not some 15q11-13 and clearly involve necessarily represent the actual imprinting, since the deletion of gametic imprint (A. Wolffe, Bethesda, the paternal or maternal sequences USA). This possibility was emphawithin this region give rise to PWS sised by W. Reik (Cambridge, UK), and AS, respectively (B. Homthemke; who determined the DNA sequence U. Francke, Palo Alto, USA; R. of genomlc regions upstream of the NichoUs, Cleveland, USA). In a frac- mouse/gf2gene and found considertion of PWS patients, there are no able heterogeneity in their methyldeletions or chromosomal abnormali- ation patterns in various tissues, ties involving this region of the including sperm. He interpreted these genome. In these instances, abnormal data by invoking the requirement for methylation patterns are observed, a finite number of clustered methylperhaps influencing the expression ated CpG residues to enable methyllevels of affected and imprinted loci. binding proteins to interact and parSpecifically, B. Horsthemke invoked ticipate in the formation of chromatin the presence of an imprinting centre; structure. This may be in contrast to by analogy with X inactivation, this the situation for the intronic CpG centre would control chromatin struc- island of the ~ gene. In this case, ture and thus regulate the expression a consistent pattern of CpG methylof the PWS and AS genes at a dis- ation could be observed in a few tance. Hence, a mutation in the residues within the intronic island of imprinting centre could prevent the the maternally inherited allele in resetting of the imprint in the germ germ-line and somatic cells (D. line. Barlow). Alternative explanations may It appears that while AS is a single therefore be required to explain gene disease, PWS is much more imprinting at different loci. complex. Importantly, the gene inFurther evidence suggesting a link volved in AS is not directly involved between imprinting and chromatin "rIG DECEMBER1994 VOL. 10 NO. 12

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structure was discussed. The Drosophila HP1 protein, a component of heterochromatin, shares a conserved structural motif, termed chromobox, with an expanding list of mammalian gene products. There may be a concerted action between chromodomain proteins and CpG methylation to modify and manifest different chromatin structures (T. James, Middletown, USA). A similar allusion was made by T. Bestor (Boston, USA), who pointed out that the mammalian DNA methyltransferase shares a sequence motif with the mammalian homologue of the fruit fly protein trithorax. In Drosophila, a family of trfthorax genes is involved in regulating homeotic selector genes z. Hence, this observation potentially links two important epigenetic regulatory systems. One aspect of how CpG methylation can modify chromatin structure was presented by A. Wolffe. The CpG methylation of specific sites has been shown to cause nucleosome phasing, which is one well-documented way of controlling gene transcription. A. Wolffe also discussed the differential modifications of histones in heteroand euchromatin. The difference in the levels of acetylated histones in the asynchronously replicating chmmatin of the X chromosome is one intriguing aspect of how different alleles can attain different chromatin structures in somatic cells. H. Cedar (Jerusalem, Israel) presented evidence that imprinted autosomai loci are also asynchronously replicated. Interestingly, regions that contain imprinted genes are replicated early when paternally inherited. Taking the PWS locus as an example, it is clear that the replicons (up to several megabases long) must contain both imprinted loci, expressed either maternally or paternally, and nonimprinted loci. Hence, a long-range function must interact with a local element, perhaps specific to each of the imprinted loci. How many imprinted genes are there? B. Cattanach and T. Mukai (Osaka, Japan) have attempted to identify imprinted genes in a more systematic way, and suggest,d that there are less than 200. Mukai and his colleagues have exploited differences in parental CpG methylation status and the restriction landmark genome scanning method3A to show that a new gene, termed U2afl-rsl, which

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has sequence similarities to U2 small nuclear ribonucleoprotein auxiliary factor, is imprinted in the mouse. As this approach links an epigenetic mark with an imprinted gene, it should be feasible to use it to detect other imprinted genes. Cattanach has identified regions that have a phenotypic effect when made maternally or paternally disomic by wanslocation. So far, this approach has provided much-needed reinforcement for investigators examining whether their favourite gene might be imprinted. However, J. Forejt (Princeton) r:,ised a cautionary note that these approaches to evaluating potentially imprinted loci bias qualitative over

quantitative aspects of imprinting. Hence, a number of imprinted genes might go undetected due to the plasticity of imprinting, whether it depends on the tissue or promoter involved, or on the particular genetic background. This meeting demonstrated that there are likely to be many roles of imprinting during development and evolution. This diversity defies the formulation of a single, unifying hypothesis. Even if the basic machinery of imprinting is universal, the applied mechanism may well vary among individual imprinted loci. Research into imprinting now needs to progress towards the

identification of the precise regulatory elements that represent the targets of the imprinting process. This is prerequisite to dissection of the molecular mechanisms that enable the transoiptional machinery to discriminate between parental alleles.

References 1 Giddings, S.T. et al. (1994) Nature Genet. 6, 310-313 2 Paro, R. (1993) Curt. OIMn. CellBiol. 5, 999-1005 3 Hatada, I., Sugama, T. and Mukai, T. (1993) Nucleic Acids Res. 21, 5577-5582 4 Hayashizaki,Y. et al. (1994) Nature Genet. 6, 33--40

LETTERS

' Se/se,nonsenseandantisense,.. . When teaching DNA transcription, a distinction must be made between the two strands of duplex DNA: one is transcribed and one is not. Two sets of terms are commonly used: coding/noncoding, and sense/antisense. According to Rieger, Michaelis and Green 1 'the coding DNA strand is the strand of duplex DNA (- sense strand) which is transcribed into a complementary RNA strand.., The DNA strand that is not transcribed, having the same sequence as mRNA, is referred to as the noncoding or antisense strand'. Similar definitions are found elsewhere2-5. However, we have found some mistaken definitions of coding and noncoding DNA strands in several textbooks, which may confuse students. The above definition of the coding and anticoding strand is reversed in A Dictionary of Genetics6: 'The coding strand has the same nucleotide sequence as mRNA... The coding strand is not the template for mRNA synthesis and is therefore the antisense strand'. Strangely, in this same book, the sense strand is defined correctly. In several other textbooks, the definitions of coding and sense strands are completely reversed 7-12. Perhaps confusion

arises from the use of 'antisense RNA' to refer to the RNA complementary to mRNA, which will be the same sequence as the 'sense DNA' strand. We would ask that in future editions of these books, some standard definition is used. Mixed definitions can only cause confusion, and these terms are surely already difficult enough! There is also confusion over correct usage of the related terms, coding region and coding sequence. Certain other textbooks 13,14avoid this problem by not mentioning either sense or antisense strand. We could not clmck many papers, but did find both correct 15 and incorrect t6 definitions among those we did check. The simplest solution may be to avoid the use of coding/noncoding and sense/antisense, and use an alternative term. One designation that may be suitable for the s¢a~c or coding strand is 'template strand', as suggested by Brown 17. Although he also suggested that the complementary strand be called the 'nontemplate strand', perhaps 'atemplate strand' is better. We can * only suggest that everyone becomes familiar enough with the various terms to make sense of coding TIG DECEMBER1994 VOL. 10 No. 12

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strands for themselves; in future, more of us may make sense of 'template' than of 'sense'!

YOSH~.ASATANAKAAND D~ MACn Instituteof BiologicalSciences, Universityof Tsukuba, Tsukuba,Ibaraki305,Japan.

References I Rieger, R., Michaelis, A. and Green, M.M.(1991) Glossary of Genettcs(Sth edn), pp. 33, 97, 348, 446, Springer-Verlag 2 Goodenough, U. (1978) Genetics (2nd edn), pp. 265-269, Holt, Rinehart & Winston 3 Hartl, D.L (1991) Basic Genetics (2nd cdn), pp. 272, 274,Jones & Bartlett 4 Oliver, S.G. and Ward, J.M. (1985) A Dictionary of Genetic Engineering pp. 18, 100, Cambridge University Press 5 Gilbert, H.F. (1992) Basic Concepts in Biochemistry, p. 39, McGraw Hill 6 King R.C. and Stansfield, W.D. (1985) A Dictionary of Genetics(3rd edn), pp. 77, 356, Oxford University Press 7 Lewin, B. (1994) Genes It, pp. 163, 1236, 1238, Oxford University Press 8 Rothwell, N.V. (1993) Understanding Genetics, pp. 262-264, 494--496, 610, 622, Wiley-Liss 9 Weaver, R.F. and Hedrick, P.W. (1992) Genetics(2nd edn), p. 174. W.M.C. Brown Publishers I0 Strier, L. (1988) Biochemistry (3rd edn). p. 705. Freeman