Variegated gene expression in mice

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RefergllO~

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Variegatedgene expression in mice KENNETH DOBIE*~j, MAJIDMEHTAH~, MARGARETMcCLENAGHAN+~AND RICHARDLATHE* [email protected] [email protected] [email protected] [email protected] *CENTREFORGENOMERESE~.RCH,UNIVERSITYOFEDINBURGH,WE.~MAINSROAD,EDINBURGH,UK EH9 3JQ. tRoSLININSTITUTE(EDINBL'RGH),ROSLIN, MIOLOTHIAN,UK EH259PS.CITI~'~SGENESA, BUEDEMOhSHEt~L67000 STRASBOERG,FRA.~CE.~SALKlxs'n'~'rE,MBVL, 10010 N. TORP,~PINESROAD,LAJOLIA,CA92027, USA. Tmnsgenic animals, particularly mice, are most-commonly generated by the microinjection of DNA into fertilized eggs. End-to-end ligation of the construct and/or homologous recombination between circularized molecules is thought to generate a tmnsgene concatemer (often 5-50 copies) that integrates at a single site in the genome. While transgene expression can be stable and heritable, the tandem-repeat nature of the insertion appears to contribute to a phenomenon akin to variegation, as described in insects and plants. This can generate, by stochastic silencing of the transgene, a variable and mosaic pattem of transgene expression, Variegation can complicate the interpretation of some experiments using transgenic animals generated by microinjection. Below, we briefly discuss the evidence for variegation in mice, mechanisms of silencing and routes by which it can be avoided, and the possibility that some endogenous genes might be subject to variegation. Several different mechanisms can lead to variations in the level of (trans)gene expression within a single

line. Variation can be due to strainspecific modifier genes, or to integration or translocation into Y chromosome heterochromatinor to the X chromosome 1. Loss of function has also been observed when cells expressing the construct have a selective disadvantage, so favouring loss of transgene DNA. For the most part, Wansgene loss appears uncommon although inverted repeats within a transgene array can favour deletion or rearrangement 2. Mosaic expression patterns have also been observed with tyrosinase fusion transgenes, although mosaic or striated expression might be an imrinsic property of this locus3. As noted above, growing evidence now suggests that some transgenes can be subject to a phenomenon akin to the variegation found in insects and plants.

Position effect variegation in Drosophila

Mosaic or 'variegated' expression of endogenous genes 4 and transgenesS-7 in Drosophila is subsumed under the term 'position-effect variegation' (PEV). This can be defined as TIG APRIL1997 VOL. 13 NO. 4

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(a) position-dependent inactivation of gene expression in a fraction of cells that generate a particular tissue, (b) heritable maintenance of this pattern, either 'on'or 'off', through subsequem cell divisions, and (c) incomplete mixing after each cell division such that cells that remain closest in space are those most-closely related by lineage. The first and best-documented example of PEV concerns a chromosome rearrangement in which an allele of the white(w +)gene was transferred to a site close to the centromi:re. Expressiorl in the eye is uniform at the original site, but after translocation to a pericentromeric region, the expression of the gene becomes variegated, resulting in patches of pigmented and unpigmented cells in the eye. Further studies have led to a paradigm, still unproved, that pericentromeric localization renders the gene susceptible to heterochromatic con• densation spreading out from the tentromere8-1°. PEV has also been documerited in yeast for gene .sequences integrated close to telomeres n or within centromere# 2. The silencing

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process in yeast can affect genes up to 20 kb away from the heterochromatineuchromatin breakpoint 13 while this extends to 2 Mb

of transgene methylation increases in step with copy number 19, consistent with progressive silencing as copy number increases. In in Drosophila14. addition, higher transgene copy numbers show morerapid silencing of expresVariegation oftransgene ~k :. ~.:.~ ~, sion20. These observations expression in mice ~ "- " " ' : As we have seen, a va. ~"~.i~:~ "~ii~ ~ " suggest, but do not prove, riety of mechanisms can ~ " ~ ~ , : "'~'' that higher copy numbers contribute to transgene contribute to variable transgene expression in mice, silencing in mouse just as riley do in Drosophila. but do any correspond to ~v~. • : ~ :~i ' ~ .~:~$ variegation as described in What might be the mechanism that triggers Drosophila? Despite many suggestive repotts, there has Fmtraz 1. Variegated gene expression in mammary gland of heterochromatic condenbeen little direct evidence, transgenicmice. In situ hybridizationwas performed to sections sariorL~In Drosophila, it has Two studies in mice now of mammarygland from line 7 mice harbouringa [3-1actoglobulin been suggested that somatic provide finn evidence for (BLG)transgenewith a pericentromericinsertionsite16. pairing can le~ad to DNA looping and, hence, sequessectoring of transgene expression, consistent with stochastic mechanism. In situ hybridization tration into a heterochromatic comsilencing and maintenance of the studies on chromosome spreads from partmentS, 7. A further possibility is expression state (on or off) through tmnsgenic animals exhibiting varie- suggested to us by the fact that mulcell division. In situ staLqing of sec- gated expression of human CD2 tiple interacting transcription factors tions of thymus from mice harbour- revealed that the transgenes were bind at many promoter elements. ing a human CD2 transgene revealed integrated dose to a centromere 15. Tandem repetition of the promoter some dustering of cells expressing Similarly, in lines displaying varie- could permit the formation of incorthe transgene15. Compelling evidence gated BLG expression the transgene rect (and unproductive) multiprotein for sectored localization of transgene array was also present close to a cen- complexes between transcription expression was also obtained in mice tromere 16. These results parallel the factors bound at adjacent promoter harbouring a transgene encoding the situation in Drosophila where prox- sites, resulting in DNA looping out ovine milk protein, [3-1actoglobulin imity to heterochromatic regions pre- between them and loss of expression. (BLG). In situ hybridization to mam- disposes to variegation. These models are consistent with Second, there are some indications a silencing process comprising three mary gland sections revealed clusters of cells strongly expressing the trans- that transgene copy number can con- steps (Fig. 2). First, the formation of gene surrounded by cells that fail to tribute to variegation, also as reported abnormal (possibly bent?) DNA strucexpress detectable transgene mRNA in Drosophila. Transgene heterochro- tures due to pairing or looping, a pro(Ref. 16; Fig. 1). In two of three lines matinization in Drosophila is not only cess favoured by sequence repetition. examined, the level of BLG expres- dependent on proximity to the cen- Second, the specific recognition of sion in the milk varied by up to ten- tromere, but also on the number and these abnormal structur~ (for instance fold between individuals of the same orientation of repeats present at the by the binding of heterochromatin line: the level of transgene product in transgene locus; silencing increases components possibly associated, in milk appeared to parallel the number in step with copy number5,7. Similar mice, with DNA methylation). Third, and size of cell clusters positive for copy-number-dependent silencing sequestration of the complexes into has been repotted in plants 17,1s. The heterochromatic regions, and faciliwansgene mRNA (Ref. 16). While in both these cases the variegating mouse lines discussed tated by proximity to preexisting clusters of expressing cells suggest above were not designed to address heterochromatin structures. clonal expansion from a committed a possible association between copy It is of note that the contention progenitor, other possibilities have number and variegation (although that silencing extends progressively not yet been formally excluded. the two variegating BLG lines con- outwards from early foci of heteroHowever, these observations indi- mined more than 17 copies of the chromatinization is consistent with a cate that individual cells are able to transgene while the nonvariegating report that the ex'tinctlon of expression suppress the expression of transgene line harboured less than five copies16). of a globin transgene increases with constructs and, subsequently, to However, there is some indirect age of the animal2°. However, it is propagate the inert state of the trans- evidence for linkage between varie- not clear that proximity to preexistgene through cell division, leading gation and copy number in mouse. ing heterochromatic regions is an to mosaic or variegated patterns of In vertebrates (but not Drosophila) absolute prerequisite for stochastic expression. DNA methylation is associated with silencing to occur. gene inactivity, although whether How might variegation arise? methylation is a cause or consequence Is variegation widely encountered? The chromosomal localization of of gene silencing in mammals is a Tandem repetition and proximity the variegating mouse transgenes matter of some debate. Nonetheless, to the centromere (or another heteroprovides a first clue to the underlying we reported previously that the extent chromatic region) both appear to TIG APRIL1997 VOL. 13 NO. 4

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that some transcription encontribute to transgene hancers can act to suppress silencing. Superficially, this variegation, although it is supports the argument that ~ [ ~ Copy1 . - . . . ~ ] Copy2 cle;w that most enhancers many transgenic animals Heterochromatic alone do not have this eshould express transgenes in region ffect. Matrix-attachment rea stable fashion, because gions (MARs; also scaffold copy numhers are often low attachment regions, SARs) and integration close to a have also been reported to centromere is unlikely. This prevent variable expression argument might be incorrect. of some transgene conAlthough no systematic studstmctseS,29 although this ic~ have been done, there is does not appear to be a evidence that transgene intesystematic feature of such gration into pericentromeric elementss0,31. regions may occur freFestenstein et aL 15 requently. Of 17 lines examport that the inclusion of ined (14 by Festenstein et tile human CD2 locus conaL iS, three by Dobie et al.I6), trol region (LCR) in a transmore than half (nine) had a gene construct could prepericentromeric localization. vent variegation while, at This might indicate that the same time, rendering variegation is rather more the level of expression procommon than previously ] Transcriptionfactorcomplex at promoter txmional to copy number. recognized, although sectorSimilar findings were ing of gene expression patO Heterochromatinprotein reported for a globin LCR terns is, of course, difficuh (Ref. 32). While a fuller disto detect in tissues (such as FIGIBIE2. A m~xlel for heterochromaticcondensatkm of tmnsgene brain and the immdne sys- an'ays precipitated by (1) repeat DNA sequences and (21 proximity cussion of tile relationship copy-numbertem) in which individual to existing heterochromatin. (a) A multimeric transcription-factor between daughter cells migrate away complex is formed ben.veen repeat transgene copies. (b) Hetero- depePdence and variefollowing cell division. How- chromatin proteins bind to the abnormal DNA strttctures. (c) The gation would be out of ever, there are abundant transgenesare mquestrated into the heterochromatincompamnent. place here, this is a potentially important finding instances of unexplained widl implications across the variable or mosaic transgene expression 21-2~. To give one exam- variegation can be avoided. First, board from transgenesis in animals to ple, five out of five transgenic lines single-copy gene insertions do not gene therapy in humans. However, it haflx~uring a housekeeping tratxs- appear to variegate. This argues for is not known whether the suppresgene 19 showed extreme variation in tile use of targeted integration of sion of variegation is a general feathe tissue levels of expression in indi- single transgene constructs by hom- ture of LCRs or whether particular vidual animals. Unpredictable swings ologous recombination in embryonal elements can only suppress variein the level of transgene expression stem cells25, or for integration by cat- gation in specific cell types. Nevertheless, the incorporation of LCRs into were obse~.'ed, ranging over two alysed recombination in microinjected orders of magnitude [M. Mehtali eggs (J.O. Bishop, pets. commun.). transgene constructs could plausibly (1986) PhD Thesis, Strasbourgl, par- Second, the use of very long con- provide a general route to ensure ticularly at high copy number, and structs (employing yeast or bacterial reproducible expression levels. artificial chromosomes) will increase most were consistent with variegation. tile di,';tance between repeat elements Do any endogenous genes and can decrease the likelihood of variegate? Can variegation in transgenic mice If the model of heterochromatic precipitating heterochromatic condenbe avoided? The possibiliW that many trans- sation (A. Colman, pets. commun.). condensation spreading from the centromere (or other heterochromatic genes are subject to stochastic silenc- Third, the incorporation of elements ing, is of concern, especially because permitting efficient site-specific re- regions) to encompass repetitive variegation can be difficult to detect combination (such as the IoxP site transgene arrays is even partly corwithout detailed analysis. The out- from bacteriophage P1) could be used rect, this would inevitably raise the come of transgenic experiments (by crossing to an animal expressing question of the fate of genes located would be considerably obscured if tile P1 recombinase, Cre, in the germ- close to the transgene or lyetween the some animals of a given line express line) to generate progeny in which transgene insertion and the centrothe transgene at high levels in a the number of transgene copies is mere. Are they stochastically silenced too, and are there genes who.~ particular organ while others do not. substantially reduced 26. The effect of multiple integration expression naturally variegates? Although screening tvansgenic Chromosome rearrangements Iomice for the chromosomal insertion and/or a pericentromeric localizsite and copy number should permit ation can also Ix~ attenuated by the cat~.M4--400 kb away from the televant the elimination of variegating lines, inclusion of specific DNA elements locus h,we Ix*en a~ociated with there are other routes by which in the construct. It was suggested 27 some human (e.g. SOX9/camponlelic TIG APRIL1997 VOL. 13 NO. 4 129

COMMENT dysplasia; PAX6/aniridia) and mouse (steel~fern,de sterility) genetic disorders, as reviewed recently33.34. Detailed molecular analysis of two aniridia (absence of iris) human pedigrees demonstrated that the disorder is associated with chromosome rearrangements greater than 85 kb downstream of the P A X 6 gene 35. Although a link bet'ween the aberrant phenotypes and the genomic rearrangements has yet to be established, it is tempting to speculate that the rearrangements could in some cases cause relocation of the affected loci into a domain subject to heterochromatic condensation, leading to variegated expression. 'Variegation' could also provide a mechanism for gene silencing during development~-39 or even for the storing of acquired information: rearrangement of neuronal centromeric satellite DNA was reported during synaptic plasticity"t° while, in Drosopbila, a protein phosphatase mutation that suppresses variegation also impairs learning and memory ~l. Finally, in plants t7.-~2 and in insects-t3 inactivation of one copy of a gene can, by a phenomenon termed co-suppression or trans-inactivation, perturb the expression of another copy situated elsewhere in the genome. Although co-suppression has not so far been documented in maremals, it would come as no surprise if the same were to occur in transgenic mice. More generally, the many examples of variegation, co-suppression and related phenomena discussed here emphasize the conclusion that mechanisms exist by which different copies of a DNA sequence can intercommunicate in ,some real sense (possibly by dir~'t physical association brought about by pairing and/ or looping) such that, iointly, their activity state can differ substantially from that of a single copy acting alone.

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