P o s itio n E ffect V a rie g a tio n
The p h e n o m e n o n o f m osaic exp ressio n o f genes p la ce d a d ja ce n t to a e u c h r o m a tin - h e te r o c h r o m a tin ju n c t io n is n a m e d P o s itio n E ffe c t V a r ie g a tio n (P E V ). S tu d ie s in D r o s o p h ila h a v e s h o w n th a t h e te ro c h ro m a tic D N A consists o f sate llite s, tra n s p o s o n -lik e repeats and fe w genes present at lo w d e n sity (G a tti and P im p in e lli, 1992; H o w e et al., 1995; W a llra th , 1998). O ne w e ll s tu d ie d exa m ple o f PEV is the m osaic expression o f the w h ite gene (co d in g fo r re d eye) in p ig m e n t cells o f the D ro s o p h ila eye. In th is case the w h ite gene, w h ic h is a ctive in n a tu ra l e u c h ro m a tic e n v iro n m e n t, is m o v e d a d ja c e n t to th e c e n tro m e ric h e te ro c h ro m a tin due to the chro m o so m a l rearrangem ents. It appears, th a t the v ic in ity o f the h e te ro ch ro m a tin affects expression o f the w h ite gene, so th a t the gene is active in some cells, b u t n o t in the others, re s u ltin g in the d iffe re n t phenotypes o f the cells w ith the same genotype. The exp erim e nts have s h o w n th a t the d e cisio n w h e th e r to express the gene o r n o t is a stochastic one and, once made it is m a in ta in e d th ro u g h ce ll d iv is io n . Such a s ta b ly in h e rite d sile n c in g is n o t caused b y a loss o f the w h ite gene o r m u ta tio n s in the c o d in g region, b u t is the consequence o f the p o s itio n in g o f the gene a djacent to the h e te ro c h ro m a tin (H e n ik o ff, 1990; W e ile r and W a k im o to , 1995).
The PEV p h e n o m e n o n has been also o b serve d w h e n genes w h ic h are active in n a tu ra l h e te ro ch ro m a tic e n v iro n m e n t are m o v e d adjacent to the e u c h ro m a tin regions. For exam ple th e D ro s o p h ila 's lig h t gene, w h ic h is
n o rm a lly expressed in h e te ro c h ro m a tic e n v iro n m e n t, e x h ib its v a rie g a te d expression w h e n is placed adjacent to e u ch ro m a tin (W a k im o to and H ea rn , 1990). I t appears th e re fo re , th a t the h e te ro c h ro m a tin and e u c h ro m a tin re p re s e n t tw o d iffe r e n t s tr u c tu r a l e n v iro n m e n ts a n d c h a n g in g one e n v iro n m e n t fo r another m a y re s u lt in gene expression va rie g a tio n .
P E V in mammals. M o s t o f the studies on PEV in m a m m a ls ha ve been d o n e u s in g tra n s g e n ic te c h n o lo g y (D o b ie et al., 1997; K io u s s is a n d F e ste n ste in , 1997; G ro s v e ld et al., 1998). In a d d itio n , e v id e n c e has a c c u m u la te d im p lic a tin g p o s itio n e ffe c t re s u ltin g fro m c h ro m o s o m a l tra n sloca tio n s in several h u m a n diseases ( M ilo t et al., 1996b; K le in ja n and van H e y n in g e n , 1998).
Variegation of transgene expression in mice. T ra n s g e n ic te c h n o lo g y has m a d e i t p o s s ib le to s tu d y re g u la tio n o f m a m m a lia n genes in v iv o (G ro sve ld and K o llia s , 1992). F rom e a rly exp erim e nts i t has becom e clear th a t the in c lu s io n o f p ro m o te rs and enhancers w as n o t s u ffic ie n t to ensure tis s u e -s p e c ific an d p o s itio n - in té g r a tio n in d e p e n d e n t e x p re s s io n o f a transgene (P a lm ite r and B rin s te r, 1986). M a n y re p o rts have suggested v a rio u s fa c to rs a ffe c tin g tra n sg en e e x p re s s io n , h o w e v e r i t w a s th e id e n tific a tio n o f Locus C o n tro l Regions (LCRs) (G ro sve ld et al., 1987) w h ic h are capable o f e n s u rin g p ro p e r transgene e xp ressio n , th a t has le d to b re a k th ro u g h s in th e u n d e r s ta n d in g o f th e tra n s g e n e e x p re s s io n m e ch an ism . The LCRs, b y d e fin itio n , are able to d ire c t e x p re s s io n o f transgene in a tissu e -spe cific, c o p y -n u m b e r d e p e n d e n t m a n n e r, so th a t expression is n o t dependent on the transgene's site o f in te g ra tio n . D e le tio n analysis has sh o w n th a t m a n y LCRs (such as the h u m a n p -g lo b in , h C D 2 ,
h A D A a n d a T C R ) h a ve m o d u la r s tru c tu re s w it h d is tin c t re g io n s responsible fo r the enhancer and p o s itio n -in té g ra tio n in d e p e n d e n t, co p y- n u m b e r dependent fu n c tio n s (T albot et al., 1990; P ru z in a et al., 1991; D iaz and W in o to , 1994; Festenstein et al., 1996; A ro n o w et al., 1995; O rtiz et al., 1997). I t has been sh o w n in o u r la b o ra to ry th a t the hC D 2 LC R (Lang et al., 1988; Greaves et al., 1989; L a ng et al., 1991) co n ta in s a re g io n w it h o u t enhancer a c tiv ity (co rre s p o n d in g to the t h ir d D N A s e I H y p e rs e n s itiv ity Site, designated HSS3) w h ic h is necessary fo r p re v e n tin g PEV (Festenstein et al., 1996b). The hCD 2 transgene c a rry in g hC D 2 LC R w it h HSS3 deleted is subjected to p o s itio n effect v a rie g a tio n re m in is c e n t to the D ro s o p h ila 's PEV. In d e e d , i t appears th a t the d e c is io n as w h e th e r to express the transgene o r n o t is a stochastic one and m a in ta in e d th ro u g h cell d iv is io n . S im ila r results have been obtained in studies on the h u m a n p -g lo b in L C R (M ilo t et al., 1996b). C entrom e re -in d u ce d v a rie g a tio n was also described in details fo r p -la c to g lo b u lin transgenic m ice (D obie et al., 1996; D ob ie et al., 1997). M o sa ic exp ressio n p a tte rn o f transgenes has been d e s c rib e d in v a rio u s system (R obertson et al., 1995; P ravtcheva et al., 1994; G u y et al., 1996).
Position effect in human diseases. I t is k n o w n th a t m a n y h u m a n diseases are caused b y chrom osom al rearrangem ent w it h b reak p o in ts as fa r as 900 kb fro m the co d in g regions o f the genes, in d ic a tin g th a t ro le p o s itio n effect m a y p la y in h u m a n genetic diseases. H o w e v e r, in these cases the p o s itio n e ffect m a y be due the re s u lt o f ch ro m o so m a l re a rra n g e m e n t le a d in g to s e p a ra tio n (o r even d e le tio n ) o f th e c is -re g u la to ry e le m e n t fr o m the tra n s c rib in g u n it o f the gene. Such a m echanism w o u ld cle a rly be d iffe re n t fro m the one causing the classical PEV in D ro s o p h ila and fu rth e r research
is needed to im p ro v e o u r u n d e rs ta n d in g o f these diseases ( M ilo t et al., 1996b; K le in ja n and va n H eyn in ge n , 1998).
M o d ifie rs o f P E V
It has been sh o w n th a t expression o f v a rie g a tin g genes can be increased o r decreased as a re s u lt o f m u ta tio n s in over 50 lo c i (c o m m o n ly re fe rre d to as suppressors o r enhancers o f PEV). Some o f the genes have been cloned and th e ir p ro te in s id e n tifie d . M a n y m o d ifie rs o f PEV are chro m o so m a l p ro te in s or m o d ifie rs o f chrom osom al p ro te in s and therefore operate on a v a rie ty o f genes (Reuter and Spierer, 1992; W e ile r and W a k im o to , 1995).
H P l and Pc. H P l is a h ig h ly conserved p ro te in coded b y Su(var)205 gene (Eissenberg et al., 1990; H ea rn et al., 1991; Eissenberg et al., 1992). The N - te rm in a l re g io n o f H P l co n ta in s a c h ro m o d o m a in , h o m o lo g o u s w it h P olycom b (Pc) p ro te in . The Pc fa m ily p ro te in s are in v o lv e d in m aintenance o f the repressive state o f the hom eotic genes. N e ith e r H P l n o r Pc appear to b in d d ire c tly to D N A , b u t ra th e r p a rtic ip a te in fo rm a tio n o f m u ltip ro te in com plexes (K o o n in et al., 1995). A p a rt fro m the ch ro m o d o m a in , the H P l p ro te in also contains a so called "shadow " ch ro m o d o m a in w h ic h alone is s u ffic ie n t to ta rg e t H P l to h e te ro c h ro m a tin (Powers and Eissenberg, 1993; A asland and Stewart, 1995). Thus, it is possible th a t H P l serves as a b rid g in g m o le cu le , b r in g in g to g e th e r p ro te in s th a t in te ra c t w it h th e ch ro m o and "s h a d o w " ch ro m o d o m ain s. H P l and some o th e r m o d ifie rs are able to cause a dosage-dependent s h ift in the v a rie g a tio n o f b o th e u chro m a tic and
h e te ro c h ro m a tic genes. F or exam ple, it has been s h o w n th a t m u ta tio n in one o f the alleles fo r the H P l p ro te in leads to increased expression o f the v a rie g a tin g w h ite gene, w hereas o v e re x p re s s io n o f H P l re s u lts in a decreased expression o f the v a rie g a tin g w h ite gene. The v a rie g a tin g lig h t gene therefore e xh ib its the converse H P l dosage-dependent response.
In spite o f the shared ch ro m o d o m a in , H P l and Pc have v e ry d iffe re n t, a lm o s t m u tu a lly e x c lu s iv e , d is t r ib u tio n p a tte rn s o n th e p o ly th e n e chrom osom es, w it h H P l p rim a ry concentrated in the p e ric e n tric regions (K o o n in et al., 1995). The lo c a lis a tio n o f H P l m a y e x p la in th e fa ct th a t m u ta tio n s in H P l o n ly a ffe c t w h ite gene e xp re s s io n w h e n th e gene integrates in to p e ric e n tric b u t n o t te lo m e ric h e te ro c h ro m a tin (W e ile r and W a k im o to , 1995; W a llra th and E lg in , 1995).
F o rm a tio n o f the PC com plexes seems to be d e te rm in e d b y the presence o f D N A elem ents designated as P olycom b Response E lem ents (PREs). P lacing th e PRE e lem ents n e x t to the G A L 4 in d u c ib le re p o rte r gene causes