I t has been s h o w n th a t a 28 kb genom ic fra g m e n t c o n s is tin g o f 5 kb 5'- fla n k in g sequences, 14 kb c o n ta in in g the gene's fiv e exons a n d 9 kb o f 3' fla n k in g sequences, can d ire c t the expression o f the C D 2 gene in T -c e ll s p e c ific , c o p y -n u m b e r d e p e n d e n t, tra n s g e n e -in te g ra tio n in d e p e n d e n t m a n ne r. The e xpression o f each c o p y o f the transgene o n T -ce lls w as s im ila r to endogenous levels (Lang et al., 1988 EM B O ). I t w as co n c lu d e d th e re fo re , th a t the 28 kb C D 2 fra g m e n t c o n ta in e d a ll th e in fo r m a tio n needed fo r h ig h le v e l, tissu e -spe cific, p o s itio n in d e p e n d e n t e xpression. F u r th e r c h a ra c te ris a tio n re v e a le d tw o T -c e ll s p e c ific D N A s e I h y p e rs e n s itiv ity sites in the 3 '-fla n k in g sequences o f the h u m a n C D 2 gene. A n o th e r site w as fo u n d im m e d ia te ly u p s tre a m o f the fir s t exon in the p ro m o te r re g io n (Greaves et aL, 1989). D r O w e n and colleagues have sh o w n th a t th e 5'- and 3'- re g io n s o f the gene are u n d e rm e th y la te d in C D 2 expressing T-cells b u t extensively m e th yla te d in other cell types (W o tto n et aL, 1989).
im m e d ia te ly d o w n stre a m o f the gene and c o n ta in in g tw o h y p e rs e n s itiv ity sites was able to confer a T-cell specific, co p y -n u m b e r dependent, p o s itio n - in t é 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 lin k e d h o m o lo g o u s a n d h e terologous genes (Greaves et aL, 1989). Subsequent d e le tio n analysis has a llo w e d us to establish the m in im u m sequences necessary fo r the co p y- n u m b e r d e pe n d e nt, s ite -in te g ra tio n in d e p e n d e n t expression. I t has been s h o w n th a t 2 kb o f the 3 '-fla n k in g sequences lin k e d to the hC D 2 m in ig e n e (genom ic hC D 2 fra g m e n t w ith a ll b u t the firs t exon re m o ve d and 5 kb 5'- p ro m o te r sequences) is s u ffic ie n t to a llo w T -ce ll specific, co p y dependent, in te g ra tio n -in d e p e n d e n t expression in transgenic m ice (L a ng et al., 1991; Festenstein et al., 1996). H o w e ve r, LC R sequences are n o t able to overcom e th e in h ib itin g effects o f p ro k a ry o tic sequences (G re e n be rg , 1992, P hD Thesis). The 2 kb re g io n appears to consist o f T -cell specific enhancer (Lake et al., 1990) w h ile a d d itio n a l sequences w ith o u t enhancer a c tiv ity co in cid e w ith the th ir d H y p e rs e n s itiv ity Site (HSS3). The HSS3 sequences appear to be re q u ire d fo r the p re v e n tio n o f PVE in transgenic m ice (Festenstein, et aL, 1996). F o o tp r in t a n a ly s is o f the h C D 2 e n ha n ce r sequences re v e a le d p o te n tia l G A T A , E lf l, CRE, L y F l and Sox4 b in d in g sites. I t has been suggested th a t Sox4 transactivates the h u m a n C D2 enhancer (W o tto n et aL, 1995).
hCD2 promoter. T w o D N A s e I h y p e rs e n s itiv ity sites w ere m a p p e d in the p ro m o te r re g io n . The fir s t site was fo u n d im m e d ia te ly u p s tre a m o f the tra n s c rip tio n in itia tio n re g io n and the second site appears to be located at a b o u t
-2
kb (W o tto n et aL, 1989; Greaves et aL, 1989; O u tra m and O w e n , 1994). The hC D 2 p ro m o te r is T A T A -le ss and uses m u ltip le sites to in itia tetra n s c rip tio n (Greenberg, 1992, P hD Thesis; O u tra m and O w en, 1994). Just u p stre a m o f the tra n s c rip tio n in itia tio n re g io n (at -20, -65; re la tiv e to the tra n s la tio n start site) the fo llo w in g sequences have been id e n tifie d : a G A T A m o tif at -260, G C -rich S P l-lik e m o tif at -160 and an E bo x m o tif C A C G T G at -95. M u ta tio n o f the E b o x abolishes tra n s c rip tio n fro m the C D 2 p ro m o te r in ce ll tra n s fe c tio n assay. A n a ly s is o f the p ro te in s in the T -c e ll e x tra c t revealed a USE p ro te in b o u n d to this site (O u tra m and O w en, 1994). A C D2 m in ig e n e w h ic h re ta in s o n ly 400 b p o f the 5 -fla n k in g re g io n s has been sh o w n to be able to express in transgenic m ice at a h ig h le ve l in a p o s itio n in d e p e n d e n t m anner. F u rth e r d e le tio n w h ic h leaves a b o u t 100 b p o f 5 fla n k in g re g io n , s ig n ific a n tly reduces the le ve l o f expression (G reenberg, 1992, PhD Thesis).
Potential regulatory elements w ith in the introns o f the hCD2. D N A s e I h y p e rs e n s itiv ity analysis in d ic a te d the presence o f a h yp e rs e n s itiv e re g io n in the fo u rth in tro n o f C D 2 gene (M a m a la k i, u n p u b lis h e d ). T h is fin d in g w as re c o n firm e d la te r and in a d d itio n , o th e r h y p e rs e n s itiv e sites w e re fo u n d in the th ir d in tro n . I t has been also s h o w n th a t a hC D 2 m axigene expresses fo u r to fiv e tim e s h ig h e r c o m p a re d to a h C D 2 m in ig e n e (co n ta in in g the hC D 2 gene w ith a ll b u t the firs t in tro n deleted, along w it h a 5 kb 5' p ro m o te r re g io n and 5.5 kb 3 '-fla n k in g regions) in transgenic m ice (Festenstein, 1996, PhD Thesis).