transcriptional regulation of interleukin 3 gene expression.pdf

8/14/2019 Transcriptional regulation of interleukin 3 gene expression.pdf

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P r o c . N a t l . A c a d . S c i . USAV o l . 8 7 , p p . 9 6 5 0 - 9 6 5 4 , D e c e m b e r 1 9 9 0B i o c h e m i s t r y

T r a n s c r i p t i o n a l r e g u l a t i o n o f i n t e r l e u k i n 3 g e n e e x p r e s s i o ni n T l y m p h o c y t e s( t r a n s c r i p t i o n f a c t o r A P - 4 / D N A b i n d i n g p r o t e i n s / t r a n s c r i p t i o n f a c t o r N F - I L 3 - A )

SARA G . S H O E M A K E R , ROBERT H R O M A S , AND KENNETH KAUSHANSKY*D e p a r t m e n t o f M e d i c i n e , D i v i s i o n o f H e m a t o l o g y a t t h e U n i v e r s i t y o f W a s h i n g t o n , S e a t t l e , WA 9 8 1 9 5C o m m u n i c a t e d b y E a r l W . D a v i e , S e p t e m b e r 6 , 1 9 9 0 ( r e c e i v e d f o r r e v i e w A p r i l 1 8 , 1 9 9 0 )ABSTRACT I n t e r l e u k i n 3 ( I L - 3 o r m u l t i - c o l o n y - s t i m u l a t -i n g f a c t o r ) p l a y s a n i m p o r t a n t r o l e i n t h e h e m a t o p o i e t i c r e -s p o n s e t o i n f l a m m a t o r y s t i m u l i t h r o u g h i t s a c t i o n o n b o t hi m m a t u r e a n d m a t u r e b l o o d c e l l s . L i k e o t h e r l y m p h o k i n e s ,I L - 3 i s p r o d u c e d i n r e s p o n s e t o a c t i v a t i o n o f t h e T - c e l l r e c e p t o ra n d p r o t e i n k i n a s e C p a t h w a y s . B y u s i n g n u c l e a r r u n - o n a s s a y so f q u i e s c e n t a n d s t i m u l a t e d T - c e l l l i n e s , w e d e m o n s t r a t e t h a tI L - 3 g e n e e x p r e s s i o n i s c o n t r o l l e d , a t l e a s t i n p a r t , a t t h e l e v e lo f t r a n s c r i p t i o n . F u n c t i o n a l r e p o r t e r g e n e a n a l y s i s w a s u s e d t od e l i n e a t e t w o r e g i o n s o f t h e I L - 3 5 ' f l a n k i n g s e q u e n c e r e s p o n -s i b l e f o r t r a n s c r i p t i o n a l s t i m u l a t i o n . DN A b i n d i n g p r o t e i n st h a t p o t e n t i a l l y m e d i a t e t h e s e r e s p o n s e s w e r e t h e n r e c o g n i z e db y m o b i l i t y - s h i f t a n d D N a s e f o o t p r i n t i n g a s s a y s . O n e r e g i o nr e s p o n s i b l e f o r t r a n s c r i p t i o n a l e n h a n c e m e n t w a s l o c a l i z e d t ot h e s e q u e n c e GATGAATAAT, t h e c o g n a t e s i t e o f a t r a n s c r i p -t i o n f a c t o r , h e r e t e r m e d N F - I L 3 - A . A s e c o n d r e g i o n o f f u n c -t i o n a l a c t i v i t y a n d p r o t e i n b i n d i n g w a s l o c a l i z e d t o a s i n g l et r a n s c r i p t i o n f a c t o r A P - 1 s i t e . I n a d d i t i o n t h r e e f u n c t i o n a l l yi n h i b i t o r y r e g i o n s w e r e i d e n t i f i e d . T h e s e r e s u l t s , a l o ng w i t h t h ef u r t h e r c h a r a c t e r i z a t i o n o f N F - I L 3 - A , w i l l c o n t r i b u t e t o t h eu n d e r s t a n d i n g o f I L - 3 g e n e r e g u l a t i o n i n s t i m u l a t e d T c e l l s .Among t h e g r o w t h f a c t o r s i n v o l v e d i n h e m a t o p o i e s i s , i n t e r -l e u k i n 3 ( I L - 3 o r m u l t i - c o l o n y - s t i m u l a t i n g f a c t o r ) a f f e c t s t h ew i d e s t v a r i e t y o f c e l l t y p e s . I n c o n j u n c t i o n w i t h o t h e r g r o w t hf a c t o r s , I L - 3 a c t s o n m a r r o w - d e r i v e d p r o g e n i t o r c e l l s t os t i m u l a t e t h e d e v e l o p m e n t o f e r y t h r o c y t e s , g r a n u l o c y t e s ,m a c r o p h a g e s , a n d m e g a k a r y o c y t e s ( 1 , 2 ) . I n a d d i t i o n , i ti n f l u e n c e s t h e g r o w t h a n d a c t i v a t i o n o f l y m p h o c y t e s ( 3 ) ,p r o m o t e s t h e s e l f - r e n e w a l o f h e m a t o p o i e t i c p r o g e n i t o r c e l l s( 4 ) , a n d a c t s u p o n m a t u r e l e u k o c y t e s t o e n h a n c e t h e i r f u n c -t i o n a l a c t i v i t y ( 5 , 6 ) . I n t h i s w a y , I L - 3 i s t h o u g h t t o b ei n v o l v e d i n t h e h o s t r e s p o n s e t o i n f e c t i o u s , i n f l a m m a t o r y ,a n d i m m u n o l o g i c s t i m u l i .C o n s t i t u t i v e e x p r e s s i o n o f I L - 3 h a s n o t b e e n o b s e r v e d i na n y n o r m a l c e l l y e t e x a m i n e d . B y u s i n g t h e p o l y m e r a s e c h a i nr e a c t i o n , mRNA s p e c i f i c f o r h u m a n I L - 3 h a s b e e n i d e n t i f i e di n a n u m b e r o f c e l l s o u r c e s ( 7 ) , b u t I L - 3 mRNA h a s o n l y b e e nf o u n d i n T l y m p h o c y t e s o r T - c e l l l i n e s t r e a t e d w i t h s t i m u l a -t o r s o f t h e T - c e l l r e c e p t o r o r o f t h e p r o t e i n k i n a s e C p a t h w a y( 8 ) . T h e m o l e c u l a r b a s i s f o r t h i s h i g h l y i n d u c i b l e a n d s e l e c t i v et i s s u e s p e c i f i c i t y i s u n c e r t a i n .To f u r t h e r u n d e r s t a n d t h e r e g u l a t i o n o f I L - 3 p r o d u c t i o n ,we h a v e c l o n e d t h e g e n e f o r h u m a n I L - 3 a n d h a v e p e r f o r m e da d e t a i l e d a n a l y s i s o f t h e 6 8 2 - b a s e - p a i r ( b p ) r e g i o n u p s t r e a mo f t h e s t a r t o f I L - 3 t r a n s c r i p t i o n . O u r r e s u l t s s u g g e s t t h a t I L - 3g e n e e x p r e s s i o n i s c o n t r o l l e d , a t l e a s t i n p a r t , a t t h e l e v e l o ft r a n s c r i p t i o n a n d t h a t b i n d i n g o f t r a n s - a c t i n g f a c t o r s a t t w od i s t i n c t s i t e s m e d i a t e much o f t h i s r e s p o n s e .

MATERIALS A N D M E T H O D SC e l l L i n e s a n d C u l t u r e . T h e T - l y m p h o c y t e c e l l l i n e J u r k a t( A T C C ) , t h e c e r v i c a l c a r c i n o m a c e l l l i n e HeLa ( A T C C ) , a n d

t h e l e u k e m i a c e l l l i n e AML-193 w e r e m a i n t a i n e d i n D u l b e c -c o ' s m o d i f i e d E a g l e ' s m e d i u m s u p p l e m e n t e d w i t h 10 ( v o l /v o l ) f e t a l c a l f s e r u m , g l u t a m a t e , a n d a n t i b i o t i c s . T h e g i b b o nT - l y m p h o c y t e c e l l l i n e MLA-144 ( A T C C ) w a s m a i n t a i n e d i nRPMI 1 6 4 0 m e d i u m s u p p l e m e n t e d a s n o t e d a b o v e . AML-193c e l l s w e r e a l s o s u p p l e m e n t e d w i t h r e c o m b i n a n t h u m a n g r a n -u l o c y t e / m a c r o p h a g e c o l o n y - s t i m u l a t i n g f a c t o r .N o r t h e r n B l o t A n a l y s i s . W h o l e c e l l u l a r RNA w a s e n r i c h e df o r p o l y ( A ) - c o n t a i n i n g t r a n s c r i p t s b y c h r o m a t o g r a p h y o no l i g o ( d T ) - c e l l u l o s e , s i z e - f r a c t i o n a t e d b y f o r m a l d e h y d e /a g a r o s e g e l e l e c t r o p h o r e s i s , a n d t r a n s f e r r e d t o n i t r o c e l l u l o s e( 9 ) . T h e b l o t s w e r e h y br i d i z ed w i t h f u l l - l e n g t h cDNA p r o b e sf o r g i b b o n I L - 3 ( 1 0 ) , m u r i n e c - j u n ( 1 1 ) , h u m a n cAMP-r e s p o n s i v e e l em en t b i n d i n g p r o t e i n (CREB) ( 1 2 ) , b o v i n e1 3 - a c t i n ( 1 3 ) , a n d a 1 . 1 - k i l o b a s e f r a g m e n t c o n t a i n i n g t h e f i r s te x o n o f t h e h u m a n c - f o s g e n e ( 1 4 ) , a l l l a b e l e d b y r a n d o mp r i m i n g ( A m e r s h a m ) , o r w i t h o l i g o n u c l e o t i d e s f o r h u m a nJUN-B [ b a s e s 7 0 9 - 7 3 8 ( s e q u e n c e k i n d l y p r o v i d e d b y R i c h a r dT u r n e r , U n i v e r s i t y o f C a l i f o r n i a , S a n F r a n c i s c o ) ] a n d h u m a nC R E - B P - 1 [ b a s e s 7 3 - 9 6 ( 1 5 ) ] l a b e l e d w i t h [ y - 3 2 P ] A T P b yp o l y n u c l e o t i d e k i n a s e . T h e b l o t s w e r e w a s h e d t w i c e a t 2 0 ' Ca n d t w i c e a t 6 5 0 C i n 0 . 2 x S S C / 0 . 1 NaDodSO4 ( 1 x SSC =0 . 1 5 M N a C I / 0 . 0 1 5 M s o d i u m c i t r a t e , pH 7 . 0 ) f o r t h e cDNAa n d g e n o m i c p r o b e s o r i n 3 M t e t r a m e t h y l a m m o n i u m c h l o r i d ea t 6 8 0 C o r 6 2 0 C f o r t h e 3 0 - o r 2 4 - b p o l i g o n u c l e o t i d e p r o b e s ,r e s p e c t i v e l y ( 1 6 ) .N u c l e a r R u n - O n A n a l y s i s . C e l l l i n e s w e r e g r o w n t o 1 X 1 0 6c e l l s p e r m l a n d t h e n s t i m u l a t e d w i t h c o n c a n a v a l i n A ( C o n A ;B o e h r i n g e r M a n n h e i m ) a t 1 0 g g / m l a n d p h o r b o l 1 2 -m y r i s i t a t e 1 3 - a c e t a t e ( P M A ; S i g m a ) ( 1 0 0 n g / m l ) f o r 1 5 m i n t o2 4 h r . C e l l s w e r e w a s h e d a n d l y s e d , a n d l a b e l e d r u n - o nt r a n s c r i p t s w e r e p r o d u c e d f r o m t h e r e s u l t a n t n u c l e i a s d e -s c r i b e d ( 9 ) . A n t i s e n s e s i n g l e - s t r a n d e d cDNA f o r g i b b o n I L - 3( 1 0 ) , h u m a n g r a n u l o c y t e / m a c r o p h a g e - c o l o n y - s t i m u l a t i n gf a c t o r ( 1 7 ) , h u m a n 8 - a c t i n ( 1 8 ) , a n d M1 3 w e r e s l o t - b l o t t e d ,p r e h y b r i d i z e d , a n d t h e n h y b r i d i z e d f o r 7 2 h r a t 6 5 0 C w i t h a ne q u a l a m o u n t o f r a d i o a c t i v i t y o f e a c h o f t h e r u n - o n R N As ( 9 ) .T h e b l o t s w e r e w a s h e d t w i c e a t 2 0 ' C a n d t w i c e a t 6 5 0 C i n0 . 1 x S S C / 0 . 1 N a D o d S O 4 a n d t h e n e x p o s e d t o f i l m .C o n s t r u c t i o n o f H y b r i d G e n e s . A 4 0 - b a s e a n t i s e n s e o l i g o -n u c l e o t i d e d e r i v e d f r o m a p u b l i s h e d I L - 3 cDNA s e q u e n c e( b a s e s 7 3 - 1 1 2 ; r e f . 1 0 ) was u s e d t o s c r e e n a h u m a n g e n o m i cf e t a l l i v e r l i b r a r y i n A C h a r o n 4 A , a s d e s c r i b e d ( 1 9 ) w i t h t h ee x c e p t i o n t h a t 3 M t e t r a m e t h y l a m m o n i u m c h l o r i d e a t 7 3 0 Cw a s u s e d t o s e t t h e w a s h s t r i n g e n c y ( 1 6 ) . A f u l l - l e n g t h c l o n eo f t h e h u m a n I L - 3 g e n e was t h u s o b t a i n e d . A 9 2 5 - b p P s t If r a g m e n t c o n t a i n i n g t h e 5 ' f l a n k i n g r e g i o n o f t h e g e n e wast h e n s u b c l o n e d i n t o M1 3 ( t e r m e d I L - 3 p r ) , s e q u e n c e d b y t h ed i d e o x y n u c l e o t i d e c h a i n - t e r m i n a t i o n m e t h o d a n d a BamHIs i t e w a s i n t r o d u c e d 4 2 b p d o w n s t r e a m o f t h e s i t e o f t r a ns c r i p t i o n i n i t i a t i o n b y s i t e - d i r e c t e d m u t a g e n e s i s ( 1 7 ) . B yA b b r e v i a t i o n s : I L - 3 , i n t e r l e u k i n 3 ; C o n A , c o n c a n a v a l i n A ; PMA,p h o r b o l 1 2 - m y r i s i t a t e 1 3 - a c e t a t e ; C A T , c h l o r a m p h e n i c o l a c e t y l -t r a n s f e r a s e ; C R E B , c A M P - r e s p o n s i v e e l e m e n t b i n d i n g p r o t e i n .* T o wh om r e p r i n t r e q u e s t s s h o u l d b e a dd r es s ed .

9650

T h e p u b l i c a t i o n c o s t s o f t h i s a r t i c l e w e r e d e f r a y e d i n p a r t b y p a g e c h a r g ep a y m e n t . T h i s a r t i c l e m u s t t h e r e f o r e b e h e r e b y m a r k e d a d v e r t i s e m e n ti n a c c o r d a n c e w i t h 1 8 U . S . C . 1 7 3 4 s o l e l y t o i n d i c a t e t h i s f a c t .


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P r o c . N a t i . A c a d . S c i . USA 8 7 ( 1 9 9 0 ) 9 6 5 1u s i n g t h i s s i t e , f r a g m e n t s o f t h e 5 ' I L - 3 f l a n k i n g r e g i o n o fs u c c e s s i v e l y l a r g e r s i z e w e r e l i n k e d t o a c l o n e o f t h e b a c t e r i a lc h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e ( C A T ) g e n e . T h e s e p r o -m o t e r - r e p o r t e r c o n s t r u c t s w e r e a s s e m b l e d i n pUC18 b yu s i n g s t a n d a r d t e c h n i q u e s a n d w e r e n u m b e r e d a c c o r d i n g t ot h e n u c l e o t i d e s i z e o f t h e p r o m o t e r c o n s t r u c t c o u n t i n g f r o mt h e c a p s i t e . A l l c o n s t r u c t i o n s w e r e s e q u e n c e d f o r c o n f i r m a -t i o n .E l e c t r o p o r a t i o n a n d F u n c t i o n a l A n a l y s i s . C e l l s w e r ew a s h e d a n d r e s u s p e n d e d i n H e p e s - b u f f e r e d s a l i n e ( 3 0 mMH e p e s , pH 7 . 0 5 / 1 3 7 mM N a C l / 5 mM K C I / 1 0 mM s o d i u mp h o s p h a t e ) a t 1 x 1 0 7 c e l l s p e r m l a l o n g w i t h 2 0 , u g o f e a c hc o n s t r u c t DNA a n d 1 0 , u g o f t h e p l a s m i d v e c t o r R S V - / 3 - G a l( t o p r o v i d e a c o n t r o l f o r t h e r e l a t i v e e f f i c i e n c y o f t r a n s f e c -t i o n ) . E l e c t r o p o r a t i o n w a s c a r r i e d o u t u s i n g t h e C e l l p o r a t o rd e v i c e ( B R L ) w i t h 7 5 0 V/cm f o r 9 m s e c a t 2 0 ' C . T h e c e l l sw e r e t h e n d i v i de d a n d c u l t u r e d i n c o n t r o l m e d i u m o r m e d i u ms u p p l e m e n t e d w i t h C o n A ( 1 0 p u g / m l ) a n d P MA ( 1 0 0 n g / m l ) .A f t e r c u l t u r e f o r 1 (MLA) o r 3 ( J u r k a t ) d a y s , c e l l l y s a t e s w e r ea s s a y e d f o r CAT a n d f B - g a l a c t o s i d a s e a c t i v i t y a n d c a l c u l a t e da s d e s c r i b e d ( 9 ) .M o b i l i t y - S h i f t a n d D N a s e I F o o t p r i n t i n g A s s a y s . N u c l e a re x t r a c t s w e r e p r ep a r e d a c c o r d i n g t o D i g n a m e t a l . ( 2 0 ) .D u p l e x o l i g o n u c l e o t i d e s r e p r e s e n t i n g v a r i o u s r e g i o n s o f t h e

I L - 3 p r o m o t e r ( s e e F i g . 2 B ) w e r e e n d - l a b e l e d w i t h [ y _ 3 2 p ] _ATP b y u s i n g p o l y n u c l e o t i d e k i n a s e . N u c l e a r e x t r a c t ( 1 0 , u g )o r p u r i f i e d r e c o m b i n a n t c - j u n p r o t e i n ( 2 0 n g ) w a s p r e i n c u -b at e d w i t h 5 0 n g o f c o m p e t i t o r d u p l e x o l i g o n u c l e o t i d e i n 1 0 A l o f b i n di n g b u f f e r [ 1 0 mM H e p e s , pH 7 . 9 / 5 0 mM N a C I / 3( v o l / v o l ) g l y c e r o l / 1 . 2 5 mM EDTA/2 mM d i t h i o t h r e i t o l /p o l y ( d I - d C ) ( 0 . 1 m g / m l ) ] f o r 1 5 m i n a n d t h e n 1 n g o f 3 2 P -l a b e l e d d u p l e x o l i g o n u c l e o t i d e w a s a d d e d a n d t h e r e a c t i o nw a s i n c u b a t e d f o r a n a d d i t i o n a l 2 0 m i n a t 2 0 C . T h e r e a c t i o np r o d u c t s w e r e f r a c t i o n a t e d b y e l e c t r o p h o r e s i s t h r o u g h 4p o l y a c r y l a m i d e g e l s ( 5 0 mM T r i s , pH 8 . 3 / 4 0 0 mM g l y c i n e / 2mM EDTA) a t 1 0 0 V . T h e g e l s w e r e d r i e d a n d e x p o s e d t of i l m . DNase I ( W o r t h i n g t o n ) p r o t e c t i o n a s s a y s w e r e p e r -f o r m e d a s d e s c r i b e d ( 2 1 ) i n 5 0 - , l d r e a c t i o n m i x t u r e s c o n t a i n -i n g m o b i l i t y - s h i f t a s s a y b u f f e r , 2 5 , u g o f n u c l e a r e x t r a c t , a n d1 n g o f a S t u I - S m a I ( p o s i t i o n s - 3 1 3 t o - 5 9 ) f r a g m en t o f t h eI L - 3 p r o m o t e r e n d - l a b e l e d w i t h [ y - 3 2 P ] A T P . D i g e s t i o n p r o d -u c t s w e r e s i z e - f r a c t i o n a t e d o n 6 a c r y l a m i d e / u r e a s e q u e n c -i n g g e l s a d j a c e n t t o s e q u e n c i n g r e a c t i o n p r o d u c t s .

RESULTSN u c l e a r Run-On A n a l y s i s . To d e t e r m i n e t h e m e c h a n i s m st h a t c o n t r i b u t e t o t h e r e g u l a t i o n o f I L - 3 g e n e e x p r e s s i o n ,N o r t h e r n b l o t a n a l y s i s w a s p e r f o r m e d o n J u r k a t a n d MLAc e l l s . I L - 3 - s p e c i f i c t r a n s c r i p t s w e r e d e t e c t e d w i t h i n 9 0 m i n t o6 h r o f m i t o g e n i c s t i m u l a t i o n ( F i g . 1 ) . B y u s i n g n u c l e a r r u n - o na n a l y s i s , b o t h T - c e l l l i n e s w e r e o b s e r v e d t o a c t i v e l y t r a n -s c r i b e t h e I L - 3 g e n e w i t h i n 2 h r o f s t i m u l a t i o n . T r a n s c r i p t i o na p p e a r e d t o p e a k a t 6 h r b u t w a s s t i l l p r e s e n t a t 2 4 h r .F u n c t i o n a l A n a l y s i s o f t h e 5 ' R e g i o n o f t h e I L - 3 G e n e . To

i d e n t i f y r e g i o n s w i t h i n t h e I L - 3 g e n e o f p o s s i b l e f u n c t i o n a ls i g n i f i c a n c e , h o m o l o g y s e a r c h e s w e r e p e r f o r m e d u s i n g 6 8 2b p o f t h e I L - 3 5 ' f l a n k i n g r e g i o n . S e v e r a l s e q u e n c e s c o m -p l e t e l y o r p a r t i a l l y m a t c h i n g t h e b i n d i n g s i t e s o f c h a r a c t e r i z e dt r a n s c r i p t i o n p r o t e i n s w e r e d e t e c t e d ( F i g . 2 A ) . T o d e t e r m i n et h e f u n c t i o n a l s i g n i f i c a n c e o f t h e s e s e q u e n c e s f o r e x p r e s s i o no f t h e I L - 3 g e n e , r e p o r t e r g e n e a n a l y s i s w a s n e x t p e r f o r m e d .A s s h o w n i n F i g . 2 B , c o n s t r u c t s c o n t a i n i n g 1 6 1 o r 3 1 3 b po f t h e I L - 3 5 ' f l a n k i n g s e q u e n c e l e a d t o t h e h i g h e s t r e p o r t e rg e n e a c t i v i t i e s i n s t i m u l a t e d J u r k a t c e l l s . A s l i t t l e a s t h e 4 4 b pu p s t r e a m o f t h e c a p s i t e c o u l d d r i v e t h e l o w l e v e l o f c o n s t i -t u t i v e p r o m o t e r a c t i v i t y n o t e d i n a l l o f t h e c o n s t r u c t s , b u t t h i s p r o m o t e r was p o o r l y i n d u c i b l e , r e s u l t i n g i n o n l y 7 o f t h em a x i m a l l e v e l o f r e p o r t e r g e n e a c t i v i t y s e e n i n c o n s t r u c t 3 1 3 .T h e s u b s t a n t i a l v a r i a t i o n s i n p r o m o t e r f u n c t i o n a m o n gc o n s t r u c t s 3 1 3 t h r o u g h 1 2 6 s u g g e s t t h e p r e s e n c e o f s e v e r a l

I L - 3A 120 O

1 2 3 4

P a c t i n.0

34

1 3B 2 _ W 4C 2 _D 2 _

3434

F I G . 1 . N o r t h e r n b l o t a n d n u c l e a r r u n - o n a n a l y s e s o f I L - 3 g e n et r a n s c r i p t i o n . ( L e f t ) P o l y ( A ) RNA ( 5 , g ) f r o m MLA c e l l s ( l a n e 1 ) o rMLA c e l l s s t i m u l a t e d f o r 1 5 m i n , 9 0 m i n , o r 6 h r w i t h Co n A a n d P MA( l a n e s 2 - 4 , r e s p e c t i v e l y ) w a s p r o b e d w i t h a 3 2 P - l a b e l e d cDNA f o rg i b b o n I L - 3 o r b o v i n e f 3 - a c t i n , a s i n d i c a t e d . S i m i l a r r e s u l t s w e r eo b t a i n e d w i t h J u r k a t c e l l s . ( R i g h t ) 3 2 P - l a b e l e d r u n - o n n u c l e a r t r a n -s c r i p t s w e r e p r o d u c e d f r o m MLA c e l l s ( g e l A ) o r f r o m MLA c e l l ss t i m u l a t e d w i t h C o n A a n d P MA f o r 2 h r , 6 h r , o r 2 4 h r ( g e l s B - D ,r e s p e c t i v e l y ) a n d h y b r i d i z e d t o a n t i s e n s e s i n g l e - s t r a n d e d cDNA f o rg i b b o n I L - 3 ( b a n d s 1 ) , h u m a n g r a n u l o c y t e / m a c r o p h a g e - c o l o n y -s t i m u l a t i n g f a c t o r ( b a n d s 2 ) , h u m a n / 8 - a c t i n ( b a n d s 3 ) , o r p a r e n t a lp h a g e M1 3 ( b a n d s 4 ) .r e g i o n s o f f u n c t i o n a l i m p o r t a n c e . T h e s t i m u l a t e d p r o m o t e ra c t i v i t y n o t e d i n c o n s t r u c t 1 2 6 w a s i n c r e a s e d 5 . 2 - f o l d w h e nt h e s e q u e n c e b e t w e e n p o s i t i o n s - 1 2 6 a n d - 1 6 1 w a s a d d e d ,a r e g i o n c o n t a i n i n g s i m i l a r i t i e s w i t h t h e b i n d i n g s i t e s f o r t h et r a n s c r i p t i o n a l r e g u l a t o r s CREB, N F - I L 2 - A , a n d N F A T - 1 .An a d d i t i o n a l 2 . 4 - f o l d i n c r e a s e i n r e p o r t e r g e n e a c t i v i t y w a sn o t e d w h e n t h e r e g i o n b e t w e e n p o s i t i o n s - 2 7 2 a n d - 3 1 3 w a sa d d e d t o c o n s t r u c t 2 7 2 . T h i s r e g i o n c o n t a i n s a t r a n s c r i p t i o nf a c t o r A P - 1 s e q u e n c e a t p o s i t i o n - 3 0 1 . A c o m p a r a b l e p a t t e r no f p r o m o t e r f u n c t i o n w a s n o t e d i n MLA-144 c e l l s b u t t h em a g n i t u d e o f t h e s e r e s p o n s e s w a s m o r e p r o m i n e n t ( F i g . 2 B ) .T h e p r e c i s e l o c a t i o n o f t h e f i r s t ( p o s i t i o n s - 1 6 1 t o - 1 2 6 )e l e m e n t a n d i t s i m p o r t a n c e t o t h e f u n c t i o n a l a c t i v i t y o f t h ep r o m o t e r w a s c o n f i r m e d b y m u t a t i n g t h e I L - 3 f l a n k i n g s e -q u e n c e b e t w e e n p o s i t i o n s - 1 5 6 a n d - 1 4 7 t o a r a n d o ms e q u e n c e ( F i g . 2 B , c o n s t r u c t 3 5 6 N F I L 3 A - ) . T h i s m u t a t i o n

n e a r l y e l i m i n a t e d i n d u c i b l e r e p o r t e r g e n e a c t i v i t y . I n c o n -t r a s t , m u t a t i o n o f t h e C R E B b i n d i n g s i t e i m m e d i a t e l y d o w n -s t r e a m o f t h i s p o s i t i o n i n c r e a s e d r e p o r t e r g e n e a c t i v i t y ( c o n -s t r u c t 1 6 1 C R E B 1 )To d e t e r m i n e w h e t h e r t h e t w o r e g i o n s t h a t a c t i n c i s t oa u g m e n t r e p o r t e r g e n e a c t i v i t y c o n f e r i n d u c i b i l i t y u p o n ah e t e r o l o g o u s p r o m o t e r , t h e 5 ' f l a n k i n g r e g i o n o f I L - 3 g e n ew a s c l o n e d u p s t r e a m o f t h e m a j o r l a t e p r o m o t e r o f a d e n o v i -r u s ( m l p ) a n d t h e CAT g e n e . I n s i x e x p e r i m e n t s , a d d i t i o n o ft h e s e q u e n c e b e t w e e n p o s i t i o n s - 3 1 3 a n d - 5 9 u p s t r e a m o ft h e c a p s i t e r e s u l t e d i n a 1 2 . 9 - f o l d i n d u c i b i l i t y o f t h e m l p i nr e s p o n s e t o T - c e l l s t i m u l a t i o n , c o m p a r e d w i t h a 1 . 6 - f o l di n d u c t i o n w i t h t h e s e a g e n t s i n a c o n s t r u c t c o n t a i n i n g t h e m l pa n d CAT a l o n e . No CAT a c t i v i t y w a s d e t e c t a b l e w h e n o n l yt h e I L - 3 s e q u e n c e a n d CAT w e r e p r e s e n t .I n a d d i t i o n t o t h e t w o e n h a n c i n g r e g i o n s , t h r e e p o t e n t i a ln e g a t i v e r e g u l a t o r y s i t e s a r e a l s o a p p a r e n t i n t h e I L - 3 5 'f l a n k i n g r e g i o n . A d d i t i o n o f t h e r e g i o n s b e t w e e n p o s i t i o n s- 3 1 3 a n d - 3 9 7 , p o s i t i o n s - 1 6 1 a n d - 2 7 2 , a n d p o s i t i o n s - 5 9a n d - 1 1 6 r e s u l t e d i n r e p r o d u c i b l e d e c r e a s e s i n r e p o r t e r g e n ea c t i v i t y i n s t i m u l a t e d J u r k a t c e l l s . E x t e n d i n g t h e p r o m o t e ra n a l y s i s t h r o u g h p o s i t i o n - 6 8 2 r e s u l t e d i n p r o m o t e r a c t i v i t ys i m i l a r t o t h a t f o r c o n s t r u c t 3 9 7 ( d a t a n o t s h o w n ) .N u d e a r P r o t e i n s t h a t B i n d t o t h e I L - 3 P r o m o t e r . O l i g o n u -c l e o t i d e s b a s e d u p o n t h e s e q u e n c e o f s e v e r a l r e g i o n s o f t h eh u m a n I L - 3 g e n e ( F i g s . 2 a n d 3 ) w e r e t e s t e d i n m o b i l i t y - s h i f ta s s a y s . S p e c i f i c r e t a r d e d b a n d s w e r e c o n s i s t e n t l y p r o d u c e db y e x t r a c t s f r o m b o t h J u r k a t a n d MLA c e l l s u s i n g a n o l i g o -n u c l e o t i d e c o n t a i n i n g t h e s e q u e n c e b e t w e e n b a s e s - 1 6 0 a n d- 1 3 0 ( F i g . 3 A ) . T h i s r e g i o n s h a r e s s e q u e n c e s i m i l a r i t i e s w i t ht h e C R E B ( 1 2 , 1 5 ) a n d t h e I L - 2 e n h a n c e r e l e m e n t s NFAT-1

B i o c h e m i s t r y : S h o e m a k e r e t a l .


3/5

9 6 5 2 B i o c h e m i s t r y : S h o e m a k e r e t a l . Po.Ni.Aa.Si S 7 90A GCCACC-ACC A G - A C C r t A . A (. \. .Az:AFTc~~~~~~~~~97 ~~~~~~~~~~~~33301

30~~~~~~~~~~ 3

P r o r n o i c - a r F r a g e n tB B a l I II )

O l i g o n u c l e o t i d e s f o r SaIm o b i l i t y s h i f t a s s a y s Sm ___ 9

A v a 4

9ivCAT A c l i y I L .L a i u at C e t i 1 L

S l i m u o t l e d4Jo n - T r o1 C0 0

FIG. 2. A) Sequence ofthe IL-3 5 ' flanking region determined from clone IL-3pr. The location ofsequences that matchorresemble putativeand known r e g u la t o ry r e g io n s are noted by boxes and include AP-1 ( r e f . 1 1 ; position -301), CREB(ref. 12 ; position -147), NFAT-1 ( r e f . 2 2;position -156), CK-1 ( r e f . 2 3 ; positions -126and -333), CK-2 (ref 23 ; position -115), and AP-2 (ref. 2 4 ; positions -74 and -90). TheTATAsequence (position -30) and the initiation codon (position +52) are also noted. The site oftranscriptional initiation (position +1) wasr ep o rte dby Yang et al. (2 5 ). ( B ) Functional activity ofthe IL-3 promoter-CAT constructs in Jurkat andMLAe lls. On the left promoter fragments linkedto theCATreporter are mapped. The numbers to the right of themapde si g n ate the location of the 5 ' end ofeach construct relative to the capsite. Constructs 3 9 7 , 3 1 3 , 2 7 2 , and 59 utilized available restriction sites shown at the l e f t ) , and constructs 1 6 1 , 161CREB- TTACGTCT mutatedto TTACGGAT , 1 2 6 , 1 1 6 , 1 0 7, and 44 utilized synthetic oligonucleotides linked to available restriction sites. Construct 356NFIL3A-ATGAATAATTmutated toCACCCGATCG wasgenerated by site-directed m utag e n esis. The relative reportergene activity SEM is shownfor Jurkat and MILA cells, as indicated. The data presentedwerenormalized soasto bring the stimulated level for construct 272 common toall assays ) toavalue of 100 and represent the meanof4-15 experiments for each construction. At least three plasmid pr e p ar ati o n s have beentested for each construct giving essentially identical r epr o ducibl e results.

and N F - I L 2 - A (22, 27). To determine whether the protein(s)that bound to this oligonucleotide represents either of thesetwo families oftranscription factors, orinstead anotherDNAbinding protein(s), mobility-shift competition experimentswerecarried out. As shown in Fig. 3A, a50-fold excessofoligonucleotide that binds either oftwoCREBsites, either oftwo AP-1 sites, ortheNFAT-1 site didnot displace the Jurkatprotein(s) from the labeled oligonucleotide. An oligonucleo-tide containing anNF -IL2-A binding sequence competedwithanaffinity -as10-fold lower than the specific sequence. Incontrast, 1-25 ng ofanoligonucleotide that contains muta-tions at four ofthe eight CREB-specific bases of the IL-3 5 'flanking region displaces specific oligonucleotide bindingwith anaffinity similar to that of native sequence (Fig. 3,affinity data not shown). Furthermore, Northern blot a n al y -si s of p o l y ( A ) - co n t ainin g RNAfrom Jurkat andMLAc e l l sfailed to detect CREB-specific transcripts ( d a t a not shown).Additional oligonucleotides containing mutations nearthe 5 'and 3 ' ends of this region weretested for co m p etitio n andallowed mapping ofthe binding site between positions -156and 147 (Fig. 3 B ) . To confirm the precise binding siteDNase I footprinting wasperformed. As shown in Fig. 3C,nucleotides between 157 and 144 bp upstream ofthe cap sitewere protected from digestion in the presence of T-cellnuclear extracts.Asecond specific band pattern wasobtained using an

o li g o n u cl e o t i de pair matching the IL-3 sequence from posi-tions -308 to -276, aregion that contains anAP-1 bindingsite ( 1 1 ). As shown in Fig. 4, stimulated Jurkat andMLAel lnuclei contain AP-1 binding protein(s) that migrate with anelectrophoretic mobility identical to that of recombinantc - j u n protein. Anidentical mobility-shift pattern wasob-tained using anoligonucleotide derived from the AP-1 regionof the metallothionein promoter. This banding pattern wasspecifically displaced by the presence ofa50-fold excessofanunlabeled oligonucleotide that matches the 308/276 se-

quence o nl y at the AP-1 site but not by irrelevant oligonu-cleotides orthose derived from any other region of the IL-3promoter. Lower orundetectable levels of this binding ac-tivity werepresent in several prepa ra ti o ns ofnuclear extractsfrom unstimulated T-cell lines. Northern blot analysis confirmed the presence of transcripts for the AP-1 proteins c-junand c-fos in stimulatedTcells (Fig. 5 ) .

DISCUSSIONTlymphocytes produce alarge number of c y t o k i n e s thataffect the proliferation and activation ofcells involved in theinflammatory reaction. The regulation of cytokine geneexpression is acomplex process that mayinvolve transcrip-tional, post-transcriptional (typically at the level ofRNAstability), ortranslational mechanisms. Inagrowing numberofinstances, transcriptional enhancementhas been shown tobe responsible for lymphocyte activation ( 2 7 ). By usingnuclear run on analysis, wedemonstrate that IL-3 geneexpression is regulated, at least in part, by transcriptionalenhancement.Afunctional analysis wasutilized tomaptworegions that substantially up -r e g ul at e reporter gene expres-sion in response to mitogenic stimulation of T-lymphocytecell lines. Thus these elements form anenhancer for aheterologous promoter. By using mobility-shift and DNasefootprinting assays, these samer e g i o n s werefound to bindproteins from stimulated T-cell nuclei and to a lesser extentfrom unstimulated cells.

Although the first of these sites, located between 161 and126 bp upstream of the IL-3 cap site, contains sequencessimilar to the binding sites for the transcription factorsCREB,NFAT-1, and NF-1L2-A, this region binds anappar-ently uncharacterized protein based onseveral criteria. ( i )MutationoftheCREBsite atfourofthe eight positions withinthe oligonucleotide probe failed to eliminate specific bindingof a nuclear protein to the remainder of the region inmobility-shift assays and inclusion of the- same mutation

;: . 1 -I 3 A - r

3 . 3 Pr' L . . . 1 . 4

P r o c . N a t i . A c a d . S c i . USA 8 7 ( 1 9 9 0 )

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P r o c . N a t l . A c a d . S c i . USA 8 7 ( 1 9 9 0 ) 9 6 5 31 2 3 4 5 6

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' 4

* L i V .t

C A T G G A T G A A T A A T T A C G T C T G T G G T T T T C T v < IL-3S 3 q u n c e- 1 6 0 -G-A 1 3 0 < C R E B Homology< W N F A T - 1 H o m o l o g y

0 - 1GA 0- 20-3TGCCTG 0 - 4

GACTGT 0 -5CCGACTGT

GCCCGACTGTGAAA-G-A-G-GTT-A-A 0 - 70 - 8

asf / - 1 5 7

ATGAATAA_ ~ ~ ~ ~ T\A_ o p p < C - 1 4 4

AP 4 w

F I G . 3. DNA b i n d i n g p r o t e i n assays o f t h e 1 6 1 / 1 2 6 r e g i o n o f I L - 3 . ( A ) M o b i l i t y - s h i f t assays. N u c l e a r e x t r a c t s f r o m u n s t i m u l a t e d J u r k a t ( l a n e1 ) , MLA ( l a n e 3 ) , HeLa ( l a n e 5 ) , o r AML-193 ( l a n e 6 ) c e l l s , or J u r k a t ( l a n e s 2 , 7 - 1 6 ) or MLA ( l a n e 4 ) c e l l s s t i m u l a t e d f o r 3 h r w i t h Co n A a n dP MA w e r e i n c u b a t e d w i t h a n e xc ess o f u n l a b e l e d i r r e l e v a n t d u p l e x o l i g o n u c l e o t i d e (CGTCAGATAAAGATCCTTCCGA, l a n e s 1 - 6 ) , ano l i g o n u c l e o t i d e ( 0 - 1 ) c o n t a i n i n g t h e 1 6 0 / 1 3 0 s e q ue n c e ( l a n e 7 ) , a mutant C R E B s i t e s e q ue n c e ( l a n e 8 , 0 - 2 ) , t h e 3 0 8 / 2 7 6 s equ en c e o f t h e I L - 3g e n e ( l a n e 9 , CAGTGAGCTGAGTCAGGCTTCCCCTTCCTGCCACG), t h e A P - 1 - c o n t a i n i n g r e g i o n o f t h e m e t a l l o t h i o n e i n promoter ( r e f . 2 6 ;l a n e 1 0 , GAGCCGCAAGTGACTCAGCGCGGGGCGTGTGCAGG), t h e s e q ue n c e u s e d t o c l o n e a C R E B cDNA ( r e f . 1 2 ; l a n e 1 1 , GATCCG-GCTGACGTCATCAAGCTA), t h e s equ en c e u s e d t o c l o n e a C R E - B P - 1 cDNA ( r e f . 1 5 ; l a n e 1 2 , TCGAGCTCCTAGCCTGACGTCA-GAGAGAGAGC), t h e NFIL-2A sequence ( r e f s . 2 2 a n d 2 7 ; l a n e 1 3 , AGTCTTTGAAAATATGTGTAATATG), t h e NFAT-1 seque n ce o f t h eI L - 2 e n h a n c e r ( l a n e 1 4 , 0 - 8 ) , o r w i t h e i t h e r o f tw o m u t a n t I L - 3 5 ' f l a n k i n g sequences ( l a n e s 1 5 a n d 1 6 , 0 - 4 a n d 0 - 5 ) . T h e b o u n d ( b a n d s B ) a n df r e e ( b a n d s F ) o l i g o n u c l e o t i d e s are n o t e d . ( B ) T h e sequence o f t h e I L - 3 5 ' f l a n k i n g r e g i o n f r o m p o s i t i o n s - 1 6 0 t o - 1 3 0 i s s h o w n , a l o n g w i t ht h e C R E B a n d NFAT-1 h o m o l o g i e s a n d t h e o l i g o n u c l e o t i d e sequences u s e d i n m o b i l i t y - s h i f t assays ( i d e n t i c a l n u c l e o t i d e s ar e u n d e r l i n e d a n dm i s m a t c h e d p o s i t i o n s ar e i n d i c a t e d b y t h e a l t e r e d b a s e ) . ( C ) DNase I f o o t p r i n t i n g a n a l y s i s . T h e r e s u l t s f r o m t h e sense s t r a n d ar e p r e s e n t e d ,i d e n t i c a l r e s u l t s w e r e o b t a i n e d u s i n g t h e a n t i s e n s e s t r a n d . L a n e s : 1 , no e x t r a c t ; 2 , c o n t r o l MLA c e l l e x t r a c t ; 3 , Co n A / P M A - s t i m u l a t e d MLAc e l l e x t r a c t .f a i l e d t o e l i m i n a t e t h e e n h a n c e m e n t o f r e p o r t e r g e n e a c t i v i t yd e t e c t e d i n t h i s r e g i o n . ( i i ) O l i g o n u c l e o t i d e s t h a t c o n t a i n t h eC R E B or t h e NFAT-1 b i n d i n g s i t e s were u n a b l e t o d i s p l a c es p e c i f i c b i n d i n g t o t h i s I L - 3 g e n e se q u e n c e a n d d i d n o tp r o d u c e s i m i l a r b a n d s when t e s t e d i n m o b i l i t y - s h i f t assays( d a t a n o t s h o w n ) . ( i i i ) B y u s i n g N o r t h e r n b l o t a n a l y s i s , n os p e c i f i c C R E B t r a n s c r i p t s c o u l d b e d e t e c t e d i n s t i m u l a t e dJ u r k a t or MLA c e l l RNA. I n a d d i t i o n t o b i n d i n g n u c l e arp r o t e i n s , t h i s s i t e i s f u n c t i o n a l l y a c t i v e as i t s m u t a t i o n

1 2 3 4 5 6 7 8 9 10 1 1

F I G . 4 . M o b i l i t y - s h i f t assay o f t h e 3 0 8 / 2 7 6 r e g i o n o f I L - 3 . N u -c l e a r e x t r a c t s f r o m u n s t i m u l a t e d J u r k a t ( l a n e 1 ) , MLA ( l a n e 3 ) , HeLa( l a n e 5 ) , or AML-193 ( l a n e 6 ) c e l l s or f r o m J u r k a t ( l a n e s 2 , 7 , a n d 8 )or MLA ( l a n e 4 ) c e l l s s t i m u l a t e d f o r 3 h r w i t h Co n A a n d PMA, o rp u r i f i e d r e c o m b i n a n t c - j u n p r o t e i n ( l a n e s 9 - 1 1 ) was i n c u b a t e d w i t ha n excess o f u n l a be l ed i r r e l e v a n t d u p l e x o l i g o n u c l e o t i d e ( l a n e s 1 - 6a n d 9 ) , w i t h o l i g o n u c l e o t i d e c o n t a i n i n g t h e 3 0 8 / 2 7 6 sequence ( l a n e s7 a n d 1 0 ) , or w i t h t h e A P - 1 - c o n t a i n i n g r e g i o n o f t h e m e t a l l o t h i o n e i np r o m o t e r ( l a n e s 8 a n d 1 1 ) . B a n d s : B , b o u n d o l i g o n u c l e o t i d e s ; F , f r e eo l i g o n u c l e o t i d e s .

e l i m i n a t e d CA T a c t i v i t y i n r e p o r t e r g e n e s t u d i e s . B a s e d o nt h e r e s u l t s o f m o b i l i t y - s h i f t c o m p e t i t i o n assays a n d DNase If o o t p r i n t i n g a n a l y s i s , we propose t h e c o g n a t e b i n d i n g s i t eGATGAATAAT. T h u s t h e s e f i n d i n g s argue s t r o n g l y f o r t h epresence o f a t r a n s c r i p t i o n a l a c t i v a t i n g p r o t e i n i n v o l v e d i nt h e r e g u l a t i o n o f I L - 3 g e n e e x p r e s s i o n i n T l y m p h o c y t e s , h e r et e r m e d N F - I L 3 - A . T h e l o w - a f f i n i t y c o m p e t i t i o n s h o w n b yt h e N F - I L 2 - A s i t e , a r e g i o n t h a t c o n t a i n s s eq u e n c e s i m i l a r i t yt o t h e i m m u n o g l o b u l i n octomer s i t e , i s i n t r i g u i n g ( 2 2 , 2 7 ) a n ds u g g e s t s t h a t N F - I L 3 - A may b e r e l a t e d t o t h e octomerb i n d i n g p r o t e i n s .A s e c o n d r e g i o n i n v o l v e d i n t h e t r a n s c r i p t i o n a l r e g u l a t i o no f I L - 3 maps t o a n A P - 1 s i t e l o c a t e d a t p o s i t i o n - 3 0 1 . T h i ssequence b i n d s a f a m i l y o f p r o t e i n s t h a t i n c l u d e s t h e p r o -t o o n c o g e n e s c - j u n a n d c - f o s a n d t h e r e l a t e d g e n es JUN-B a n dJUN -D ( 1 1 , 1 4 ) . S e v e r a l l i n e s o f e v i d e n c e p o i n t t o t h eA

1 2 3 4

B

.12

1 2 3

C

4 1 2 3 4F I G . 5 . N o r t h e r n b l o t a n a l y s i s o f M L A - c e l l t r a n s c r i p t i o n f a c t o r s .P o l y ( A ) RNA ( 5 j i g ) f r o m u n s t i m u l a t e d MLA c e l l s ( l a n e 1 ) or c e l l ss t i m u l a t e d w i t h Co n A a n d PMA f o r 1 5 m i n ( l a n e 2 ) , 2 h r ( l a n e 3 ) , or6 h r ( l a n e 4 ) was h y b r i d i z e d w i t h cD N A p r o b e s f o r m u r i n e c - j u n ( A )

or f o r b o v i n e , - a c t i n ( C ) or w i t h a g e n o m i c p r o b e f o r human F O S ( B ) .

A C 1 2 3 0 , _ ..

F iB

B i o c h e m i s t r y : S h o e m a k e r e t a l .


5/5

9 6 5 4 B i o c h e m i s t r y : S h o e m a k e r e t a l .i n v o l v e m e n t o f A P - 1 p r o t e i n s i n t h e t r a n s c r i p t i o n a l r e g u l a t i o no f I L - 3 i n l y m p h o c y t e s . M o b i l i t y - s h i f t a s s a y s s h o w e d t h a t a no l i g o n u c l e o t i d e d e r i v e d f r o m t h e s e q u e n c e b e t w e e n p o s i t i o n s- 3 0 6 a n d - 2 7 6 , a n d o n e d e r i v e d f r o m t h e m e t a l l o t h i o n e i np r o m o t e r A P - 1 s i t e , b o t h b i n d a n u c l e a r p r o t e i n ( s ) p r e s e n t i nT c e l l s , c r o s s - c o m p e t e , a n d m i g r a t e w i t h a n e l e c t r o p h o r e t i cm o b i l i t y i d e n t i c a l t o t h a t o f r e c o m b i n a n t c - j u n p r o t e i n . F u r -t h e r m o r e , we d e m o n s t r a t e d t h a t R N A f r o m b o t h J u r k a t a n dMLA c e l l s c o n t a i n s c - j u n a n d c - f o s t r a n s c r i p t s . M u e g g e e t a l .( 2 8 ) h a v e a l s o s h o w n t h a t A P - 1 p r o t e i n s a r e i n v o l v e d i n t h er e g u l a t i o n o f I L - 2 t r a n s c r i p t i o n . T h e s e d a t a s t r o n g l y s u g g e s tt h a t A P - 1 p r o t e i n s a r e i n v o l v e d i n t h e t r a n s c r i p t i o n a l r e g u -l a t i o n o f I L - 3 i n h u m a n T c e l l s . A s e c o n d p o t e n t i a l s i t e f o rA P - 1 r e g u l a t i o n o f I L - 3 g e n e e x p r e s s i o n i s p r e s e n t b e t w e e np o s i t i o n s - 1 4 7 a n d - 1 4 0 . T h i s o c t a n u c l e o t i d e m a t c h e s t h ec o g n a t e C R E B s i t e a t s i x p o s i t i o n s , c a n c o m p e t e w i t h l o wa f f i n i t y f o r b i n d i n g o f n u c l e a r p r o t e i n s t o t h e A P - 1 s i t e o f t h eI L - 3 g e n e ( d a t a n o t s h o w n ) , c a n b i n d p u r i f i e d c - j u n p r o t e i n i nDNase f o o t p r i n t i n g a s s a y s ( Ri c h a r d T u r n e r , p e r s o n a l c o m -m u n i c a t i o n ) , a n d a p p e a r s t o b e p r o t e c t e d i n o u r f o o t p r i n t i n ga s s a y s u s i n g T - c e l l n u c l e a r e x t r a c t s ( F i g . 3 C ) , T h e f u n c t i o n a ls i g n i f i c a n c e o f t h i s r e g i o n , h o w e v e r , i s u n c e r t a i n a n d w a r -r a n t s f u r t h e r s t u d y .T h e p r e s e n c e o f t h e s e b i n d i n g a c t i v i t i e s i n u n s t i m u l a t e dc e l l s , a l b e i t a t a l o w e r l e v e l t h a n i n s t i m u l a t e d T - l y m p h o c y t en u c l e a r e x t r a c t s , d o e s n o t n e c e s s a r i l y m i l i t a t e a g a i n s t a r o l ef o r t h e s e p r o t e i n s i n t h e t r a n s c r i p t i o n a l a c t i v a t i o n o f t h e I L - 3g e n e . A d d i t i o n a l a l t e r a t i o n s , s u c h a s p r o t e i n p h o s p h o r y l a t i o no r e n h a n c e d b i n d i n g a f f i n i t y , n o t d e t e c t a b l e b y m o b i l i t y - s h i f to r D N a s e f o o t p r i n t i n g a s s a y s may b e r e s p o n s i b l e f o r t r a n -s c r i p t i o n a l a c t i v a t i o n ( 2 7 , 2 9 , 3 0 ) .F i n a l l y , d e s p i t e t h e p r e s e n c e o f a n u m b e r o f c o n s e n s u ss e q u e n c e s f o r c h a r a c t e r i z e d DNA b i n d i n g p r o t e i n s , t h e f i r s t1 2 6 b p o f t h e I L - 3 g e n e 5 ' f l a n k i n g r e g i o n d i s p l a y s a m i n i m a lr e s p o n s e i n t h e p r e s e n c e o f m i t o g e n i c s t i m u l a t i o n . Two o ft h e s e s e q u e n c e s , t e r m e d C K - 1 a n d C K - 2 b y S h a n n o n e t a l .( 2 3 ) , a r e p r e s e n t b e g i n n i n g a t p o s i t i o n s - 1 2 6 a n d - 1 1 5 ,r e s p e c t i v e l y . A s e c o n d C K - 1 s i t e i s a l s o p r e s e n t a t p o s i t i o n- 3 3 3 o f t h e I L - 3 5 f l a n k i n g r e g i o n . I n a d d i t i o n , t h e d e c a n u -c l e o t i d e s e q u e n c e b e g i n n i n g a t p o s i t i o n - 7 4 m a t c h e s t h ec o n s e n s u s t r a n s c r i p t i o n f a c t o r A P - 2 b i n d i n g s i t e p r o p o s e d b yI m a g a w a e t a l . ( 2 4 ) a t a l l 1 0 p o s i t i o n s , a n d t h e d e c a n u c l e o t i d es e q u e n c e s b e g i n n i n g a t p o s i t i o n s - 9 6 a n d - 9 0 m a t c h t h i sc o n s e n s u s s e q u e n c e a t 8 p o s i t i o n s . C o m p a r i s o n o f t h e r e -p o r t e r g e n e a c t i v i t i e s o f c o n s t r u c t s t h a t f l a n k t h e C K - 1 a n dC K - 2 s i t e , c o n s t r u c t s 1 2 6 v s . 1 1 6 a n d c o n s t r u c t s 1 1 6 v s . 1 0 7 ,r e s p e c t i v e l y , o r o f t h e c o n s t r u c t s t h a t f l a n k t h e c o n s e n s u sA P - 2 s i t e s , c o n s t r u c t s 1 0 7 v s . 5 9 , r e v e a l e d m i n i ma l m o d u l a -t i o n o f CAT a c t i v i t y a c r o s s t h e s e s i t e s i n t h e 5 ' f l a n k i n g r e g i o no f t h e I L - 3 g e n e . T h u s , t h e f u n c t i o n a l s i g n i f i c a n c e o f t h eC K- 1 , C K- 2 , a n d A P - 2 s i t e s c a n n o t b e d e t e r m i n e d f r o m t h e i rp r e s e n c e a l o n e . I t i s p o s s i b l e t h a t t h e s e e l e m e n t s r e q u i r ea d d i t i o n a l u p s t r e a m s e q u e n c e s t o e x e r t a n i n f l u e n c e o nt r a n s c r i p t i o n . A d d i t i o n a l d e l e t i o n m u t a t i o n s o f t h e s e r e g i o n sw i l l b e r e q u i r e d t o d e t e r m i n e w h e t h e r C K - 1 , C K - 2 , a n d A P - 2a r e f u n c t i o n a l l y i n v o l v e d i n t h e r e g u l a t i o n o f I L - 3 g e n ee x p r e s s i o n i n T l y m p h o c y t e s .I n t h i s s t u d y we h a v e f o c u s e d u p o n t h e r e g i o n s r e s p o n s i b l ef o r u p - r e g u l a t i o n o f I L - 3 t r a n s c r i p t i o n i n T l y m p h o c y t e s . Weh a v e d e s c r i b e d s e q u e n c e s p r e s e n t i n t h e 5 ' f l a n k i n g r e g i o n o ft h e I L - 3 g e n e t h a t a c t i n c i s a n d n u c l e a r p r o t e i n s t h a t b i n d t ot h e s e r e g i o n s a n d a p p e a r t o p a r t i c i p a t e i n t h i s p r o c e s s . O n eo f t h e s e f a c t o r s , A P - 1 , h a s b e e n e x t e n s i v e l y c h a r a c t e r i z e d i no t h e r s y s t e m s . T h e o t h e r f a c t o r i s a p r o t e i n p r e s e n t i n b o t hc o n t r o l a n d m i t o g e n - s t i m u l a t e d T c e l l s . A m o r e c o m p l e t ec h a r a c t e r i z a t i o n o f t h i s p u t a t i v e t r a n s c r i p t i o n a l r e g u l a t o ra w a i t s s t u d i e s i n n o n l y m p h o i d c e l l s y s t e m s , i n v i v o c r o s s l i n k -i n g o f n u c l e a r p r o t e i n s t o t r a n s f e c t e d DNA f r a g m e n t s , a n d , o fc o u r s e , i t s g e n o m i c c l o n i n g . A l o n g w i t h f u r t h e r c h a r a c t e r -

i z a t i o n o f t h e d o w n - m o d u l a t i n g e le me n t s , t h e se s t u d i e ss h o u l d p r o v i d e a b e t t e r u n d e r s t a n d i n g o f t h e r e g u l a t i o n o fI L - 3 g e n e e x p r e s s i o n i n h u m a n T l y m p h o c y t e s .N o t e . M a t h e y - P r e v o t e t a l . ( 3 1 ) h a v e a l s o r e p o r t e d o n t h e r e g u l a t i o no f I L - 3 g e n e e x p r e s s i o n i n T l y m p h o c y t e s . I n t h i s s t u d y m e t h y l a t i o n -i n t e r f e r e n c e a s s a y s d e m o n s t r a t e p r o t e i n - D N A c o n t a c t i n t h e samer e g i o n a s t h e DNase f o o t p r i n t i n g e x p e r i m e n t s shown i n F i g . 3 C .We a c k n o w l e d g e P e t e r L o c k h a r t ( Z y mo g e n e t i c s , S e a t t l e ) f o r s y n -t h e s i z i n g t h e o l i g o n u c l e o t i d e s , D r . S t e v e n C l a r k ( G e n e t i c s I n s t i t u t e ,C a m b r i d g e , MA ) f o r s u p p l y i n g t h e g i b b o n I L - 3 cDNA, D r . R i c h a r dT u r n e r ( U n i v e r s i t y o f C a l i f o r n i a , S a n F r a n c i s c o ) f o r s u p p l y i n g t h er e c o m b i n a n t c - j u n p r o t e i n a n d t h e cDNA s e q u e n c e f o r humanJUN-B, D r . T e r r y Meyer ( M a s s a c h u s e t t s G e n e r a l H o s p i t a l , B o s t o n )f o r s u p p l y i n g t h e C R E B cDNA, a n d D r . W i l l i a m Lamph ( S a l kI n s t i t u t e , L a J o l l a , CA) f o r s u p p l y i n g t h e c - j u n cDNA. T h i s w o r k was

s u p p o r t e d b y N a t i o n a l I n s t i t u t e s o f H ea l t h G r a n t s CA 3 1 6 1 5 a n d 5T 3 2 H L 0 7 0 9 3 . K . K . i s t h e r e c i p i e n t o f a J u n i o r F a c u l t y R e s e a r c hA w a r d o f t h e A m e r i c a n C a n c e r S o c i e t y .1 . L o p e z , A . F . , T o , L . B . , Y a n g , Y . - C . , G a m b l e , J . R . , S h a n n o n , M . F . ,B u r n s , G . F . , D y s o n , P . G . , J u t t n e r , C . A . , C l a r k , S . C . V a d a s , M . A .( 1 9 8 7 ) P r o c N a l d . A c a d . S c i . USA 8 4 , 2 7 6 1 - 2 7 6 5 .2 . M e s s n e r , H . A . , Y a m a s a k i , K . , J a m a l , N . , M i n d e n , M . M . , Y a n g , Y . - C . ,W o n g , G . G . C l a r k , S . C . ( 1 9 8 7 ) P r o c . N a i t l . A c a d . S c i . USA 8 4 ,6 7 6 5 - 6 7 6 9 .3 . K i m o t o , M. , K i n d l e r , V . , H i g a k i , M . , O d y , C . , l z u i , S . V a s s a l l i , P .( 1 9 8 8 ) J . I m m u n o l . 1 4 0 , 1 8 8 9 - 1 8 9 4 .4 . K o b a y a s h i , M . , V a n L e e u w e n , B . H . , E l s b u r y , S . , M a r t i n s o n , M . E . ,Y o u n g , I . G . H a p el , A . J . ( 1 9 8 9 ) B l o o d 7 3 , 1 8 3 6 - 1 8 4 1 .5 . R o t h e n b e r g , M . E . , O w e n , W. F . , S i l b e r s t e i n , D . S . , W o o d s , J . , S o b e r -m a n , R . J . , A u s t en , K . F . S t e v e n s , R . L . ( 1 9 8 8 ) J . C l i n . I n v e s t . 8 1 ,1 9 8 6 1 9 9 26 . V a l e n t , P . , S c h m i d t , G . , B e s s e m e r , J . , M a y e r , P . , Z e n k e , G . , L i e h l , E . ,H i n t e r b e r g e r , W . , L e c h n e r , K . , M a u r e r , D . B e t t e l h e i m , P . ( 1 9 8 9 )B l o o d 7 3 , 1 7 6 3 - 1 7 6 9 .7 . E r n s t , T . J . , R i t c h i e , A . R . , S t o p a k , K . S . G r i f f i n , J . D . ( 1 9 8 9 ) B l o o d7 4 , S u p p l . 1 , 1 1 6 .8 . N i e m e y e r , C . M . , S i e f f , C . A . , M a t h e y - P r e v o t , B . , W i m p e r i s , J . Z . ,B i e r e r , B . E . , C l a r k , S . C . N a t h a n , D . G . ( 1 9 8 9 ) B l o o d 7 3 , 9 4 5 - 9 5 1 .9 . K a u s h a n s k y , K . ( 1 9 8 9 ) J . I m m u n o l . 1 4 3 , 2 5 2 5 - 2 5 2 9 .1 0 . Y a n g , Y . - C . , C i a r l e t t a , A . B . , T e m p l e , P . A . , C h u n g , M . P . , K o v a c i c , S . ,W i t e k - G i a n n o t t i , J . S . , L ea r y , A . C . , K r i z , R . , D o n a h u e , R . E . , W o n g ,G . G . C l a r k , S . C . ( 1 9 8 6 ) C e l l 4 7 , 3 - 1 0 .

1 1 . L a m p h , W. W . , W a m s l e y , P . , S a s s o n e - C o r s i , P . V e r m a , I . M . ( 1 9 8 8 )N a t u r e ( L o n d o n ) 3 3 4 , 6 2 9 - 6 3 1 .1 2 . H o e f f l e r , J . P . , M e y e r , T . E . , Y u n , Y . , J a m e s o n , J . L . H a b e n e r , J . F .( 1 9 8 8 ) S c i e n c e 2 4 2 , 1 4 3 0 - 1 4 3 3 .1 3 . D e g e n , J . L . , N e u b a u e r , M . G . , D e g e n , S . J . F . , S e y f r i e d , C . E . M o r r i s , D . R . ( 1 9 8 3 ) J . B i o l . C h e m . 2 5 8 , 1 2 1 5 3 - 1 2 1 6 2 .1 4 . C u r r e n , T . , M c C o n n e l l , W. P . , v a n S t r a a t e n , F . V e r m a , I . M . ( 1 9 8 3 )M o l . C e l l . B i o l . 3 , 9 1 4 - 9 2 1 .1 5 . M a e k a w a , T . , S a k u r a , H . , K a n e i - l s h i i , C . , S u d o , T . , Y o s h i m u r a , T . ,F u j i s a w a , J . , Y o s h i d a , M . I s h i i , S . ( 1 9 8 9 ) EMBO J . 8 , 2 0 2 3 - 2 0 2 8 .1 6 . W o o d , W. I . , G i t s c h i e r , J . , L a s k y , L . A . L a w n , R . M . ( 1 9 8 5 ) P r o c .N a t l . A c a d . S c i . USA 8 2 , 1 5 8 5 - 1 5 8 8 .1 7 . K a u s h a n s k y , K . , O ' H a r a , P . J . , H a r t , C . E . , F o r s t r o m , J . W. H a g e n ,F . S . ( 1 9 8 7 ) B i o c h e m i s t r y 2 6 , 4 8 6 1 - 4 8 6 7 .1 8 . P o n t e , P . , N g , S . - Y . , E n g e l , J . , G u n n i n g , P . K e d e s , L . ( 1 9 8 4 ) N u c l e i cA c i d s R e s . 1 2 , 1 6 8 7 - 1 6 .1 9 . K a u s h a n s k y , K . , O ' H a r a , P . J . , B e r k n e r , K . , S e g a l , G . M . , H a g e n , F . S . A d a m s o n , J . W . ( 1 9 8 6 ) P r o c N a t i . A c a d . S c i . US A 8 3 , 3 1 0 1 - 3 1 0 5 .2 0 . D i g n a m , J . D . , L eb ov i t z , R . M . R o e d e r , R . G . ( 1 9 8 3 ) N u c l e i c A c i d sR e s . 1 1 , 1 4 7 5 - 1 4 8 9 .2 1 . B o h m a n n , D . T j i a n , R. ( 1 9 8 9 ) C e l l 5 9 , 7 0 9 - 7 1 7 .2 2 . D u r a n d , D . B . , B u s h , M . R . , M o r g a n , J . G . , W ei s s , A . C r a b t r e e , G . R .( 1 9 8 7 ) J . E x p . M e d . 1 6 5 , 3 9 5 - 4 0 7 .2 3 . S h a n n o n , M . F . , G a m b l e , J . R . V a d a s , M . ( 1 9 8 8 ) P r o c . N a t I . A c a d .S c i . US A 8 5 , 6 7 4 - 6 7 8 .2 4 . I m a g a w a , M . , C hi u , R . K a r i n , M . ( 1 9 8 7 ) C e l l 5 1 , 2 5 1 - 2 6 0 .2 5 . Y a n g , Y . - C . C l a r k , S . C . ( 1 9 8 7 ) L y m p h o k i n e s 1 5 , 3 7 5 - 3 8 7 .2 6 . T u r n e r , R. T j i a n , R . ( 1 9 8 9 ) S c i e n c e 2 4 3 , 1 6 8 9 - 1 6 9 4 .2 7 . C r a b t r e e , G . R . ( 1 9 8 9 ) S c i e n c e 2 4 3 , 3 5 5 - 3 6 1 .2 8 . M u e g g e , K . , W i l l i a m s , T . M . , K an t , J . , K a r i n , M . , C h i u , R . , S c h m i d t , A . ,S i e b e n l i s t , U . , Y o u n g , H . A . D u r u m , S . K . ( 1 9 8 9 ) S c i e n c e 2 4 6 , 2 4 9 - 2 5 1 .2 9 . C h i u , R . , B o y l e , W. J . , M e e k , J . , S m e a l , T . , H u n t e r , T . K a r i n , M .( 1 9 8 8 ) C e l l 5 4 , 5 4 1 - 5 5 2 .3 0 . M o n t m i m y , M . R. B i l e z i k j i a n , L . M . ( 1 9 8 7 ) N a t u r e ( L o n d o n ) 3 2 8 ,1 7 5 - 1 7 8 .3 1 . M a t h e y - P r e v o t , B . , A n d r e w s , N . C . , M u r p h y , H . S . , K r e i s s m a n , S . G . N a t h a n , D . G . ( 1 9 9 0 ) P r o c . N a t l . A c a d . S c i . US A 8 7 , 5 0 4 6 - 5 0 5 0 .

P r o c . N a t l . A c a d . S c i . USA 8 7 ( 1 9 9 0 )

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