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UMI
A B ell &Hbwell Infonnation Compaiy
300 North Zeeb Road, A nn Arbor MI 48106-1346 USA 313/761^700 800/521-0600
by
TATYANA H A M ILTO N
B. Sc., N ovosibirsk State U niversity, Russia, 1990
A D issertatio n S u b m itted in Partial F ulfillm ent o f th e R equirem ents for the D egree of
DOCTOR OF PHILOSOPHY
in th e D e p artm en t of Biochemistry a n d M icrobiology
We accept this thesis as co n form ing to ih e i^ b u ired sta n d a rd
rvisor (D ept, o f B iochem istry & Ibiology)
O lafson, D ep artm en tal M em ber 'o f B io c h e n jis ^ & N/p:Sob^ology) D r. F ra n d s E. N ano, Deps (Dept, of Bi M em ber logy) chael J. A s h m (Dept, o f Biology) TJenry P earson, D ep artm en tal M em ber (Dept, of BigdaggUjtry & M icrobiology)
Dr. D av id Setzer, E ^ m a l E xam iner Case W estern Reserve U n iv ersity )
© TATYANA B. H A M ILTO N , 1997 U n iv ersity of Victoria
A ll rig h ts reserv e d . This thesis m ay not be re p ro d u c e d in w h o le or in p a rt, by m im e o g ra p h o r o th e r m eans, w ith o u t th e p e rm issio n o f the a u th o r.
G C G -TG G -G C G -(T /G )(G /A /T)(T /G ). These sequences have a fo u r-fo ld h ig h e r affin ity for th e p ro tein th a n the n o n se lec te d se q u e n c e G C G - T G G -G C G -C C C , as m ea su re d b y a q u a n tita tiv e n itro cellu lo se filter b in d in g assay.
The effects of D enys-D rash syndrom e (DOS) point m u ta tio n s o n th e D N A b in d in g activ ity of W T l w ere d e te rm in e d . SAAB a ssa y re v e a le d th a t n o n e of the DOS m u ta n t p ro te in s give rise to a new seq u en ce sp e c ifid ty . O ne m u tatio n , R394W abolishes specific b in d in g o f th e p ro te in . T he re m a in in g m u ta tio n s re s u lt in re d u c e d D N A - b in d in g activity, ran g in g from 1.4 to 14-fold, w h ich suggests th a t ev en sm a ll ch an g es in D N A -binding activity m a y p re d p ita te th e clinical p h e n o ty p e o f D enys-D rash syndrom e.
C o m p a ra tiv e an aly sis of th e D N A b in d in g c h a ra c te ristic s of W ilm s' tu m o u r a n d Early g ro w th response p ro te in s w as c o n d u c te d . T h e sto ich io m etry o f th e D N A -p ro tein co m p lex es, th eir s ta b ility to d isso d a tio n , a n d the effects of p H , tem p eratu re a n d salt c o n ce n tra tio n o n th e e q u ilib riu m b in d in g o f th ese p ro tein s to th eir c o g n a te D N A
seq u en ces h av e been d e te rm in e d . U n d e r th e co n d itio n s o f 0.1 M sa lt, p H 7.5, a n d 22 * C W T l-Z F h as an a p p a re n t disso ciatio n c o n stan t (K d) of 1.14± 0.2 X 1 0 ^ M, a n d EGR-1 protein h as a Kd of 3.55 ± 0.4 x 10"^ M .
In a d d itio n , w e tested re la tiv e co n trib u tio n of each b a se p a ir in th e co n sen su s b in d in g site to th e h ig h affinity b in d in g by p o in t m u ta tio n a l an aly sis, a n d identified im p o rta n t differences th at ex ist in th e b in d in g m odes o f th e tw o proteins.
T ra n s c rip tio n fa c to r UIA c o n tro ls th e e x p re s s io n o f th e 5S rib o so m a l R N A genes d u r in g d e v e lo p m e n t of X enopus laevis., a n d specifically interacts w ith b o th 5S D N A a n d 5S rR N A m olecules. T h e p re s e n t s tu d y assesses c o n trib u tio n s o f th e cen tral z in c fin g ers f o u r th ro u g h s e v e n to specific D N A and R N A b in d in g a c tiv itie s of th e p ro tein . T h e resu lts d e m o n stra te th a t each zinc fin g e r in the zf 4 -7 re g io n c o n tr ib u te s to b o th th e h ig h a ffin ity D N A a n d R N A in te ra c tio n s: the largest effect on T FlllA -D N A b in d in g (10-fold) w a s p ro d u c e d w h e n zinc fin g er 5 o f TFIHA w as rep la ce d w ith the d o n o r sequences o f eith er p43 or W T l. H ow ever, w hile all th e zinc fingers 4-7 c o n trib u te to th e high affin ity 5S rR N A b in d in g , s u b s titu tio n of a n a - helical p o r tio n of zinc fin g er 6 w ith th e e q u iv a le n t se q u en c es fro m W T l a b o lish e d R N A -binding activity o f TFIILA, su g g e stin g th a t z in c finger 6 p la y s a particularly im p o rtan t role in b in d in g to RNA.
D r. 4rerry|
S nuth
Dr. M ichael J. Ashwi M em W r (D ept, o f Biology)
Dr. D a v id Setzer, E xternal E xam iner (Case W este rn R eserve U n iv e rsity )
A bstract...ii
Table of C o n te n ts... v
List of Tables...x
List o f Figures...xii
L ist of A b b rev iatio n s... x v ii A cknow ledgm ents... xx
C h a p te r 1.0 E arly g ro w th resp o n se p ro te in (EGR-1): a p ro to ty p ic a l m em ber of a C2H2 zinc finger fam ily of tran scrip tio n factors 1.1 In tro d u ctio n ... 1
1.1.1 O verview of the EGR-1 fam ily of tran scrip tio n fa c to rs... 1
1.1.2 Gene targ ets of EGR-1 re g u la tio n ...6
1.1.3 Identification of EGR-1 cDNA, an d characterization o f its p ro tein p r o d u c t... 7
1.1.4 D N A -binding function o f EGR-1...14
1.1.5 Structures of other zinc finger-D N A com plexes... 32
1.1.6 Studies to w a rd a zinc finger recognition code...43
C h a p te r 2.0 T he W ilm s' tu m o u r gene p ro d u c t: a tu m o u r su p p re sso r in v o lv ed in re g u la tio n of k id n e y d e v elo p m e n t 2.1 In tro d u ctio n ... 47
2.1.1 The concept of tum or su p p resso r g e n e s ... 47
2.1.2 Biology o f W ilm s' tu m o r a n d associated sy n d ro m e s... 47
2.1.3 Identification and characterization o f the W Tl gene a n d its p ro tein p ro d u ct... 50
3.2.2 O ther M aterials...75
3.2.3 C o n stru c tio n a n d P u rifica tio n of R e co m b in a n t W T l-Z F P a n d W TIA FI-ZFP P r o te in s ... 76
3.2.4 Selection A m plification a n d B inding (SAAB) A ssa y ...80
3.2.5 C o n stru ctio n of plasm ids containing sequence elem ents o f th e insulin-like g ro w th factor II fetal pro m o ter, a n d the non-selected sequence w ith th e CCC subsite for finger 1 b in d in g ... 86
3.2.6 Purification o f olig o n u cleo tid es... 87
3.2.7 End-labeling of DNA...88
3.2.8 N itrocellulose Filter B inding A ssa y ... 88
3.2.9 T ransient Transfection A ssay s...89
3.3 R esults... 90
3.3.1 Isolation o f DNA b in d in g su b site fo r fin g e r 1 o f W T l-Z F by S A A B ...90
3.3.2 Q u an titativ e Binding of W Tl-ZFP to V arious D N A S eq u en ces...93
3.3.3 A n In vitro Selected W T l B inding Site A cts as a Strong T ranscriptional R egulator...97
3.4 Discussion...101
4.1 In tro d u c tio n ...108
4.2 M aterials a n d M eth o d s...113
4.2.1 C o n stru ctio n a n d expression of D enys-D rash m u ta n t p ro te in s ...113
4.2.2 Selection am plification a n d b in d in g (SAAB) assay... 114
4.2.3 N itrocellulose Filter B inding A ssa y ... 117
4.3 R e s u lts ... 118
4.3.1 Selection o f D N A binding sites for D eny s-D rash m u ta n t p r o te in s 118 4.3.2 M e asu rin g th e b in d in g affinities of th e m u ta n t p e p tid e s for the selected DN A seq u en ces... 121
4.4 D isc u ssio n ... 125
C h a p te r 5.0. C o m p arativ e an aly sis of the D N A b in d in g ch aracteristics o f W ilm s' tu m o u r a n d Early G ro w th R esponse Proteins 5.1 In tro d u c tio n ...129
5.2 M aterials a n d M ethods... 130
5.2.1 C o n stru ctio n a n d p u rificatio n of reco m b in an t W T l-Z F a n d E G R l-ZF p ro tein s...130
5.2.2 C o n stru ctio n of m u ta n t W T l-Z F an d E G R l-Z F DN A b in d in g s e q u e n c e s ... 130
5.2.3 End-labeling o f DNA... 132
5.2.4 N itrocellulose Filter B inding A ssa y ... 132
5.3 R esu lts... 132
5.3.1 E quilibrium b in d in g c o n sta n ts ... 132
5.3.2 M onovalent sa lt dependence of the Ka for D N A b in d in g ...139
5.3.3 p H d ep en d en ce o f K a ...1 4 1 5.3.4 Effect of d iv a le n t m etal io n concentration o n D N A b in d in g ...144
6.1 T he C2H2 zinc fin g er d o m a in ... 163
6.2 S tru ctu re / fu n ctio n analysis o f TFIHA... 173
6.2.1 P urification a n d ch aracterizatio n of TFIHA g en e p ro d u c t...173
6.2.2 TFIIIA gene e x p re ssio n ...179
6.2.3 R oles of TFIHA in tran scrip tio n an d storage o f 55 rR N A ... 180
6.2.4 S tru ctu re a n d fu n ctio n of 5S rR N A and its g e n e ... 182
6.2.5 R ole of TFIIIA zin c fingers in R N A rec o g n itio n ... 183
6.2.6 R ole of TFIIIA zin c fingers in DN A re c o g n itio n ... 194
C h a p te r 7.0 The s tu d y o f th e nucleic acid interactions of zinc fin g er 4-7 region o f TFIHA 7.1 I n tr o d u c tio n ... 206
7.2 M aterials a n d M e th o d s ... 207
7.2.1 C o n stru ctio n of TFIIIA finger sw ap m u ta n ts ... 207
7.2.2 E xpression a n d p u rification of TFIHA p r o te in s ... 213
7.2.3 R adiolabeling o f 5S D N A ... 218
7.2.4 Synthesis a n d R adiolabeling o f 5S rRNA... 218
7.2.5 N itrocellulose filter b in d in g a ssa y s... 221
7.3.1 E ffects o f zinc fin g e r s u b s titu tio n m u ta g e n e sis o n th e D N A
b inding activ ity of TFIIIA...221
7.3.2 E ffects o f zinc fin g e r s u b s titu tio n m u ta g e n e sis o n th e R N A b inding activ ity of TFIIIA...228
7.4 D iscussion...230
8.0 C o n c lu s io n s... 236
T ab le 4.3 Frequencies o f each nucleotide selected at the finger 3 su b site positions by W Tl-ZFP a n d the finger 3 p o in t m u tan ts...120 T a b le 4.4 F in g er 3 su b s ite sequences selected from a ra n d o m iz e d tem plate by w ild type W T l-Z FP and the finger 3 point m u ta n ts ...122 T ab le 4.5 B inding affiiüties of w ild ty p e a n d finger 2 m u ta n t W T l-Z F P for selected D N A sequences... 124 T ab le 4.6 B inding affinities o f w ild type a n d finger 3 m u ta n t W T l-Z F P for selected D N A sequences... 124 T ab le 5.1 Sequences o f m u ta n t oligonucleotides h arbouring in d iv id u a l base pair su b stitu tio n s in th e EGRl-ZF a n d W Tl-ZF consensus D NA
b in d in g site... 133 T a b le 5.2 Effect of th e d ifferen t m o n o v alen t salts on th e b in d in g of W T l-Z F a n d EG Rl-ZF to D N A consensus sequences... 148 T ab le 5.3 D issociation con stan ts for W T l-Z F a n d EG R l-ZF b in d in g to w ild -ty p e a n d m u tan t D N A consensus seq u en ces... 150 T ab le 7.1 Sequences of oligonucleotides u se d in construction of TFIIIA su b stitu tio n m u ta n ts...208
T a b le 7.2 S e q u e n c e s o f se le c tio n p rim e rs u s e d in tr a n s fo r m e r m u tag en esis p ro to co l to construct s u b s titu tio n m u ta n ts o f TFIHA...216 T ab le 7.3 The effects o f TFIHA z in c fin g er su b stitu tio n m u ta tio n s o n the D N A a n d RN A b in d in g of the fac to r... 223 T a b le 7.4 T he effects o f TFIIIA z in c fin g er su b stitu tio n m u ta tio n s o n the D N A a n d RN A b in d in g of the facto r... 227
F ig u re 1.4 S u m m ary of EGR-1 fu n ctio n al d o m ain s... 13 F ig u re 1.5 Sequence of the EGR-1 zinc finger p ep tid e a n d of th e D N A b in d in g site u se d in cocrystallization... 16 F ig u re 1.6 The o v erall arra n g em e n t of the three zinc fingers of EGR-1 in the m ajor g ro o v e of DNA (Pavletich & Pabo, 1991)...17 F ig u re 1.7 Sketch su m m arizin g th e principal am in o ad d -b ase contacts as seen in the o rig in al EGR-1 X-ray stru ctu re (Pavletich & Pabo, 1991)...19 F igure 1.8 S u m m a ry of direct base an d p h o sp h ate contacts in
EGR-1-D N A com plex...20 F ig u re 1.9 S u m m ary of w ater-m ed iated and v an d er W aals contacts to bases an d p h o sp h a tes in EG R -l-D N A com plex... 21 F ig u re 1.10 D raw ings of am ino ad d -b ase p air contacts of the EG R-l-D N A com plex...22 F ig u re 1.11 S chem atic diagram of EGR-1 zinc fingers in teractin g w ith the p ro p o sed o v erlap p in g , 4-bp D N A subsites (Isalan et al., 1997)... 27 F ig u re 1.12 S chem atic re p re se n ta tio n of h y d ro g en b o n ding b e tw ee n Z n -co o rd in ated h istid in e an d DN A backbone...29 F ig u re 1.13 Sequences of the GLI zinc finger d o m ain a n d the D N A - b in d in g site u se d for cocrystallization...33
F ig u re 1.14 Sketch su m m arizin g base and p h o sp h a te contacts m a d e by
the GLI p ep tid e... 34
F ig u re 1.15 The su m m ary of T ram track-D N A contacts... 36
F ig u re 1.16 Schem atic sum m ary of the principal protein-D N A contacts o b serv ed in the cocrystal structures of the EGR-1 a n d T ram track...37
F ig u re 1.17 Schem atic representation of the Y Y l-D N A in teractio n s... 40
F ig u re 1.18 C om parison of YYl w ith other zinc finger stru c tu re s...41
F ig u re 2.1 The am in o acid sequence of W Tl p ro te in ... 52
F ig u re 2.2 Schematic diagram of the stru ctu re of the W Tl m R N A a n d p r o te in s ... 54
F ig u re 2.3 DNA sequences w hich b in d W Tl p ro tein s (R ed d y & Licht, 1996)... 58
F ig u re 2.4 Protein-D N A contacts m ade by EGR-1 protein, an d p ro p o se d D N A contacts for W T l zinc fingers... 62
F ig u re 2.5 Proposed W Tl zinc finger-D N A contacts (Reddy & L icht, 1996)... 64
F ig u re 3.1 DNA sequence of the peptide en co d in g insert in p la s m id pET-W TZFP, w ith the am ino a d d sequence of the p ep tid e ...73
F ig u re 3.2 Schem atic representation of the recom binant W T l-Z F p lasm id , pU C 18/W T l-Z F . Restriction sites u sed in subsequent c lo n in g ste p s are show n... 78
F ig u re 3.3 Plasm id m a p of pET-16b expression v ecto r... 79
F ig u re 3.4 C oom assie blue-stained 15% SD S-polyacrylam ide gel sh o w in g purifiied W T l-Z FP an d W TIA FI-ZFP p ro te in s...81
F ig u re 3.5 Protocol for the SAAB (Selection a n d A m plification of B inding site assay)...82
elem ents... 95 F ig u re 3.10 (A ) S equences of W T l e le m e n ts id en tifie d in th e fetal p ro m o te r o f th e IGF-11 gene. (B) R esu lts o f a n n itro c ellu lo se filter b in d in g a s s a y m e a s u rin g th e e q u ilib riu m b in d in g of W T l-Z F P to different D N A elem en ts...98 F ig u r e 3 .1 1 T r a n s ie n t tr a n s f e c tio n a s s a y s m e a s u r in g W T l re s p o n s iv e n e s s ...100 F ig u re 3.12 T he h y d ro g e n -b o n d d o n o rs a n d accep to rs p re s e n te d by W atson-C rick base p airs to the m ajor gro o v e a n d the m inor g ro o v e ... 103 F igure 4.1 W T l m u ta tio n s associated w ith D enys-D rash sy n d ro m e ...109 F ig u re 4.2 C o m p a ris o n of th e se q u en c es o f W T l a n d EGR-1 zinc fingers...112 F ig u re 4.3 C o o m a ssie b lu e -s ta in e d 15% S D S -p o ly a c ry la m id e gel sh o w in g p u rifiie d W T l-Z F P D enys-D rash m u ta n ts ... 115 F ig u re 4.4 T h e SAAB te m p la te o lig o n u c le o tid e s c o n ta in in g ra n d o m iz e d se q u en c es fo r th e WT1[-KTS] fin g er 2 (A) or fin g e r 3 (B) re c o g n itio n ...116 F ig u re 4.5 T h e e q u ilib riu m b in d in g o f th e W T l elem en t G C G TGG G AG TGT to W T l-Z F P , R366H an d R366C...123
F ig u re 5.1 C oom assie b lue-stained 15% SD S-polyacrylam ide gel sh o w in g EG R l-ZF p ro te in purified b y affinity ch ro m ato g rap h y ...131 F ig u re 5.2 E q u ilib riu m binding cu rv es of W T l-Z F and E G R l-Z F p ro te in s to their targ et D N A sequence... 135 F ig u re 5.3 Scatchard analysis of W T1-2T (A) an d E G R l-ZF...137 F ig u re 5.4 Tim e d ep en d en ce of the b in d in g of W T l-Z F an d E G R l-Z F to D N A co n sen su s se q u en c es...140 F ig u re 5.5 KCl co n cen tratio n d ep en d en ce of the b in d in g of W T l- Z F a n d E G R l-Z F to D NA consensus seq u en ces... 142 F ig u re 5.6 p H d ep en d en ce of the b in d in g of W T l-Z F an d E G R l-Z F to D N A co n sen su s se q u en c es...143 F ig u re 5.7 Effect of the m ag n esiu m ion concentration on the b in d in g of W T l-Z F (closed circles) a n d EG Rl-ZF (open circles) to DNA c o n s e n s u s se q u e n c e s... 145 F ig u re 5.8 T e m p e ra tu re d ependence of the bin d in g of W T l-Z F a n d E G R l-Z F to DNA con sen su s sequences... 146 F ig u re 6.1 Schem atic rep resen tatio n of a C2H2 zinc fin g er... 165
F ig u re 6.2 R epresentative stru ctu res from various zinc finger fam ilie s...170 F ig u re 6.3 Schem atic re p re se n ta tio n of the interfinger o rie n ta tio n s o f
MBP-1 zin c fingers a n d EGR-1 fingers 1 a n d 2...174 F ig u re 6.4 D iagram s of the fu n ctio n al dom ains of eukaryotic zin c- c o n ta in n g tran scrip tio n factors. 175
F ig u re 6.5 A m ino a d d sequence of the nucleic a d d b in d in g d o m a in o f T FIIIA ...177 F ig u re 6.6 Schem atic d iag ra m illu stra tin g alig n m en t of TFIIIA z in c fin g er reg io n along ICR... 178 F ig u re 6.7 O rg an izatio n of the Xenopus 5S rRNA g en es...184
Figure 7.1 S chem atic d iagram illustrating construction of (A) pUC19-zf4-7; (B) pU C 19-W Tl-B X ...210
F igure 72. Schem atic chart of the PC R -m ediated site-directed
m u tag en esis p ro to co l used to create TFIIIA su b stitu tio n m u tan ts... 212 Figure 7.3 S chem atic diagram show ing the strategy for in tro d u c in g
m u ta tio n s u sin g the T ransform er site-directed m utagenesis k it
(Clontech Inc., 1994)... 214
Figure 7.4 C oom assie blue-stained 15% SD S-polyacrylam ide gel
sh o w in g TFIIIA w ild type and m u tan t p ro tein s... 219
Figure 7.5 C oom assie blue-stained 15% SD S-polyacrylam ide gel
sh o w in g TFIIIA w ild type and m utant p ro tein s... 220 Figure 7.6 C o m p ariso n of the am ino acid sequences of zinc fingers 4-6 of the d o n o r p ro te in p43 w ith the zinc fingers 4-6 of TFIIIA...222 Figure 7.7 S am p le n itrocellulose filter b in d in g curves of TFIIIA w ild type a n d m u ta n t p ro tein s w ith the 58 rR N A gene...225 F igure 7.8 C o m p ariso n of the am ino a d d sequences of zinc fingers 1-4 of the d o n o r W T l-Z F w ith the zinc fingers 4-7 of TFIIIA... 226 Figure 7.9 C o m p e titio n assay of TFIIIA an d TF(4-7)WT for 5 S rR N A b in d in g u s in g tRNA^*^® as a com petitor...229
List o f Abbreviations
bp: base p a ir
BSA: b ovine se ru m album in
BW S: B eckw ith-W iedem ann syndrom e CAT: C h lo ram phenicolA cetylT ransferase cD N A : co m p lem en tary deoxyribonucleic acid cpm : co u n ts p e r m inute
D D S: D enys-D rash syndrom e deo x y n u cleo tid e trip h o sp h ates:
d A T P , deoxyadenosine triphosphate dC T P, deoxycytidine triphosphate d G T P , deoxyguanine triphosphate d T T P , deoxythym idine triphosphate DEPC: d ieth y pyrocarbonate
D N A : deoxyribonucleic acid D TT: d ith io treito l
E .coli: Escherichia coli
EDTA: ethylenediam ine-tetraacetic acid E G R l: Early G ro w th Response 1
F.U.P.: F o rw ard U niversal P rim er
HEPES: N -2-h y d ro x y eth y lp ip erazin e-N '-2 -eth an esu lp h o n ic acid IC R : in tern al control region
IE: in term ed iate elem ent
IG FII: Insulin-like G ro w th Factor II
IPTG : isopropyl-P-D -thiogalactopyranoside K TS: lysine, th reonine, serine
G T P, g u a n in e trip h o sp h ate CTP, cy tid in e trip h o sp h ate A TP, a d e n o sin e trip h o sp h ate U TP, u rid in e trip h o sp h ate nucleotide bases: G , g u a n in e C, cytosine A, ad en in e T, th y m id in e N , either A , A , G, or T
PAGE: p olyacrylam ide gel electrophoresis PD GF-A : P latelet-D eriv ed G ro w th Factor A PEG: polyethylene glycol
PM SF: p h en y lm eth y lsu lfo n y l flu o rid e PPG : 2,5-diphenyloxazole
R .U .P.: R everse U n iv ersal P rim e r RB: retin o b lasto m a
R N A : ribonucleic acid R N ase: ribonuclease
R N P : rib o n u cleo p ro tein rR N A : rib o so m al nucleic acid S: S v ed b erg u n it
SAAB: selected am plification a n d binding
SAAB: S electio n A n d A m plification B inding a ssa y SD S: so d iu m dodecyl sulphate
TBE: Tris b ase, borate, EDTA T FIIIA : tran scrip tio n factor UIA TFIIIB: tran scrip tio n factor UIB TFIIIC: tran scrip tio n factor HIC
T ris-H C l: tris-(hydroxym ethyl) am inom ethane h y d ro ch lo rid e tR N A : tra n sfe r ribonucleic acid
TTK : T ram T racK protein
W A G R : W ilm s' tum our. A n irid ia , u ro G e n ita l m a lfo rm a tio n s, m e n ta l R e ta rd a tio n
W T l: W ilm s' T u m o u r 1 X bo: Xenopus borealis oocyte X bs: Xenopus borealis som atic X lo: Xenopus laevis oocyte X ls: Xenopus laevis som atic XIt: Xenopus laevis trace-oocyte
T erry W . Pearson a n d Dr. M ichael J. A shw ood-S m ith, for w h ich I a m g ratefu l. I w o u ld also like to m e n tio n Dr. A1 T. M atheson, Dr. E d E. Ish ig u ro a n d Dr. Ju an A usio for th eir assistance.
M y sincere th an k s to all th e m em bers o f D r. R om aniuk's lab - K athy B arilla, Frank Borel, John F erris, Steve H e n d y , M aya Isk a n d a r, C olleen N elson, N ik V eldhoen, Ju d y W ise, Q im in You, an d W ei-Q ing Z an g - fo r their h e lp a n d frien d sh ip . My th a n k s also to th e o th e r g ra d u a te stu d en ts a t the d e p artm e n t w h o d id n 't h e sitate to help: G erry B earon, S io b h an C o w ley , Ben F o rw a rd , Lee A n n H ow e, A rm a n d o Jardim , K izzy M dluli, D m itrii R odionov, C aroline Stebeck.
I a m grateful to A lbert L abossiere an d Scott Scholz, w h o w ere alw ays th e re to help w ith all m an n e r of technical assistance from fixing a c o m p u te r to setting u p a photo room .
I w o u ld very m u ch like to acknow ledge th e h e lp of th e office: R osanne Poulson, M aree Roome, a n d Claire T ugw ell.
I w o u ld also like to th a n k M ario n a n d A r th u r F o n tain e for co n n ectin g m e to Dr. P au l R om aniuk, for their in fin ite k in d n ess, a n d for b ein g like second p aren ts to m e.
Finally, I w o u ld like to th an k A rth u r H a m ilto n , m y h u s b a n d a n d best frie n d , for his lo v e, patience a n d su p p o rt. W ith o u t h im m y life w o u ld n e v e r be com plete.
D edication
tra n s d u c tio n e v e n ts occu rrin g a t th e level o f the p lasm a m e m b ra n e . The im m e d ia te -ea rly genes are the e a rlie st d o w n stre a m n u clear ta rg e ts for these e v e n ts. The a ctiv a tio n of these genes is g en erally very r a p id , tran sie n t, a n d in d e p e n d e n t of de novo p ro tein synthesis. A subclass of th e s e g en es encodes tr a n s c r ip tio n fa c to rs, w h ich fo rm the firs t ste p in in itia tio n o f g en etic p ro g ra m s th at w o u ld lead to a n a p p ro p ria te cellu lar response.
T he best characterized m em b ers of th is g ro u p o f im m e d ia te -ea rly genes in c lu d e c -ju n , c-fos, a n d Egr-1. E ach of th e se genes, in tu r n , re p re se n ts a p ro to ty p e for a fam ily of closely related p ro tein s. N u m e ro u s stu d ie s hav e d e m o n s tra te d th a t EGR-1 in d u ctio n is un iv ersal. V arious s tim u li th a t induce EGR-1 expression, as well as d iv erse cell ty p es in w hich EGR-1 ex p ressio n has b e e n d e scrib e d , a re su m m arize d in F igure 1.1. E x tra c e llu la r stim u li th a t in d u c e EGR-1 c an be g ro u p ed in to the follow ing categories: (1) m itogens; (2) d e v e lo p m e n ta l o r d iffe re n tia tio n cues; (3) tis su e o r r a d ia tio n in ju ry ; (4) sig n als th a t cause n eu ro n al excitation. In p ractically every cell ty p e exam ined, EGR-1 ex p ressio n is rap id ly in d u c e d by su c h m itogens as h o rm o n e s , g ro w th fa c to rs, a n d th e tu m o r p ro m o te r TP A (p h o rb o l) (S u k h a tm e e t al., 1987; S u k h atm e e t al., 1988; L au an d N ath an s, 1987; L em aire et al., 1988). V arious
POGF EGF rr .i lasulin p tty fo h em a g g lu iin in and jdenoune diphofptuic PDCF. »»sopcesiin tenim bFGF. PDCF BB p in ijl hcpafectomir GM-CSF. LPS endochelin angiotensin (I ntvobiasu hqsalocylcs
penptieral blood lymphocytes B lymphocytes
kidney epithelial cells kidney ittesjngial cells skeletal muscle Sol* cells skeletal muscle Sol* cells liver
pentooeal macrophages astrocytes
vascular smooth muscle cells
H y p ertro p h ic
O ille re n tia tiv e
endothelin. angiotensin II
NGF
retinoic acid. DSISO retinoacacid TP A. DMSO
myocyte
pheochrofflocytoma PCI 2 (neunll embryonal carcinoma PI9
embryonal calvarial cells. RCT-1 (osteoblast) myeloid leukemia HL60 and U 9)7
T issue!
radiation in ju ry ischemia ioniang tadialion
kidney 29). SQ-20B
N euronal ecciCatioo potassium ions metrazole NMDA visual stimuli efccnooonvTilstve shock therapy, dopattiine receptor activation, opiate withdrawal
peripheral nervous system
PCI 2 (depolaricatioo) seizures in vivo hippocampus visual ctxtci CNS
sciativc nerve transecirao
O ther MOCK. IXC-PKi leital epithelial celli
F ig u re 1.1 Biological processes m ed iated b y EGR-1 in response to d iv erse stim u li (C ashier & Sukhatm e, 1995).
im p o rta n t role o f EGR-1 in n e u ro n a l signaling. For ex am p le, EGR-1 lev els increase d ram atically in the b ra in follow ing seizu re activity (S ukhatm e e t al., 1988). A d ra m a tic in c re a s e in EGR-1 lev els is o b s e rv e d fo llo w in g e le ctro co n v u lsiv e sh o ck th e ra p y , d opam ine rec ep to r activ atio n , a n d o p ia te w ith d ra w a l (Bhat e t al., 1992). Finally, the expression of EGR-1 in d ev elo p in g a n d a d u lt b ra in em p h asizes th e im portance o f EGR-1 in neu ro p h y sio lo g ical processes (W atson a n d M ilb ran d t, 1990). Thus, EGR-1 m ed iates resp o n ses of e n o rm o u s co m p lex ity . I t acts in different cellu lar co n tex ts a n d is a b le to re sp o n d to a m u ltitu d e o f extracellular signals.
All m em bers o f th e EGR fam ily share h ighly sim ilar, C2H2 zinc fin g er
D N A -b in d in g m otifs. A t p re se n t, the closest m em bers o f th e EGR fam ily of tran scrip tio n al reg u la to rs are: EGR-2/Krox20 (C havrier e t al., 1988; Josep h e t al., 1988), EGR-3 (P a tw a rd h a n e t al., 1991), a n d E G R -4 /N G F I-C /p A T 1 3 3 (P a tw a rd h a n e t al., 1991; C ro sb y e t al., 1991; M uller e t al., 1991). A ll o f the above p ro tein s h av e zinc finger dom ains th at are v irtu ally id en tical to th a t of EGR-1 (Figure 1.2). T he EGR-1 zinc-finger d o m ain is o v e r 95% id en tica l to th a t o f EGR-2 (Joseph e t al., 1988) a n d 91% identical to th a t o f EGR-3 a t the am in o a d d level (P a tw ard h a n e t al., 1991). The resid u es im p o rta n t for specific
d g r i WTI Spl K p r R P R K r p H R P s K T P L H E R P H A r. R f F K 1, S H L 0 K H s R K H T C E K P r 0 T c p Y c K 0 s E c R C s C D P C K K K 0 H ( - - I S L« 2 1 . -Egr-1 CD c p V E S C o R R F S R S D E L T R K I R I H T C Q K P F O E ff-2 CD c p A E C C o R R F S R S 0 E L T R K I R I H T C H K P F O Egr-Î CD c p A E C C 0 R R F S R 5 0 E C, T R H E R I H T C H K P F O WTI C2) c 0 F K 0 C E R R F S R S 0 Q L K R H Q R R H T C V K P F Q Spl CD c K C Q C C c K V r c £] T S K L R A H E R W H T C E R P F H - . I S LS 21 • Egr-1 C2) c R t - - c K R N F S R 5 0 H L T T H I R T H T C E K P F A Egf-2 (2 ) c R c - - c K R N F S R S D K L T T H I R T K T C E K P F A Egr-3 (2 ) c R I - - c K R S F S R S 0 K L T T K I R T K T C E R P F A WTI O ) c K T - - c Q R K F S R S 0 H L K T K T R T H T C Q R P F S Spl C2) c T W S V C C K R F T R S 0 E L Q R K R R T H T C E R R F A M IG l (1 ) c P I - - c K R A F H R L E K 0 T R K K R I H T C E R P H A . . L S 1 1 2 1 * . • . . . . £ g r-l C3) c - - o r e C R K F A K S O e R R R H T R I H E R Q K O K R A O R s V V Egf-2 ( 3) c - - o r e C R K F A R S 0 E R R R K T R I H E R Q R E R R S S A p S A Egr-3 ( 3) c - - E F c C R K F A R S D E R R R K A R I H E R Q R E R R A E R c c A WTI W c R w e s c Q K K F A R S 0 E t V R H H H K H Q R N K T K L Q L A L • Spl (3) c - - P E C p K R F K R S 0 K L S R H X R T H Q H R R C C P C V A L s V M IGl (2) c 0 F p c c V K R F S R S 0 E L T R K R R I H T M S K P R C R R C R RK a - h d i x
F ig u re 1.2 C om parison of D N A -b in d in g d o m ain s of the EGR-1 fam ily of proteins. 25nc fingers a n d adjacent sequences o f the EGR-1 fam ily of proteins a re alig n ed . C onserved cysteine a n d histid in e residues are m ark e d (•); the a~ helical reg io n is u n d erlin ed ; c o n serv ed resid u es im p o rtan t fo r d eterm in in g b in d in g speciH dty are enclosed; b asic resid u es are d en o ted (+) (C ashier & S ukhatm e, 1995).
T he W ilm s' tu m o r g en e p ro d u c t h as fo u r zinc fin g ers, th re e o f w h ich p o ssess r e la te d b u t less h o m o lo g o u s z in c finger D N A -b in d in g d o m a in s (ap p ro x im ately 65% id en tity to the EGR-1 zinc finger d o m ain ) (G essler e t al., 1990; C all e t al., 1990). T he m am m alian ubiq u ito u s tran scrip tio n a l a ctiv a to r S p l has th re e related zinc fingers (K adonaga e t al., 1987). S p l fin g er 2 is m ost sim ilar to EGR-1 fingers 1 a n d 3 (K adonaga e t al., 1987). A nother d is ta n t
m em b er o f th e EGR fam ily o f p ro tein s is a yeast p ro te in M IG l in v o lv e d in resp o n ses to glucose re p re ssio n (N eh lin a n d Ronne, 1990). It c o n ta in s tw o zinc fin g ers th a t are m o st sim ilar to th e second a n d th ird fingers o f EGR-1 p ro tein , w ith 60% id en tical resid u es (N e h lin an d R onne, 1990). O u ts id e the zinc fin g ers, MIG-1 has n o obvious sim ila rity to other p ro tein s. T his absence o f seq u en ce co n serv atio n o u tsid e the z in c finger m otifs is a c o m m o n fin d in g a m o n g C2H2 zinc fin g er p ro tein s. T h erefo re, a c o m p a riso n o f th e fam ily
m em b ers m u s t rely o n a n a lig n m e n t o f fin g er seq u en ces. S tu d ie s o f th e im m e d ia te -e a rly p ro tein s a n d d o w n s tre a m p ro m o te r elem en ts th e y ta rg e t w ill c o n tin u e to en h an ce o u r k n o w le d g e o f p ro tein -D N A in te ra c tio n s a n d general m ech an ism s of tran scrip tio n a l a ctiv a tio n an d rep ressio n .
C o n s is te n t w ith its role in c ellu la r p ro life ra tio n a n d d ifferen tiatio n , EGR-1 b in d s a n d re g u la te s genes in v o lv e d in th e s e fu n ctio n s. Som e d isc u ssio n follow s o n th e best d o cu m en ted instances th a t involve re g u la tio n by EGR-1.
P latelet-d eriv ed g ro w th factor A (PDGF-A) is a p o te n t m itogen w h ich is p resen t a t elev ated lev els in response to g ro w th factors o r cytokines (W ang a n d D eu el, 1992). A n EGR-1 b in d in g site h as been d e fin e d in the p ro m o te r reg io n o f th e PD G F-A g en e (W ang a n d D euel, 1992). T h e DNA fra g m e n t b o u n d b y EGR-1 h a s th e follow ing seq u en ce: G A G -G A G -G A G -G A G G A . A lth o u g h th is site d e v ia te s from th e co n sen su s EGR-1 b in d in g seq u en ce, w h ic h is G C G -G /T G G -G G G , it c o m p e te s e q u a lly w e ll in th e g e l-sh ift c o m p e titio n e x p erim en ts. Sim ilar m o tifs h av e been id e n tifie d in p ro m o te rs o f o th er g ro w th -re la te d genes, such as th e insu lin recep to r, th e tu m o r g ro w th factor P, th e ep id erm al g ro w th factor recep to r, c-myc a n d c-Ki-ras (W ang an d Deuel, 1992).
A n o th e r gene w h o se exp ressio n is reg u la te d b y EGR-1 is th y m id in e kinase (tk), a n im p o rta n t p lay er in D N A biosynthesis. U sin g m onoclonal anti - EGR-1 a n tib o d ies, it w as show n th a t EGR-1 w as one o f th e com ponents of th e tk p ro m o te r co m p lex o b tain ed fro m se ru m -s tim u la te d n u clear e x tra ct (M o ln a r e t a l., 1994). F u rth e r tr a n s ie n t tr a n s f e c tio n e x p e rim e n ts d e m o n s tra te d th at EGR-1 activates a rep o rte r d riv en by a tk p ro m o ter elem ent (M olnar e t al., 1994).
EGR-1 has b een sh o w n to be re q u ire d for d ifferen tiatio n of m yeloblasts a lo n g th e m a c ro p h a g e lin eag e (N g u e n e t al., 1993). T h e use o f EGR-1 a n tis e n s e o lig o m e rs in th e cell c u ltu r e m e d iu m r e s u lte d in b lo c k e d m a c ro p h a g e d iffe re n tia tio n in n o rm a l m y elo b lasts a s w e ll as m y e lo id
M H C in d u c tio n a p p e a rs to be tissue-specific since it w as o b se rv e d in the m yogenic Sol8 cell line, b u t n o t in N IH 3T3 fibroblasts (G upta et al., 1991).
EGR-1 is ex p ressed a t high level in the rat a d re n a l g lan d a n d in PC12 cells as d em o n strated b y E lbert et al., (1994). A pro p o sed function for EGR-1 in a d ren erg ic d ifferen tiatio n m ay be via reg u latio n o f p h e n y le th a n o la m in e N - m e th y ltra n sfe ra s e (PN M T) (PN M T is a n a d re n a l e n zy m e t h a t co n v erts n o re p in e p h rin e to e p in e p h rin e ) (E lb ert e t al., 1994). T he a n a ly sis o f the PN M T p ro m o te r rev ealed th a t it co n tain s tw o p o ten tial EGR-1 b in d in g sites, o n e of them differing o n ly b y one n u cleo tid e from the EGR-1 co n sen su s DNA b in d in g sequence. T ran sien t transfections show ed th a t EGR-1 c an stim u la te a PN M T rep o rter by fourfold (Elbert e t al., 1994).
D esp ite th e a b u n d a n c e of d a ta o n EGR-1 in d u c tio n b y m ito g en ic sig n als, a d d itio n a l EGR-1 gene ta rg e ts a w a it elu cid atio n . F in ally , in vitro stu d ies of th e EGR-1 in v o lv em en t in cellu lar p ro liferatio n an d d ifferen tiatio n n e e d to be correlated w ith th e in vivo role o f EGR-1.
U sin g d iffe re n tia l sc re e n in g tec h n iq u e s, se v era l g ro u p s se t o u t to id e n tify novel g e n es w hich h a v e a low level o f e x p ressio n in n o n d iv id in g cells b u t w hich a re rap id ly u p -re g u la te d in cells stim u lated b y m itogen. T he fo llo w in g criteria w ere used to isolate su ch novel genes: (1) tran scrip ts sh o u ld
be ra p id ly and tran sien tly in d u ced b y serum stim u latio n of q u iescen t cells; (2)
these genes sh o u ld be in d u ced w ith o u t in terv en in g p ro tein sy n th esis, i.e. th e in d u c tio n sh o u ld n o t be affected by inhibitors o f p ro tein sy n th esis, su c h as cy clo h ex im id e; (3) e x p ressio n s h o u ld be in d u c e d by a w id e sp e c tru m o f m ito g en s, such as g ro w th factors, horm ones, a n d o th e r ligands; (4) ex p ressio n sh o u ld b e in d u ced in a broad a rra y o f cell types; a n d (5) the genes sh o u ld be h ig h ly co n serv ed in evolution.
T h e novel im m e d ia te -e a rly g en e has b e e n clo n ed by a n u m b e r of research groups. Sukhatm e e t al. (1987, 1988) id en tified a gene d esig n ated Egr- 1 . T he au th o rs u se d a d ifferential screening tech n iq u e to screen a lib ra ry fro m B A L B /c 3T3 cells s tim u la te d w ith s e r u m in th e p re s e n c e o f cyclohexim ide. C lones w hich preferen tially h y b rid iz ed to cD N A from se ru m a n d cyclohexim ide-treated fibroblasts w ere id en tifie d by co m p arin g th e m to cD N A from q u iesce n t cells. A 3.4-kb tra n sc rip t a p p e a re d u p o n m ito g e n ic stim u la tio n of a v a rie ty of cell ty p e s, a n d w as d e sig n a te d Egr-1. U sin g a s im ila r d iffe re n tia l sc ree n in g s tra te g y , M ilb ra n d t (1987) in d e p e n d e n tly i s o la te d a t r a n s c r ip t n a m e d N G F I - A , w h ic h w as a c tiv a te d in r a t p h eo ch ro m o cy to m a PC12 cells b y n erv e g ro w th factor, an d is a ra t an alo g of the m o u se Egr-1. The sam e g en e h as been in d e p e n d e n tly clo n ed b y o th e r g ro u p s, u sing a sim ila r approach: zif268 w as c lo n ed from se ru m -stim u la te d 3T3 fib ro b la sts fro m B A LB/c m ice (L au a n d N a th a n s, 1987); tis8 w a s id e n tifie d as a p h o rb o l-in d u c ib le g en e in 3T3 cells (Lim e t al., 1987); th e
seq u en ce o f th e p ro te in is highly c o n se rv e d across sp ecies, in d ic a tin g the im p o rtan ce o f th e Egr-1 gene p ro d u ct. C o m p le m en tary D N A s firom h u m a n (Suggs e t al., 1990), r a t (M ilbrandt, 1987), m o u se (S u k h a tm e e t a l., 1988; Lem aire e t al., 1988; C h risty et al., 1988), chicken (S im m o n s et al., 1989), and zebrafish (D ru m m o n d e t al., 1994) are h ig h ly sim ilar. T he p ro d u c t o f th e Egr- 1 gene is a p ro te in o f 80-82 kOa (Cao e t al., 1990). C ell firactionation stu d ies an d im m u n o c y to c h e m is try d e m o n s tra te d th a t EGR-1 is lo ca liz ed to the nucleus, w h ic h is co n sisten t w ith its p u ta tiv e D N A -b in d in g fu n ctio n (C ao et al., 1990; D ay e t al., 1990). Further ex am in atio n of th e a m in o acid se q u en c e of the EGR-1 p ro te in sh o w e d it to c o n ta in basic resid u es c lu ste red in th e zinc fingers a n d th e a d ja c e n t sequence (F ig u re 1.3B). T he a m in o -te rm in a l am ino a d d s are rich in p ro lin e a n d serin e-th reo n in e resid u es, o rg a n iz e d in stretch es o f tree to five c o n se c u tiv e am in o a cid s. T here is o n e series o f se v en consecutive se rin e -th re o n in e resid u es, w h ic h is fo llo w e d b y se v en g ly d n e s (Figure 1.3B). T he carb o x y l term in u s o f th e EGR-1 p r o te in is also ric h in pro lin e a n d s e rin e /th re o n in e residues. T he regions o f th e p ro te in c o n tain in g proline am in o a d d s a re predicted to lack a-h elical se c o n d a ry stru c tu re ,
100 Egr-1 residues ZOO 300 400 SOO $33 1 1 p isn 1--- r Sci/Thr-nch T
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A la ^ y s ) Asp I l a 6 ^ ) c l y Arg Lys Pbo A la Acg t o r Asp c l u Arg Lys Arg ^ i ^ Tbr Lys Lou A rc C l a L ys Asp Lys Lys A la As p Lys t e r V I v » l Ala t e r Pro A la ^ a t e r t e r Lew t o r t a r Tyr Pro t o r Pro P al A la Tbr t e r Tyr Pro t e r Pro A la t b r t b r
t e r Pbe Pro t o r P ro v a l Pro Tbr t e r Tyr t o r t e r Pro CLy t e r t e c Tbr Tyr Pro Pro A la His t a r C l y Pba Pro t o r Pro t o r V al A la Tbr Tbr fbe Ala t e r v » l pro Pro
A la Pbe Pro Tbr C la V a l t a r t o r Pbo Pro t o r A la C ly Val t e r t o r to r Pbo t e r Tbr
SN
t e c Tbr C ly Lau t a r Asp Mac Tbr Ala Tbr Pbo t o r Pro Arg Tbr l i e Clu t l o Cys $31
EGR-1 m ay b e subject to phosphorylation. In d eed , alkaline p h o sp h a ta s e w as sh o w n to c o n v ert slow m igrating fo rm of EGR-1 to the faster m ig ra tin g fo rm as se e n o n SDS-PAGE (Cao e t al., 1990; Day et al., 1990).
F u rth e r s tru c tu re -fu n c tio n a n aly sis o f EGR-1 p r o te in d e fin e d its a c tiv a tio n , r e p re s s io n , D N A b in d in g a n d n u c le a r lo c a liz a tio n d o m a in s (G ash ler e t al., 1993), su m m a riz e d in a d ia g ra m in F ig u re 1.4. T hus, th e o rg an izatio n o f EGR-1 is m odular in nature, w h ich is ch aracteristic of m any classical tran scrip tio n factors. A p o te n t activational d o m ain w a s m a p p e d to th e se rin e - a n d th re o n in e -ric h a m in o te rm in a l d o m a in , u s in g d e le tio n a n aly sis of EGR-1 (G ashler e t al., 1993). T hese EGR-1 a c tiv a tio n seq u en ces w e re sh o w n to b e in d e p e n d e n t d o m ain s by p la c in g th em in th e context o f o th e r proteins. W h en residues 3-281, or 3-138, o r 138-281 w e re fu se d to th e D N A -b in d in g d o m a in o f the y east factor GAL4, th ey a c tiv a te d tra n sc rip tio n 100-fold as GAL4 fusions (Gashler e t al., 1993). T hese findings w e re confirm ed b y stu d y in g N G FI-A protein, the r a t hom olog o f EGR-1 (R usso e t al., 1993). A w e a k tra n sa c tiv a tio n d o m ain w as m a p p e d by sev eral la b o ra to rie s to the C - te rm in u s of EGR-1: am in o acids 420-533 (R usso e t al., 1993; G a sh le r e t al., 1993).
A re p re ssio n d o m ain of EGR-1 was id e n tifie d w h e n a sm a ll in te rn al d e le tio n 5' to th e zinc-finger d o m ain (am ino acids 284-330) re s u lte d in alm o st 5-fold increased tran sactiv atio n in H eLa cells (G ashler e t al., 1993). This w as a n u n ex p ected fin d in g . Further experim ents h av e sh o w n th a t th is rep ressio n d o m a in is also m o d u la r in nature, since it could b e fused to th e D N A -binding d o m a in of GAL4, a n d represses tran scrip tio n 7- to 10-fold (G ash ler e t al., 1993). T h is s e r in e / th r e o n in e - r ic h d o m a in is h ig h ly c o n s e rv e d i n e v o lu tio n (D ru m m o n d e t al., 1994), an d is d istin c t from rep ressio n d o m a in s o f other tran scrip tio n al re g u la to rs, such as th e alanine- a n d g ly d n e -ric h rep re ssio n
4 1 7 *1» L K Q K D K K K O K f f V V X f f
too
Egr*I am ino acids
200 300 S M S3) 1 1 ^ PWT "T r S c r/T h r-rich rZTE A c t i r a d o a Repression DNA blading N u d e s r localization s tr o n g m U l 314 331 413 313 33# 331 413
F ig u re 1.4 S u m m ary of EGR-1 functional d o m ain s.
(A ) A m ino a d d sequence o f EGR-1 re p re ssio n dom ain a n d zin c fingers. (B) Schem atic m a p o f EGR-1 d o m a in s (Gashler & Sukhatm e, 1995).
d o m a in in K ru p p e l (Licht e t al., 1990), hydrophobic- a n d proline-rich Even- s k ip p e d re p re s s o r (H a n a n d M anley, 1993), or p ro lin e - an d g ly c in e -ric h re p re s s o r o f W T I (M ad d en e t al., 1993). The fact th a t 7 o u t of 24 a m in o a d d re s id u e s o f th e EGR-1 re p re ssio n do m ain are serine o r th reo n in e su g g e sts th at the re p re s s io n function m a y be regulated by phosphorylation.
C o n s is te n t w ith its tra n sc rip tio n a l re g u la to ry fu n ctio n , EGR-1 w as sh o w n to localize to the n u d e u s (Cao e t al., 1990; D ay e t al., 1990). G enerally, n u d e a r lo calizatio n signals are short stretches of 8 - 1 0 a m in o a d d s rich in basic
a n d p ro lin e re sid u e s (Silver e t al., 1984). In EGR-1, th e only reg io n s rich in basic resid u es are the th ree zinc finger reg io n and the ad jacen t stretch o f basic a m in o a d d s , su g g e stin g th a t the n u d e a r localization sig n a l resid es in those seq u en ces. U sin g a series o f deletion m u ta n ts of EGR-1 alo n g w ith su b cellu lar frac tio n a tio n a n d W estern b lo t analysis, am ino a d d s 315 to 429 w ere sh o w n to be im p o rta n t for p ro p er targ e tin g to the n u d e u s (Day e t al., 1990). H o w ev er, the z in c fin g er reg io n a lo n e (amino a d d s 331 to 419) w as not su fficie n t for n u d e a r lo ca liz atio n of th e protein. The basic seq u en ce 5' to the z in c finger d o m a in (am in o a d d s 315 to 330) was also required fo r nuclear targ e tin g (Day e t a l., 1990). T h u s, EGR-1 has a b ip a rta te nuclear lo calizatio n sig n a l, the la rg e st p o rtio n o f w hich c o in d d e s w ith the D N A -binding dom ain.
1.1.4 D N A -b in d in g fu n ctio n o f EGR-1
T he D N A sequence to w hich EGR-1 binds w as in itially id en tified based o n th e a s s u m p tio n th a t EGR-1 re g u la te s its o w n e x p re ssio n a n d m ig h t, th erefo re, b in d to the 5' u p stre a m flanking sequence o f th e Egr-1 gene (C hristy a n d N a th a n s , 1989). U s in g bacterially ex p ressed EGR-1 p ro tein , s p e d fic b in d in g to o n e p ro m o te r frag m en t w as observed in g el m obility sh ift assays (C h risty a n d N a th an s, 1989). This b in d in g site w as lo ca te d w ith in 650 base
EGR-1 is the first C2H2 zinc fin g er p ro tein fo r w h ich a high reso lu tio n
stru c tu re w as obtained. The stru ctu re o f the com plex consisting of th e cloned th re e zin c finger p e p tid e and the 12-bp DNA c o n ta in in g the specific 9-bp se q u e n c e h as b een so lv e d at 2.1-Â reso lu tio n (P a v le tic h a n d P ab o , 1991) (F ig u re 1.5). The stru c tu ra l in fo rm atio n o b tain ed in this stu d y se rv e d as a topological blueprint for other m em bers of the zinc fin g er fam ily. T his EGR-1 - D N A com plex w as recently refined a t 1.6 Â (Elrod-E rickson et al., 1996). The n ew stru c tu re confirm s all the basic features of th e 2 .1 Â m odel, a n d reveals
a d d itio n a l critical details o f the com plex.
T he crystal stru c tu re s show th a t the th ree fin g ers w ra p a ro u n d the d o u b le helix, describing a C shape (Figure 1.6). T h e overall alig n m en t o f the fingers o n the DNA is antiparallel (finger 1 binds n e a r the 3' end, a n d finger 3 b in d s n e a r the 5’ en d o f the 5 -GCGTGGGCG-3' c o n sen su s b in d in g site). [The m o d e o f TFIUA - D N A in teractio n is also a n tip a ra lle l, w ith m o st o f the c o n tacts involving the guan in e-rich stra n d of th e D N A (Sm ith e t al., 1984; V ran a e t al., 1988)]. T he a-h elices o f the zinc fin g e rs a re tip p e d a t ca.45° rela tiv e to th e plane o f the base p airs; therefore, th e y are only ap p ro x im ately a lig n e d w ith the m ajor groove. T he N -term inal e n d o f each a -h e lix m ak es specific contacts w ith th e base pairs, each helix in te ra c tin g p rim arily w ith the 3-bp su b se t o f the 9-bp consensus b in d in g site.
- 1 1 2 3 6 M E R P Y A C P V E S C D R R F S R I S D E L T R H I RI H TIg Q K 1 5 10 15 20 25 30 P F Q C R I - - C M R N F S R IsD H L T T H I R T HtI G E K 35 40 45 50 55 60 P F A C D I - - C G R K F A R IS D E R K R H T K I H L k o K D 65 70 75 80 85 P-sheets a-helix
B
1 2 3 4 5 6 7 8 9 10 11 A G C G T G G G C G T C G C A C C C G C A TF ig u re 1.5 Sequence of the EGR-1 zinc finger p ep tid e and o f th e DNA binding site u se d in cocrystallization. (A) Sequences of the three zinc fingers are alig n ed by conserved residues a n d secondary stru ctu re elem ents, a-helices a re enclosed in boxes, and P-sheets are indicated by arrow s. T he conserved cysteine an d h istid in e residues a re h ighlighted in bold. (B) Sequence of the d u p lex oligonucleotide used in cocrystallization (Pavletich & Pabo, 1991).
Hnger 1
F ig u re L6 T he o v e ra ll arran g em en t of the th ree zinc fingers o f EGR-1 in the
T he N -term in al s tra n d o f each P-sheet m akes n o contact w ith D N A , w h ile th e m o re C -term in al s tra n d contacts the s u g a r-p h o sp h a te b ackbone a lo n g o n e D N A stra n d . T he am in o acid side ch ain s d irec tly in v o lv e d in contacts w ith the D N A bases lie at a-helical positions -1, 2 ,3 , a n d 6 (num bered
relativ e to th e first resid u e in each a-helix). Base specific contacts id en tified in th e o rig in al crystal stru c tu re are prim arily h ydrogen b o n d s w ith the G -rich stra n d o f th e D N A (Figure 1.7). The higher resolution stru ctu re p ro v id es a m o re d e ta ile d view o f th e EG R -l-D N A interface w ith m ore d irec t a n d w ater- m e d ia te d contacts. T he su m m a ry of d irect base a n d p h o sp h a te contacts is sh o w n in F igure 1.8. W ater-m ed iated an d v an d e r W aals co n tacts to bases a n d p h o sp h a te s are su m m arize d in Figure 1.9.
M an y fea tu res o f th is com plex w ere correctly p re d ic te d by sequence a n a ly s e s a n d m u ta tio n a l s tu d ie s (N a rd elli et al., 1991). S ite -d ire c te d m u ta g e n e sis ex p erim e n ts w ith D N A -binding d o m ain s of Krox-20 a n d S p l p ro te in s su b s titu te d the n o n h o m o lo g o u s am ino acids in the rec o g n itio n a - helices a n d resu lte d in in terco n v ersio n of the specificity of the m u ta n t finger (N a rd elli e t al., 1991). T hus, ev en p rio r to the so lu tio n o f the EGR-1 crystal stru c tu re , som e of the base-contacting positions had alread y been identified.
Fingers 1 an d 3 have identical residues at positions -1, 2, 3 a n d 6 of the
a-h elix : R, D, E, R, w h ereas finger 2 has R, D, H, a n d T a t the c o rresp o n d in g p o sitio n s. Fingers 1 a n d 3 m ake tw o p rim ary contacts to the g u a n in e s of the G C G su b site u sin g the g u a n id in iu m g ro u p o f the arginines to h y d ro g e n bon d w ith th e N 7 a n d 0 6 of th e g u a n in e s in each su b site (Figure l.lO A ). The in te ra c tio n o f arg in in e w ith g u an in e w as predicted to p lay a n im p o rta n t role in s e q u e n c e -s p e c ific re c o g n itio n b a se d o n its u n iq u e s te re o c h e m ic a l c o m p le m e n ta rity (Seem an e t al., 1976). Indeed, these contacts a re the m ost p ro m in e n t ones in the EGR-1 - D N A complex. These arg in in es also in teract
Finger 2
His 49
Arg 46
Finger 1
Arg 24
Figure 1.7 Sketch su m m arizin g the principal amino ad d -b ase contacts as seen in the original EGR-1 X -ray structure (Pavletich & Pabo, 1991).
F in g e r 3 ( ,6 1 Arg 8 0 His 5 3 |(,2 ) Asp 76 N ^ r g T i A n g e r 2 Arg 4 2 -— ■ His 2 5 . S a r 4 5 (+3) His 49 |( * 2 ) A s p ^ - ( - l ) A f g R n g e r l (♦6) Arg 24
) (
|( » 2 ) A s p 2 0 - ( - l) A /g 1g (Finger 3)Figure 1.8 S um m ary o f d irect base a n d phosphate co n tacts in E G R -l-D N A com plex. T h e n u m b e rs in p a re n th e sis in fro n t o f a m in o acids a r e th eir positio n s w ith in a -h e lic es. Boxes in d ic a te coupled re s id u e pairs. A rro w s in d icate h y d ro g e n b o n d s; d o tte d a rr o w s rep resen t b o n d s w ith m a rg in a l geom etry (derived fro m Elrod-Erickson e t al., 1996).
( -2 )S e r 17 (♦€) T h r5 2 ( ♦ » « i s 4 9 -— (♦^2) A sp 48 (-1) Arg 46 M l S e r 47 (r-2) Asp 48 R o g e r 1 R o g e r t 48) Arg 24 (*3) Glu 21 (♦2) A sp 20 (-1) Arg 18
c
(♦S) Thr 23 ( 4 l ) S e r 19Figure 1.9 S u m m ary o f w ater-m ed iated a n d vsm d er W aals contacts to bases an d p h o sp h a tes in EGR-1 - D N A com plex. The n u m b ers in p a re n th esis in front of am in o a d d s a re th eir p o sitio n s w ith in a -h e lic e s . Boxes in d ic ate coupled re s id u e p a irs. A rro w s in d ic ate w a ter-m ed iated contacts. Sm all n um bers sh o w n o v e r th e a rro w s in d ic a te th e n u m b er o f w a te r-m e d ia te d bonds. D otted arro w s re p re se n t van d e r W aals contacts.
B ase pair 4 b r Finger 3 )
020 in Finger I (0 4 8 InFingefZ: RI8 In Fingar 1 076 in Finger 3 ) (R46 InFIngerZ:
R74 In F in g e rs )
B B esepeir'S br Finger I
( Base pair2 b r Bnger 3 )
n H 8eeepelr6 N.—
•H----/
---(R80 lnR na«r3)Figure 1.10 D raw ings o f am ino a d d -b a se p a ir contacts o f the EGR-1-DNA complex: (A ) D raw ing o f th e A sp-A rg-guanine in teractio n that occurs in all three zinc fingers; (B) D raw ing of the A rg-guanine interaction that is p resen t in fingers 1 a n d 3; (C) D raw in g of the H is-guanine interaction seen in finger 2 (Pavletich & Pabo, 1991).
The n e w 1.6 Â stru c tu re helps e x p lain th e ro les of the a s p a rtic a d d residues a t p o sitio n 2 of th e recognition helices. The crystal stru c tu re show s th a t the A rg -1 /A sp 2 re sid u e pairs m ake w a te r-m e d ia te d c o n tacts w ith the cytosine w h ic h is base p a ire d to the g uanine co n tacted by A rg -1 (as w ell as w ith the p h o sp h a te on th e 5' side of this guanine). T hese contacts a re seen in all three zinc fingers. In fingers one an d th ree, th ese a sp a rta te s also m ake w ater-m ed iated contacts w ith the neighboring base 5' o f the critical guanine. For instance. A sp +2 in fin g er 1 m akes a w ater-m ed iated contact w ith cytosine
9, and an analogous contact is m ade by Asp +2 of finger 3 to the N 4 o f cytosine 3 (Figure 1.9). The cry stal stru ctu re show s th a t A sp +2 of fin g er 2 clearly contacts cytosine 8', an d tentatively suggests th a t A sp +2 residues firom fingers
1 and 3 form s w eak in teractio n s w ith a base p o sitio n e d o u tsid e th e canonical triplet on the secondary, C -rich strand of the D N A (Figure 1.8). In th e original X-ray stru c tu re one of the oxygens of the aspartic a d d w as sh o w n to be w ithin hydrogen b o n d in g distance to a neighboring base o n th e parallel, C -rich strand o f the DN A. H ow ever, since the H -b o n d in g g eo m etry w as n o t id e a l, these contacts w e re p resu m ed u n im p o rta n t for rec o g n itio n (P avletich a n d Pabo, 1991). M ore favorable g eo m etries w ere o b se rv ed in th e re fin e d stru c tu re (Elrod-Erickson e t al., 1996). For exam ple, w h en A rg -1 o f finger 3 co n tacts G4,
c o n ta c ts As* (th e c o m p lem en tary base to T5 o n the o p p o s ite stra n d ) (F igure
1.8). Such c o n ta ct w as clearly d e m o n stra te d for finger 2 (A sp48 in te rac tin g w i t h Cg ). T he c o rre sp o n d in g in teractio n s fo r fin g ers 1 a n d 3 h a d less fav o ra b le stereochem istry.
T h ere a re sev eral p ie c e s o f e v id en ce su g g e s tin g t h a t th is c o n ta c t c o n trib u te s to reco g n itio n in o th e r zinc fin g er p ro te in s . I n th e co cry stal s tru c tu re o f th e Drosophila re g u la to ry p ro te in T ram track c o m p le x e d w ith its D N A the h o m o lo g o u s a sp artic acid in th e seco n d zinc fin g e r w a s sh o w n to m a k e d irec t h y d ro g e n b onds w ith the seco n d ary D N A s tr a n d (F airall e t al., 1993). R eplacem ent o f the asp artic acid by alanine in the se c o n d zin c finger o f th e S. cerevisiae A D R l p ro te in resu lte d in sig n ific an t r e d u c tio n o f p ro te in b in d in g to its cognate site (T hukral e t al., 1991; T hukral e t al., 1992).
Several biochem ical stu d ie s also p ro p o se the in te ra c tio n o f A sp +2 w ith th e p a ra lle l s tra n d o f the D N A . Zinc fin g er p h ag e d isp la y se lec tio n stu d ie s p ro v id e d o rig in al su p p o rt for th is interaction. The tech n iq u e o f p h a g e d isp lay m a k e s u se o f th e e x p re ssio n of zinc fin g e r reg io n s t h a t c a rry p a rtia lly ra n d o m iz e d rec o g n itio n h elices as p a rt o f a p h a g e c o a t p ro te in . By th e n a llo w in g the p h a g e carrying z in c finger p e p tid e s on its s u rfa c e to eq u ilib ra te w ith a targ et D N A sequence, it is possible to isolate a n d a m p lify th e specific zin c finger p ro tein s th at recognize desired D N A targ et sites. In th e stu d y by C h o o a n d K lu g (1994a) the seq u en ce of th e m id d le f in g e r o f EGR-1 w a s ra n d o m iz e d to test new b in d in g specificities. Strikingly, in a lm o st all of th e se lec te d fingers in w h ich a rg in in e interacts w ith the 3' g u a n in e , a sp artic acid w a s selected a t p o sitio n +2. W h e n position -1 w as n o t a rg in in e , asp artic a d d a t p o sitio n +2 w as practically n e v e r selected. This su g g e sts th a t th e asp artic a d d resid u e + 2 p lay s an im p o rta n t role in co n trib u tin g to th e s p e d f id ty of th e
co n tact. Sw irnoff a n d M ilb ran d t (1995) carried o u t b in d in g s ite selections to d e riv e th e high-affinity consensus b in d in g site fo r EGR-1 fam ily o f proteins.
T h e ir fin d in g s s u p p o r t the im p o rta n c e o f a s p a rtic a c id fo r se q u en c e re c o g n itio n by EGR-1 p ro tein fam ily m em bers. T he a u th o rs n o te d a v ery s tr o n g selectio n for thym ine a t p o sitio n 5 a n d less fre q u e n tly for g u an in e, w h ile a d e n in e o r cy to sin e w e re n ev er se le c te d . B oth A a n d C, th e c o m p le m e n ta ry bases to T o r G a t p o sitio n 5 h a v e h y d ro g e n b o n d d o n o r g ro u p s (N6 and N4) th a t can in te rac t w ith the carboxylate o x y g e n o f A sp +2.
T h e se re s u lts s u p p o r t the id ea th a t the h y d ro g e n b o n d p o te n tia l o n th e s e c o n d a ry D N A s tra n d co n trib u tes to reco g n itio n . A n o th e r im p lica tio n of th ese fin d in g s is th a t a single z in c finger can actu ally sp e cify n o t 3 bp , as o rig in a lly p ro p o sed , b u t 4 bp D N A sites, a n d th e s e b in d in g site s o v erlap by o n e b a s e pair. R ecently p u b lish ed w o rk by Isalan , e t al. (1997) d em o n strates th a t EGR-1-hke zinc fingers can, in fact, specify o v erlap p in g , 4 -b p subsites. In this s tu d y , the th ird zinc finger o f EGR-1 p ro tein w a s m o d ified to rem ove the f in g e r's p o te n tia l fo r DN A in te rac tio n s. N o rm a lly , the t h ir d z in c fin g er in te ra c ts w ith the first triplet o f the EGR-1 con sen su s sequence. H ow ever, as a re s u lt o f finger th ree alterations, changes w ere o b serv ed in th e 5 ’ p o sitio n of the m id d le trip le t o f th e DNA b in d in g site. T h u s, d eletin g th e c o n tact from fin g e r 3 affects the specificity for th e 5' base o f th e b in d in g site o f finger 2.
F u rth e r selectio n a n d gel m o b ility sh ift e x p e rim e n ts p o in te d to th e sam e co n clu sio n (Isalan e t al., 1997). T h u s, the p o te n tia l o f zinc fin g ers to specify o v e rla p p in g , 4-bp subsites w as d em o n strated , a n d the role o f a sp artic acid a t helical p o sitio n 2 w as em phasized. The specificity of each reco g n itio n subsite is, th ere fo re , m e d ia te d b y tw o ad jacen t fingers th a t are sy n e rg istic in th eir m ode o f binding. This has led th e authors to red efin e the b in d in g subsites for each zin c finger, a n d the new schem atic d ia g ra m o f reco g n itio n is sh o w n in Figure 1.11.
T h e roles of th e th ird a -h e lic a l resid u es are also refin ed in the 1 .6 Â
stru c tu re (Elrod-Erickson e t al., 1996). The role of His49 (the th ird residue in finger 2 a-helix) w as prev io u sly discussed in the o rig in al EGR-1 crystal
stru c tu re (Pavletich a n d Pabo, 1991). This re sid u e w as p rev io u sly sh o w n to m ake a d irec t h y d ro g e n bond to N 7 of g u an in e 6. In the n ew stru ctu re, this
h istid in e m ay be contacting 0 6 of th e guanine in stea d . In a d d itio n , His49 also m akes a series of v a n der W aals in te rac tio n s w ith the m e th y l g ro u p of th y m in e 5. The sig n ifican ce o f th e h is tid in e -th y m in e in te ra c tio n w a s d e m o n stra te d by Sw irnoff an d M ilb ran d t (1995) in th eir b in d in g site selection ex p erim en ts. T hus, h istid in e co n trib u tes to th e reco g n itio n o f tw o bases o n the G -rich DN A stran d .
G lu ta m ic acid resid u es w h ic h occur a t a -h e lic a l p o s itio n th ree of fingers 1 a n d 3 w ere suggested to contribute to specificity because biochem ical site se lectio n an d b in d in g stu d ie s all show a p referen ce for cy to sin e a t the center o f trip lets reco g n ized b y fingers one a n d th re e (C hristy a n d N ath an s, 1989; Jam ieso n e t al., 1994; S w irnoff & M ilb ran d t, 1995). As rev e ale d in the refined crystal stru c tu re , the carboxylate g ro u p s o f Glu21 a n d G lu77 do n o t m ake a n y base-specific contacts. H ow ever, the sid e chains o f th ese glutam ates do h y d ro g e n bond to the backbone am ides of th e arginines im m ed iately
F ig u re 1 .1 1 Schem atic diagram of EGR-I zinc fingers in te rac tin g w ith the
