Molecula basis o FIR-media ed c-myc ansc ip ional con ol
Cyp ian D. Cukie 1, Da id Hollingwo h1, S ephen R. Ma in2, Geo Kelly3, I ene Díaz-
Mo eno1,4, and And es Ramos1
1Molecula S uc u e Di ision, MRC Na ional Ins i u e o Medical Resea ch, The Ridgeway, Mill Hill,
London NW7 1AA, UK
2Physical Biochemis y Di ision, MRC Na ional Ins i u e o Medical Resea ch, The Ridgeway, Mill
Hill, London NW7 1AA, UK
3MRC Biomedical NMR Cen e, The Ridgeway, Mill Hill, London NW7 1AA, UK
4Ins i u o de Bioquimica Vege al y Fo osin esis, US-CSIC, A da. Ame igo Vespucio 49, 41092,
Se illa, Spain
Abs ac
The Fa UpS eam Elemen (FUSE) egula o y sys em p omo es a peak in he concen a ion o c-
Myc du ing cell cycle. Fi s , he FBP ansc ip ional ac i a o binds o he FUSE DNA elemen
ups eam o he c-myc p omo e . Then, FBP ec ui s i s speci ic ep esso (FIR) which ac s as an on/
o ansc ip ional swi ch. He e we desc ibe he molecula basis o FIR ec ui men showing ha he
andem RNA ecogni ions mo i s o FIR p o ide a pla o m o independen FUSE DNA and FBP
p o ein binding and explaining he s uc u al basis o he e e sibili y o he FBP-FIR in e ac ion.
We also show ha he physical coupling be ween FBP and FIR is modula ed by a lexible linke
posi ioned sequen ially o he ec ui ing elemen . Ou da a explain how he FUSE sys em egula es
p ecisely c-myc ansc ip ion and sugges ha a small change in FBP–FIR a ini y leads o a
subs an ial e ec on c-Myc concen a ion.
The c-myc p o o-oncogene egula es he p oli e a ion, g ow h and di e en ia ion o soma ic
cells, in eg a ing a numbe o in acellula and ex acellula p og ams o gene con ol. c-Myc
a ge s he PolII-dependen p omo e o 10–15% o genes, including ansc ip ion ac o s,
mRNA me abolism p o eins, DNA epai ac o s, elome ases and cy okines 1,2. Fu he , i
modula es he ansc ip ion o a numbe o non-coding RNAs 3. c-myc mis- egula ion has been
co ela ed wi h a b oad ange o cance pa hologies 4.
The Fa Ups eam Sequence Elemen (FUSE) ups eam o he c-myc p omo e media es a as
ansc ip ion- esponsi e mechanism esponsible o an upsu ge o c-Myc le els du ing he cell
cycle. FUSE-based c-Myc up egula ion is media ed by he unwinding and opening o an AT-
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Co espondence should be add essed o A.R. ([email p o ec ed]).
Au ho con ibu ions
Cloning was pe o med by D.H. and C.C. Exp ession and pu i ica ion o he FBP and FIR cons uc s we e pe o med by C.C. and D.H.
NMR spec a we e eco ded by C.C. and G.K. and analyzed by C.C. The s uc u es in his pape we e calcula ed by C.C. All NMR
i a ions we e pe o med by C.C. SIA analysis was pe o med by I.D.M. CD da a we e eco ded by A.R. and analyzed by S.M. BLI da a
we e eco ded and analyzed by C.C. and S.M. The pape was w i en by C.C. and A.R. All au ho s we e in ol ed in planning he
expe imen s.
Accession Codes: P o ein Da a Bank: Coo dina es o he s uc u e o FIR RRM1-RRM2 alone and in complex wi h FBP Nbox ha e
been deposi ed wi h accession codes 2kx and 2kxh espec i ely.
Supplemen a y In o ma ion accompanies he pape on www.na u e.com/na u e.
UKPMC Funde s G oup
Au ho Manusc ip
Na S uc Mol Biol. Au ho manusc ip ; a ailable in PMC 2011 Ma ch 1.
Published in inal edi ed o m as:
Na S uc Mol Biol. 2010 Sep embe ; 17(9): 1058–1064. doi:10.1038/nsmb.1883.
UKPMC Funde s G oup Au ho Manusc ip UKPMC Funde s G oup Au ho Manusc ip
ich s e ch ( he FUSE sequence) loca ed abou 1.7 kb ups eam o he c-myc p omo e as a
consequence o he nega i e supe coiling ha accumula es du ing p e-exis ing lowe -le el
ansc ip ional ac i i y 5,6 (Fig. 1a). The FUSE DNA non-coding s and (hence o h e e ed
o as ssFUSE o ssFUSE DNA) ec ui s he FUSE-Binding P o ein (FBP) ha , in u n, in e ac s
wi h T ansc ip ional Fac o IIH (TFIIH) – a complex mul i-componen machine in ol ed in
ansc ip ional ini ia ion and p omo e escape – and inc eases he a e o p oduc i e c-myc
ansc ip ion 7,8. This inc ease in ansc ip ion leads o u he nega i e supe coiling and o
u he opening o he FUSE. The FBP–FUSE complex ec ui s he FBP-In e ac ing Rep esso
(FIR) p o ein ha in e ac s wi h bo h FBP and ssFUSE DNA. Upon binding o FBP and
ssFUSE, FIR in e ac s wi h TFIIH 7 educing FBP-media ed ansc ip ion and he e o e
nega i e supe coiling. FIR-media ed ansc ip ional ep ession leads o e-winding o he
FUSE, ejec ion o FBP and, e en ually, ejec ion o FIR i sel , b inging he sys em back o a
basal ansc ip ion s a e (Fig. 1a).
The FUSE egula o y sys em can be ep esen ed by a simple h ee-componen mechanism o
con ol c-myc ansc ip ion and is a p omising ool in colo ec al cance he apy 9,10. Cen al o
FUSE egula ion is he FIR–FBP in e ac ion ha media es FIR ec ui men and ansc ip ional
shu -o . I has been shown ha ssFUSE DNA binds o he andem RNA Recogni ion Mo i s
(RRMs) o FIR (RRM1-RRM2) and o he ou K-Homology (KH) domains o FBP (Fig. 1b)
11-13 bu also ha a di ec FBP–FIR in e ac ion akes place ia a 26-aa elemen in he amino-
e minus o FBP (called he Nbox, Fig. 1b) and he FIR RRM1-RRM2 andem domains 11.
The impo ance o his in e ac ion has been es ablished by Chung and co-wo ke s 11 who
analyzed he ac i i y o FIR in he FBP–DNA and FBP3–DNA ansc ip ional ac i a ion
sys ems. FBP3 (Fig. 1b), a p o ein o he FBP amily, has been shown o be exp essed a
di e en imes han FBP du ing he cell cycle, and no o be a ec ed by FIR-media ed
ep ession 11,14. Chung and co-wo ke s ha e shown ha FBP Nbox media es an in e ac ion
be ween FBP and FIR while FBP3 Nbox lacks his capabili y. They ha e also shown ha when
he low e iciency o FIR ec ui men o he DNA–FBP3 sys em is by-passed by using FIR o
a high a ini y DNA binding domain, FIR can ep ess FBP3. This indica es ha Nbox-media ed
ec ui men o FIR o he DNA is a c ucial s ep o con ol o FIR ep essional ac i i y 11.
Despi e he a ailabili y o a b oad ange o unc ional in o ma ion, ou molecula
unde s anding o FUSE media ed egula ion – and he e o e ou capabili y o in e e e wi h i
– is se e ely limi ed by he lack o s uc u al da a on he FBP–FIR in e ac ion and by he pauci y
o quan i a i e da a on he p o ein–p o ein and p o ein–DNA in e ac ions ha build FUSE
egula ion. He e we ocus on he molecula mechanism o FIR ec ui men and on he FIR–
FBP in e ac ion ha de ines he leng h and in ensi y o c-myc up egula ion du ing cell cycle.
We use human (Homo sapiens) FIR and FBP p o eins and FUSE DNA o explain why FBP is
ec ui ed be o e FIR in he FUSE unc ional cycle and how he necessa y combina ion o
speci ici y and e e sibili y is achie ed by he Nbox–FIR in e ac ion. Fu he , we quan i y
di ec ly he e ec o he Nbox in a model FBP–FIR–FUSE sys em and he decoupling e ec
o an un olded linke loca ed be ween he Nbox and he DNA-binding egion o FBP. We
conclude ha he swi ch o he FUSE sys em om an ac i a ing o a ep essing s a e is based
on a ela i ely modes inc ease in he a ini y o FIR o he FBP–FUSE DNA complex.
Resul s
S uc u e, dynamics and DNA binding p ope ies o FIR RRM1-RRM2
To unde s and he molecula basis o FIR ec ui men we need o cha ac e ize he s uc u e o
he FIR RRM1-RRM2 andem domains and hei in e ac ions wi h FBP and wi h FUSE DNA.
We ha e sol ed he solu ion s uc u e o FIR RRM1-RRM2 alone and in complex wi h he
FBP Nbox and showed ha in FIR RRM1-RRM2 he helical ace o RRM1 packs on o he β-
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shee ace o RRM2, c ea ing a s able in e ace (1,100 Å2 o bu ied su ace) (Figu e 2a,
Supplemen a y Fig. 1 and Table 1). This o ien a ion is simila o wha is obse ed in he
s uc u e o he DNA-bound FIR RRM1-RRM2 15 (Supplemen a y Fig. 1b). NMR elaxa ion
da a con i m ha binding o FIR o he FBP and FUSE binding pa ne s does no lead o
con o ma ional ea angemen s in he p o ein (Supplemen a y Fig. 2).
The a angemen o RRM1 and RRM2 desc ibed abo e indica es ha only he β-shee nucleic
acid binding su ace o RRM1 is a ailable o DNA binding. Ou NMR Chemical Shi
Pe u ba ion (CSP) da a con i m ha DNA binding in ol es canonical nucleic acid binding
RiboNucleoP o ein (RNP2 and RNP1) mo i s plus esidues in he α2/β4, β1/α1 and β2/β3 loops
o RRM1, including he esidues making con ac wi h he single nucleo ide isible in he
published s uc u e o he DNA-bound FIR RRM1-RRM2 15 (Fig. 2b). Based on he known
RRM–nucleic acid s uc u es, his su ace is likely o accommoda e a 4–5 nucleo ides long
DNA sequence 16 and he e o e we ha e explo ed he nucleobase p e e ence o FIR o ou
successi e nucleo ides posi ions using Sca old Independen Analysis (SIA) 17. We show ha
FIR RRM1-RRM2 has a mode a e sequence p e e ence o T o T/G in all o he ou posi ions
analyzed by SIA (Fig. 2c, Supplemen a y Table 1). We use ollow-up NMR binding assays
wi h DNA 5-me s o di e en base composi ions o con i m his sequence p e e ence and o
de ine a ange o a ini ies o FIR in e ac ion wi h op imal and non-op imal DNA sequences
(Fig. 2c). Knowing FIR's sequence p e e ence and he ange o a ini ies o di e en DNA
sequences allows a mo e accu a e unde s anding o FIR–FUSE in e ac ions.
FIR–FUSE and FBP–FUSE in e ac ions
In o de o de ine accu a e models o FBP and FIR ec ui men and ejec ion i is necessa y o
quan i y he s eng h o he FIR–FUSE in e ac ion and o compa e i wi h he FBP–FUSE
in e ac ion. A p elimina y model o FBP ec ui men pu o wa d by Liu and co-wo ke s
p edic s ha ssFUSE has a highe a ini y o FIR han o FBP and p oposes ha he ini ial
ec ui men o FBP – a he han FIR – o he ssFUSE is due o he highe concen a ion o
FBP in he cell 8 while a ecen pape published du ing e ision o ou wo k indica es ha he
a ini y o FIR o he FUSE is he same o lowe by a ac o o 5 han he one o FBP, depending
on he FUSE DNA used 18. Conside ing ha FIR and FBP can bind o o e lapping sequences
in he FUSE 12 a simila a ini y o he wo p o eins o he DNA would indica es ha he choice
o ec ui ing FBP a he han FIR is dependen on he a ia ions o hei concen a ion in he
cell. To cla i y i he ea lie binding o FBP o he FUSE is dependen on he concen a ion o
FBP and FIR, we ha e eco ded BioLaye In e e ome y (BLI) expe imen s using an
immobilized 40-me ssFUSE (ssFUSE40, Fig. 1a) and inc easing concen a ions o FIR (Fig.
2d). This DNA has been designed o ecapi ula e he a eas o in e ac ions o FIR and FBP as
de ined by Benjamin e al. 12 . We show ha FIR binds o he FUSE wi h a Kd alue o ~7
μM, as de e mined om he a io o he dissocia ion and associa ion a e cons an s, and
independen ly om analysis o he dependence o he ampli ude o he BLI signal on FIR
concen a ion. Ins ead, BLI expe imen s epo ha he complexes be ween ssFUSE40 DNA
and wo FBP KH domains cons uc s wi h and wi hou he Nbox (Fig. 1b) ha e dissocia ion
cons an s in he ~1.5–2 nM ange (Fig. 2d, Supplemen a y Fig. 3, Supplemen a y Table 2). So,
FBP–ssFUSE binding is h ee o de s o magni ude s onge han FIR–ssFUSE binding and
he e o e he ini ial ec ui men o FBP in he cell is d i en by i s highe a ini y o he ssFUSE.
The associa ion o FBP wi h he DNA is long las ing (ko ~4 × 10−4 s−1, Supplemen a y Fig.
3), consis en wi h he p olonged TFIIH-media ed enhancemen o p oduc i e c-myc
ansc ip ion obse ed upon elimina ing FIR down egula ion o TFIIH 8.
FIR dime iza ion on he FUSE has been p oposed o lead o FBP ejec ion 15 o o guide
ea angemen o he FUSE–FBP–FIR complex 18. FIR was p oposed o o m a dime while
bound o ssDNA based on he X- ay s uc u e o ha complex (whe e he p o ein c ys allizes
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as a dime in he asymme ic uni , Fig. 3a) and on he 2:1 s oichiome y ob ained om Size
Exclusion Ch oma og aphy coupled wi h Ligh Sca e ing, Re ac i e Index and Ul aViole
abso bance (SEC-LS/RI/UV) da a. We used NMR o in es iga e his possibili y. Ou assays
show ha wo molecules o FIR bind o a single ssFUSE-de i ed 29-me used in unc ional
s udies 11 (ssFUSE29, Fig. 1a) and ha he a ini y o he wo equi alen o quasi-equi alen
p o ein binding si es is in he low mic omola ange (Fig. 3b), consis en wi h he BLI da a and
wi h he as exchange egime o he shi ing esonances. This is consis en wi h he 2:1
s oichiome y ha has been epo ed o a simila FIR–FUSE DNA complex 15. Howe e , ou
da a also show ha FIR does no dime ize upon binding he FUSE (ssFUSE29). We analyzed
he chemical shi s o all o he amide p o ons wi hin 10 Å o he p o ein–p o ein in e -
molecula in e ace, as de i ed om he X- ay s uc u e (Fig. 3a). In RRM2, which is impo an
o dime iza ion and is a away om he ac ual DNA-binding su ace, none o he well dispe sed
p o ons equencies o he monome ic ee RRM1-RRM2 is a ec ed upon ssFUSE29 binding,
sugges ing ha he dime is no o med. In RRM1 he p oposed dime in e ace is in close
p oximi y o he DNA binding su ace and some chemical shi changes om DNA binding
a e likely o a ec esidues in he pu a i e dime in e ace. To decouple dime iza ion om
DNA binding, we compa ed he CSP eco ded upon RRM1–ssFUSE29 binding o he ones
eco ded upon RRM1 in e ac ion wi h he DNA pen ame s desc ibed in he p e ious pa ag aph
– ha bind wi h a 1:1 FIR:DNA s oichiome y. Ve y simila chemical shi changes a e ob ained
o he wo DNA molecules indica ing ha RRM1 is no in ol ed in dime iza ion ei he (Fig.
3c, Supplemen a y Resul s). Fu he , NMR elaxa ion da a show ha he o a ional co ela ion
ime o he DNA-bound p o ein is lowe han he one expec ed i he wo p o eins would make
con ac o ming a compac 50 kDa complex (Supplemen a y Fig. 2a, Supplemen a y Resul s).
The binding o mo e han one FIR molecule on he FUSE is consis en wi h he elaxed T/TG
sequence p e e ence o FIR, as desc ibed abo e bu he chemical shi changes we obse e
upon FIR–DNA in e ac ions show unambiguously ha dime iza ion does no ake place. I is
unclea whe he he binding o mo e han one FIR on he ee FUSE DNA ha we obse e has
any physiological ele ance, as in he cell FBP binds be o e FIR educing he ssDNA a ailable
o FIR binding and one copy only o FIR can be ec ui ed by he FBP Nbox.
FIR–FBP in e ac ion
We hen examined he in e ac ion be ween FIR RRM1-RRM2 and he FBP Nbox ha is
essen ial o FIR ec ui men o FUSE DNA 11. Ou NMR da a e eal ha he FBP Nbox
assumes a helical con o ma ion and docks on a hyd ophobic su ace de ined by α1, α2 and he
β1/α1 and α2/β4 loops (Fig. 4 and Supplemen a y Fig. 4 and 5) o he RRM2 domain o FIR
(Kd ~15 μM, Fig. 4a and Supplemen a y Table 2). The binding o a helical elemen in an
o ien a ion quasi-pa allel o RRM1 α1 has ne e been obse ed be o e in p o ein–RRM
in e ac ions and he FBP–FIR s uc u e p esen ed he e de ines a no el p o ein in e ac ion
su ace on he mul i- unc ional RRM pla o m (Fig. 5) 16. The pep ide–p o ein con ac s a e
mos ly hyd ophobic (Fig. 4d and Supplemen a y Fig. 4b) and an o e iew o he in e ace
shows ha wo alanine esidues om he Nbox (Ala34 and Ala38) occupy he cen al pa o
he pep ide ecogni ion su ace and a e lanked by bulkie hyd ophobic esidues (e.g. Phe31,
Leu35, Ile41) (Fig. 4c and Supplemen a y Fig. 4b). This a angemen esul s in a e y limi ed
in e -digi a ion be ween side chains o FBP and FIR esidues and in low equency mo ions a
he FBP–FIR in e ace (Supplemen a y Fig. 2 and Supplemen a y Resul s). The posi ion o
hese wo alanine esidues explains he esul s o a ecen mu a ional s udy 11 in which a e y
s ong binding impai men was obse ed o A34V and A38V mu an s. The mu a ions place
a la ge hyd ophobic chain in he cen e o he in e -molecula in e ace, esul ing in s e ic
hind ance (Supplemen a y Fig. 4b). In he same s udy, Chung and co-wo ke s showed ha
mu a ion o Leu35 o Phe31 o alanine has a subs an ial bu smalle e ec , which can be
explained by he loss o he speci ic in e ac ions media ed by hei wo pa ially packed side
chains (Fig. 4c and Supplemen a y Fig. 4b). By con as , mu a ion o Ala42 (A42V) – ha we
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show o be e y loosely packed agains he RRM2 su ace (Supplemen a y Fig. 4b) – does no
lead o subs an ial loss o binding. Ou s uc u e e eals ha he side chains a angemen on
he FBP–FIR in e ac ion su ace is designed o p o ide a low a ini y bu speci ic ecogni ion.
The d ama ic loss o binding ob ained by inse ing a bulky hyd ophobic side chain in he
sol en -excluded pa o he in e ace indica es ha he helix, al hough in a dynamic in e ac ion,
canno e-o ien eely on he RRM2 su ace. I seems likely ha he global speci ici y is
p o ided by he sum o he hyd ophobic con ac s made by he di e en side chains o he
hyd ophobic ace o he Nbox helix. Indeed when Chung and co-wo ke s mu a e
simul aneously bo h Leu35 and Phe31 o alanine he loss o binding is s onge ha he one
obse ed o he single mu an s. Relaxa ion da a on he bound RRM1-RRM2 and he
b oadening o se e al pep ide esonances a he p o ein–pep ide in e ace (Supplemen a y
Me hods, Supplemen a y Fig. 2) a e consis en wi h he s uc u al in o ma ion on he complex
and wi h he ansien na u e o he FIR–FBP con ac s in he FUSE-media ed c-myc egula ion
– in which he ejec ion o FBP p ecedes FIR ejec ion and is dependen on he dis up ion o he
FBP–FIR in e ac ion (Fig. 1a).
The FBP–FIR–FUSE sys em
Wha is he ela ionship be ween p o ein binding and DNA binding on he FIR RRM1-RRM2?
DNA and p o ein in e ac ion su aces a e on di e en domains and on opposi e sides o he
RRM1-RRM2 p o ein (Fig. 6a). The chemical shi changes induced by he wo binding
pa ne s a e addi i e (Fig. 6b), indica ing ha he wo binding e en s a e independen . Indeed,
he a ini y o RRM1-RRM2 o he sho TGTGT DNA oligo does no change when he FBP
Nbox pep ide is also bound and ice e sa (Supplemen a y Fig. 6, Supplemen a y Table 2).
This con i ms ha no allos e ic coope a i i y is p esen be ween Nbox and ssFUSE DNA
binding. The e o e he epo ed e ec o he Nbox on FIR ec ui men is due o he physical
e he ing ha exis s be ween FBP and FIR in he la ge h ee-componen FBP–FIR–ssFUSE
sys em. The lack o an allos e ic e ec in RRM1-RRM2 is consis en wi h he lack o
con o ma ional changes upon binding he FBP Nbox pep ide and he ssFUSE DNA
(Supplemen a y Fig. 1b).
The e ec o physical e he ing by FBP on FIR DNA binding ac i i y was di ec ly measu ed
using BLI and a la ge FBP–FIR–DNA h ee-componen sys em (Fig. 7a). 150 nM FIR RRM1-
RRM2 does no show de ec able binding o he ee DNA o o he DNA bound by he ou
KH domain FBP cons uc , while he addi ion o he Nbox ec ui ing elemen o he FBP
cons uc s imula es he in e ac ion (Fig. 7a). The Kd o FIR in he h ee-molecule complex, as
es ima ed om he associa ion and dissocia ion a e cons an s, is lowe by a ac o o 10–50
han he Kd o FIR o he ee DNA (7 μM). This inc ease in a ini y is modes , much lowe
han he one expec ed i he Nbox was s uc u ally coupled o he i s o he DNA binding
modules (KH1) o FBP. The pa ial decoupling be ween DNA binding by FBP and FIR is
explained by he p esence o a ~50-amino acid uns uc u ed linke ha sepa a es he Nbox
om KH1 (Fig. 1b). The decoupling e ec es ima ed o he FUSE sys em (by a ac o o
~5,000) is consis en wi h he one calcula ed based on a model commonly used o wo
in e ac ing egions sepa a ed by a lexible linke 19,20. Such a decoupling e ec has been o en
epo ed o RNA ecogni ion by mul i-domain p o eins 21.
In o de o alida e he model abo e we p o ide di ec expe imen al e idence ha FIR RRM1-
RRM2 and FBP Nbox do no in e ac wi h he KH egion o FBP and ha he Nbox-FIR RRM1-
RRM2 uni is s uc u ally decoupled om he DNA binding egion o FBP. We ha e i a ed
an unlabeled sample o he FBP cons uc comp ising bo h he Nbox and he KH domains in o
a 15N labeled sample o FIR RRM1-RRM2 and eco ded inge p in NMR 15N-1H co ela ion
NMR spec a. We ha e compa ed hese spec a wi h he ones eco ded du ing an equi alen
i a ion pe o med using an unlabeled sample o he Nbox-only cons uc . The same pa e n
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o chemical shi changes is obse ed in bo h i a ions (Supplemen a y Fig. 7a). This indica es
ha he Nbox–FIR in e ac ion does no change whe he he KH domains a e p esen o no in
he FBP cons uc and ha nei he he KH domains no he linke in e ac wi h RRM1-RRM2.
Fu he , he mode a e inc ease in he line-wid h o he RRM1-RRM2 esonances
(Supplemen a y Fig. 7b) p o ides independen e idence ha he lexible linke be ween he
Nbox and he KH domains s uc u ally decouples FIR RRM1-RRM2 om he FBP KH domain
egion – i ha was no he case a e y se e e b oadening would be obse ed upon binding o
he ~45 kDa FBP cons uc . Nex , we ha e added FUSE DNA o he FIR RRM1-RRM2–FBP
Nbox KH1-KH4 complex desc ibed abo e and eco ded again a inge p in NMR 15N-1H
co ela ion NMR spec um. A d ama ic b oadening is obse ed o FIR RRM1-RRM2
esonances upon addi ion o DNA. The b oadening is mo e se e e o esonances in he mo e
igid pa s o he molecules (e.g. in he β-shee ) and is absen o esonances o he lexible C-
e minus (Supplemen a y Fig. 7c). This indica es ha bo h FBP and FIR bind a he same ime
o he FUSE DNA. Simul aneous binding es ablishes a second DNA-media ed physical link
be ween FBP and FIR – s ongly impai ing he capabili y o he wo molecules o umble
independen ly.
FBP3 in e ac ions wi h FUSE and FIR
In o de o in es iga e whe he a 10–50 old di e ence in a ini y can jus i y ec ui men o
FIR in a cellula en i onmen , we compa ed p o ein–p o ein and p o ein–DNA in e ac ions in
he FBP and FBP3 sys ems. Using BLI we show ha a FBP3 Nbox-KH1-KH4 cons uc binds
o he FUSE elemen (ssFUSE40) wi h an a ini y compa able o he one o FBP (Kd = 1.1 nM)
(da a no shown, Supplemen a y Table 2). Howe e , when we added FIR o he FBP3 Nbox-
KH1-KH4–FUSE sys em no Nbox-dependen binding o FIR o he DNA was obse ed (Fig.
7b). This is consis en wi h he published da a ha indica e ha FBP3 Nbox does no ec ui
FIR in he cell, and con i ms ha he Nbox-dependen ec ui men o FIR we obse e by BLI
mi o s he unc ionally ele an ec ui men o FIR. Nex , we es ablished he di e ence in
a ini y o FBP and FBP3 Nboxes o FIR RRM1-RRM2 ha leads o he loss o unc ional
ec ui men . We measu ed he a ini y o he FBP3 Nbox–FIR RRM1-RRM2 in e ac ion: he
Kd o he complex is 280 ± 36 μM and is highe han he one o he FBP Nbox–FIR RRM1-
RRM2 complex by a ac o o ~20 (Fig. 7c). Tha is, a d op by a ac o o ~20 in a ini y
co esponds o a loss o unc ion in he FBP(s) Nbox–FIR in e ac ion.
Discussion
The wo k p esen ed he e explains he molecula basis o FIR-media ed c-myc egula ion. We
show ha he ini ial ec ui men o FBP by he pa ially open FUSE is jus i ied by i s high
a ini y o a speci ic si e on he DNA. FIR is hen ec ui ed by concu en FBP and DNA
binding, and sepa a e FIR–FBP and FIR–DNA in e ac ions a e es ablished on FIR RRM1-
RRM2. I has been shown ha a single RRM domain (RRM2 o Ra e 1) can bind
simul aneously o p o ein and RNA a ge s 22,23. The use o wo RRM domains in FIR is
he e o e no an absolu e equi emen imposed by he small size o he RRM domain bu a
cha ac e is ic o his speci ic sys em. In FIR, he physical sepa a ion o he DNA and FBP
binding su aces (Fig. 6a) minimizes in e e ence be ween binding e en s in he h ee-subuni
complex. This sepa a ion is unc ionally impo an , because he FBP–FIR in e ac ion mus be
dis up ed upon FBP ejec ion while FIR emains bound o he DNA. The use o a cen al pa ch
o alanines su ounded by la ge hyd ophobic esidues o c ea e a ansien and ye speci ic
in e ac ion is, as a as we a e awa e o , desc ibed he e o he i s ime. I will be o in e es
o explo e whe he such a pa e n de ines o he ansien p o ein–p o ein in e ac ions.
Assembly o he FIR-con aining complex (and he e o e ansc ip ional ep ession) is con oled
by he ela i ely low a ini y, high speci ici y FBP–FIR in e ac ion 11, which is based on a
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no el, non-canonical mode o RRM–p o ein ecogni ion. The sequence o FBP and FIR
dissocia ion in he cell mus be, a leas pa ially, con oled by o he p o ein–p o ein
in e ac ions. The binding a ini y we measu e o he FIR–FUSE in e ac ion (Kd ~7 μM) is
al oge he no su icien o main ain he obse ed FIR–DNA binding a e FBP has dissocia ed
8.
The FUSE mechanism is based on a single ac i a o (FBP) ec ui ing i s own (single) ep esso
(FIR). This allows he es ablishmen o a unc ional ela ionship be ween he leng h o he
ac i a ion s ep and he weak coupling be ween FBP and FIR binding o ssFUSE. A s ong
coope a i i y o FBP–FIR binding on he DNA would speed FIR ec ui men beyond wha is
desi able and would educe he peak o c-myc exp ession (Fig. 7d). Ins ead, he long lexible
linke and he weak Nbox–FIR in e ac ion c ea e a weak coupling be ween he wo p o eins
ha can be egula ed mo e sub ly. In e es ingly, he analysis o he in e ac ion be ween FBP3
Nbox and FIR shows ha a d op by a ac o o 20 in he s eng h o he in e ac ion impai s
unc ional FIR ec ui men in he cell, con i ming a egula o y model whe e a change in a ini y
o one– wo o de s o magni ude egula es an impo an ansc ip ional swi ch. Recen wo k
has asse ed he po en ial o a ge ing c-myc as an an icance s a egy 24 sugges ing ha c-
myc ep ession could be explo ed as a he apeu ic s a egy. Ou wo k e eals how he
modula ion o FBP–FIR coupling allows an ini ial pe iod o unhinde ed FBP-p omo ed
ansc ip ion. This opens he exci ing possibili y ha compounds ha s abilize he Nbox–
RRM2 in e ac ion could speed FIR ec ui men and educe p oduc i e ansc ip ion o c-myc
(Fig. 7d).
Me hods
P o ein p epa a ion
FIR RRM1-RRM2 (amino-acids 103–297, NP_055096) we e cloned in o pETM-30 ec o
(EMBL-Heidelbe g, P o ein Exp ession Facili y), in oducing TEV p o ease-clea able
HisTag-GST usion N- e minal o he inse . The HisTag-GST usion p o ein was pu i ied om
he soluble ac ion by nickel-a ini y ch oma og aphy (Qiagen) ollowed by gel il a ion. The
inal p o ein was concen a ed o 0.4–0.6 mM and s o ed in 10 mM T is-HCl pH 8.0 (o 7.4),
50 mM NaCl, 2 mM T is(2-ca boxye hyl)phosphine (TCEP), 0.05% (w/ ) NaN3 in he
p esence o p o ease inhibi o s (Roche) a −80 °C.
Unlabeled FBP Nbox pep ide (amino-acids 27–52, NP_003893) wi h and wi hou an N-
e minal Y esidue and unlabeled FBP3 Nbox (amino-acids 15–40, NP_003925) we e
chemically syn hesized (Pep ide Syn hesis Facili y, Uni e si y o B is ol and in-house,
espec i ely). The addi ion o Y esidue does no a ec FBP Nbox–FIR RRM1-RRM2
in e ac ion as assessed by NMR i a ions (da a no shown). Labeled (15N o 15N13C) FBP
Nbox pep ide (wi h N- e minal Y) was cloned and exp essed as desc ibed o FIR RRM1-
RRM2 and hen pu i ied om he soluble ac ion by nickel-a ini y ch oma og aphy (Qiagen)
ollowed by HPLC pu i ica ion. The pep ide was s o ed in 10 mM T is-HCl pH 5.0 (o 8.0),
0.05% (w/ ) NaN3 in he p esence o p o ease inhibi o s (Roche) a −80 °C.
FBP Nbox-KH1-KH4 (amino-acids 27–455, NP_003893), FBP KH1-KH4 (amino-acids 85–
455, NP_003893) and FBP3 Nbox-KH1-KH4 (amino-acids 13–431, NP_003925) we e cloned,
exp essed and pu i ied by nickel-a ini y ch oma og aphy as desc ibed o FIR RRM1-RRM2.
The p o ein was u he pu i ied on anion exchange and hepa in a ini y columns. The p o eins
we e s o ed in 10 mM T is-HCl pH 7.4, 50 mM NaCl, 2 mM TCEP, 0.05% (w/ ) NaN3 in he
p esence o p o ease inhibi o s (Roche) a −80 °C.
Fo de ails o he p o ein p epa a ions see he online Supplemen a y Me hods sec ion.
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ssDNA oligonucleo ides
All ssDNA oligonucleo ides and SIA oligonucleo ides pools we e chemically syn hesized
(Sigma and In eg a ed DNA Technologies). ssFUSE40 co esponds o nucleo ides 769–808 in
he complemen a y s and o X00364 en y.
NMR spec oscopy
All NMR expe imen s we e eco ded a 37 °C o 45 °C on Va ian Ino a and B uke A ance
spec ome e s equipped wi h c yop obes and ope a ing a 600, 700 and 800 MHz 1H
equencies. The spec a we e p ocessed wi h he NMRPipe package 27 and analyzed wi h
Spa ky 28. Fu he de ails can be ound in Supplemen a y Me hods.
S uc u e calcula ions
Dis ance and angle es ain s we e used o pe o m s uc u e calcula ions wi h ARIA 1.2 29.
Expe imen al dis ance es ain s we e achie ed by in eg a ing Spa ky assigned NOE peaks
wi h XEASY 30. Dihed al es ain s (φ and ψ) we e ob ained om he chemical-shi -based
TALOS da abase 31. Fo he ee and bound p o eins, H-bond es ain s we e added in
subsequen calcula ions i an exchange-p o ec ed 1HN was H-bonded in a leas 50% o he
p elimina y s uc u es gene a ed in a gi en un. Fo he pep ide, he H-bond es ain s o a
s anda d α-helix we e added in he helical egion (Ala30–Lys44).
In i e a ions 0–7 o ARIA calcula ions i y andomized con o me s we e subjec o simula ed
annealing wi h a s anda d CNS p o ocol 32. The en lowes -global-ene gy s uc u es we e used
o assignmen a he nex i e a ion. In i e a ion 8 he numbe o gene a ed s uc u es was
inc eased o 200. Finally, he 20 lowes -ene gy ob ained s uc u es we e wa e e ined 33. The
quali y o each gene a ed amily was e alua ed wi h PROCHECK_NMR 34. Ramachand an
plo s a is ics o FIR RRM1-RRM2: Mos a o ed egions 83.9%, Addi ional allowed egions
14.3%, Gene ously allowed egions 0.9%, Disallowed egions 0.9%. Ramachand an plo
s a is ics o FIR RRM1-RRM2–FBP Nbox complex: Mos a o ed egions 82.2%, Addi ional
allowed egions 15.3%, Gene ously allowed egions 1.0%, Disallowed egions 1.5%. The
s uc u es we e displayed and analyzed wi h MOLMOL 26, PYMOL (www.pymol.o g) and
Insigh II (Accel ys).
Ci cula Dich oism (CD)
Fa -UV CD spec um o 50 μM FBP Nbox in 10 mM T is-HCl pH 7.4, 20 mM NaCl was
eco ded a 5 °C on a Jasco J-715 spec opola ime e (Jasco) equipped wi h a PTC-348 Pel ie
empe a u e-con ol sys em. The CD signal is epo ed as he mean esidue CD ex inc ion
coe icien (ΔεMRW).
Binding assays: NMR
NMR i a ions we e pe o med by i a ing unlabeled in e ac ing pa ne in o solu ion o 15N-
labeled sample ( o de ails see Supplemen a y Me hods). 15N so as HMQC spec a 35 we e
eco ded a each i a ion poin a 37 °C o 45 °C on Va ian Ino a and B uke A ance
spec ome e s equipped wi h c yop obes and ope a ing a 600, 700 and 800 MHz 1H
equencies. To ob ain Kd alues he a e age chemical shi pe u ba ions (Δδa g = ((ΔδN/
10)2 + ΔδH2)1/2) o 7–10 peaks we e plo ed as a unc ion o ligand:p o ein a io, ollowed by
wo-pa ame e nonlinea leas -squa es i o he da a o each peak in he p og am O igin
(O iginLab), using a one-si e binding model which co ec s o dilu ion e ec 36. Kd alues
a e epo ed as a e age ± 2 s anda d de ia ions.
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Binding assays: SIA
SIA analysis was pe o med as desc ibed in Beu h e al. 17 wi h 16 pools o 5-me DNA
oligonucleo ides (nANNN, nGNNN, nCNNN e c.). In b ie , solu ions o 25 μM 15N-labeled
FIR RRM1-RRM2 in 10 mM T is-HCl pH 7.4, 50 mM NaCl, 2 mM TCEP we e i a ed wi h
indi idual DNA pools ( a ios 1:0, 1:1 and 1:4). 15N so as HMQC spec a 35 we e eco ded a
each i a ion poin a 37 °C on a Va ian Ino a spec ome e equipped wi h c yop obe and
ope a ing a 800 MHz 1H equency. A e age chemical shi pe u ba ions (Δδa g = ((ΔδN/
10)2 + ΔδH2)1/2) o 15 peaks we e analyzed o ob ain SIA sco es. The esul s we e isualized
wi h WebLogo (h p://weblogo.be keley.edu/logo.cgi).
Binding assays: BLI
All BLI expe imen s we e pe o med in 10 mM T is-HCl pH 7.4, 150 mM NaCl, 2 mM TCEP,
0.5 mg ml−1 BSA on an Oc e Red ins umen (Fo eBio, Inc., Menlo Pa k, CA) ope a ing a
25 °C. S ep a idin coa ed biosenso s wi h immobilized bio inyla ed ssFUSE40 (Sigma) we e
exposed o di e en concen a ions o FIR RRM1-RRM2, FBP Nbox-KH1-KH4, FBP KH1-
KH4 o FBP3 Nbox-KH1-KH4 o combina ion o hem as desc ibed in he Supplemen a y
Resul s sec ion.
Supplemen a y Ma e ial
Re e o Web e sion on PubMed Cen al o supplemen a y ma e ial.
Acknowledgmen s
We would like o hank D s A. O eggioni and T. F enkiel o help in eco ding NMR expe imen s, D s C. deChia a
and G. Nicas o o ad ice on he ARIA p o ocols used in s uc u e calcula ions, D . A. M. Candel o help wi h
spec oscopic da a and S. K alo ico a o gene al suppo in he lab. We would also like o hank D Pe e Rosen hal
o use ul discussions. All NMR spec a we e eco ded a he MRC Biomedical NMR Cen e. We would like o hank
D S. Kindle (Uni e si y o Hambu g) o he gi o a plasmid wi h he FIR RRM1-RRM2 gene. This wo k has been
unded by he MRC G an -in-aid U117574558.
Re e ences
1. Le ens D. Disen angling he MYC web. P oc Na l Acad Sci U S A 2002;99:5757–5759. [PubMed:
11983876]
2. Wie s a I, Al es J. The c-myc p omo e : s ill Mys e Y and challenge. Ad Cance Res 2008;99:113–
333. [PubMed: 18037408]
3. Kenne h NS, Whi e RJ. Regula ion by c-Myc o ncRNA exp ession. Cu Opin Gene De 2009;19:38–
43. [PubMed: 19179065]
4. Meye N, Penn LZ. Re lec ing on 25 yea s wi h MYC. Na Re Cance 2008;8:976–990. [PubMed:
19029958]
5. Kouzine F, Liu J, San o d S, Chung HJ, Le ens D. The dynamic esponse o ups eam DNA o
ansc ip ion-gene a ed o sional s ess. Na S uc Mol Biol 2004;11:1092–1100. [PubMed:
15502847]
6. Kouzine F, San o d S, Elisha-Feil Z, Le ens D. The unc ional esponse o ups eam DNA o dynamic
supe coiling in i o. Na S uc Mol Biol 2008;15:146–154. [PubMed: 18193062]
7. Liu J, e al. De ec i e in e play o ac i a o s and ep esso s wi h TFIIH in xe ode ma pigmen osum.
Cell 2001;104:353–363. [PubMed: 11239393]
8. Liu J, e al. The FUSE/FBP/FIR/TFIIH sys em is a molecula machine p og amming a pulse o c-
myc exp ession. EMBO J 2006;25:2119–2130. [PubMed: 16628215]
9. Ma sushi a K, e al. An essen ial ole o al e na i e splicing o c-myc supp esso FUSE-binding p o ein-
in e ac ing ep esso in ca cinogenesis. Cance Res 2006;66:1409–1417. [PubMed: 16452196]
10. Ma sushi a K, e al. c-myc supp esso FBP-in e ac ing ep esso o cance diagnosis and he apy.
F on Biosci 2009;14:3401–3408. [PubMed: 19273283]
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Figu e 5.
FIR–FBP in e ac ion ep esen s a no el ecogni ion mode in he RRM amily. Ribbon
ep esen a ions o he s uc u es o p o ein–p o ein complexes be ween RRM domains (g ey)
and hei binding pa ne s (blue and g een): Y14–Mago–PYM (1 k8), UPF3b–UPF2 (1uw4),
p14–SF3b155 (2 9d), Ra e 1–Vinculin (3h2u), U2AF35–U2AF65 (1jm ), U2AF65–SF1
(1o0p), SPF45–SF3b155 (2peh), U2B″–U2A′ (1a9n) and FIR–FBP ( his s udy). In he PTB–
Ra e 1 complex he Ra e 1 pep ide posi ion is epo ed (blue) on he s uc u e o PTB RRM2
in he complex wi h RNA (2adb), acco ding o he published model 22. U2AF35, U2AF65 and
SPF45 RRM domains belong o he UHM sub amily, ha bind a conse ed T p esidue in he
ligand pep ides and includes he hi d RRM o PUF60, an iso o m o FIR p o ein 25. The ca oon
ep esen a ion o he di e en s uc u es we e gene a ed using he p og am MOLMOL 26.
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Figu e 6.
FIR RRM1-RRM2 independen ly binds FBP Nbox and ssFUSE on wo physically sepa a ed
si es loca ed on opposi e sides o he molecule. (a) Su ace ep esen a ion o FIR RRM1-
RRM2. Residues showing subs an ial CSP upon addi ion o ssFUSE29 o FBP Nbox pep ide
a e colo ed in blue and ed espec i ely. This s uc u al ep esen a ion was gene a ed using he
p og am PYMOL (www.pymol.o g). (b) Supe imposed 15N-1H co ela ion spec a show ha
FIR RRM1-RRM2 in e ac s independen ly wi h a ssFUSE29 and he FBP Nbox pep ide. The
spec a o RRM1-RRM2+ssFUSE DNA, RRM1-RRM2+FBP Nbox and RRM1-RRM2
+ssFUSE DNA+FBP Nbox a e in blue, ed and g een espec i ely. A ep esen a i e egion
con aining one RRM1 esonance pe u bed by DNA binding (Val114) and one RRM2
esonance pe u bed by pep ide binding (Asp222) is displayed. The chemical shi changes o
RRM1-RRM2 peaks in he p o ein–DNA and p o ein–pep ide complexes a e addi i e in he
h ee molecule complex, indica ing independen binding.
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Figu e 7.
FBP Nbox ec ui s FIR o ssFUSE DNA. (a) BLI binding assays show ha he p esence o he
Nbox in FBP cons uc inc eases he a ini y o FIR o he DNA. The BLI biosenso s we e
de i a ized wi h ssFUSE40 and exposed o di e en combina ions o p o ein cons uc s
(indica ed on he igh ), as epo ed in he igu e. RU changes in h ee pa allel expe imen s a e
displayed. (b) FBP3 Nbox does no media e ec ui men o FIR o he DNA. BLI biosenso s
we e de i a ized wi h ssFUSE40 and exposed o di e en combina ions o p o ein cons uc s
(indica ed on he igh ), as epo ed in he igu e. The RU changes obse ed in h ee pa allel
expe imen s a e displayed. (c) Iso he ms o FBP and FBP3 Nbox binding o FIR RRM1-
RRM2 (NMR). Kd a e epo ed ± 2 × s.d. (d) Th ee di e en modes o FBP–FIR coupling and
hei e ec on FIR binding and c-myc ansc ip ion. Le – In he absence o a coupling be ween
FBP and FIR he a ini y o FIR o he ssFUSE (Kd ~7 μM) would be oo low o FIR o bind
he ssFUSE. FBP ac i a ion o c-myc ansc ip ion (bo om) would he e o e con inue
unpe u bed. Middle – Boxed, he physiological scena io. The weak binding o he Nbox o
FIR RRM2 and he 50-amino acid linke be ween he Nbox and KH1 a e esponsible o he
physiological weak coupling be ween FBP and FIR. This coupling is necessa y o c ea e he
equi ed peak in c-myc exp ession (bo om) and egula e he cell cycle. Righ – A s onge
coupling be ween FBP and FIR c ea ed by, o example, a small molecula weigh compound
binding a he in e ace be ween he wo molecules ha would inc ease wo o de s o magni ude
he appa en a ini y o FIR o he FBP–ssFUSE complex, would speed FIR ec ui men and
lead o a sho e peak in c-myc exp ession (bo om).
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Table 1
NMR and e inemen s a is ics o p o ein s uc u es
FIR RRM1–RRM2
(103–297) FIR RRM1–RRM2
(103–297) – FBP
Nbox (27–52)
complex
NMR dis ance and dihed al cons ain s
Dis ance cons ain s 4,431 4,647
To al NOE 4,368 4,611
In a- esidue 1,588 1,632
In e - esidue 2,780 2,979
Sequen ial (|i – j| = 1) 906 878
Medium- ange (|i – j| < 4) 487 546
Long- ange (|i – j| > 5) 1,387 1,483
In e molecula – 72
Hyd ogen bonds 63 36
To al dihed al angle es ain s 236 280
φ118 140
ψ118 140
S uc u e s a is ics
Viola ions (mean and s.d.)
Dis ance cons ain s (> 0.3 Å) (Å) 0.32 ± 0.02 0.34 ± 0.03
Dihed al angle cons ain s (> 5°) – *6.37 ± 0.98
Max. dihed al angle iola ion (°) – *8.65
Max. dis ance cons ain iola ion (Å) 0.35 0.42
De ia ions om idealized geome y
Bond leng hs (Å) 0.0036 ± 0.0001 0.0036 ± 0.0001
Bond angles (°) 0.480 ± 0.011 0.507 ± 0.008
Imp ope s (°) 1.46 ± 0.08 1.41 ± 0.06
A e age pai wise .m.s. de ia ion** (Å)
Hea y 1.91 ± 0.23 2.03 ± 0.24
Backbone 1.28 ± 0.20 1.40 ± 0.21
*No iola ions > 5°
**Pai wise .m.s. de ia ion was calcula ed among 20 e ined s uc u es ( esidues 105-288 o FIR RRM1-RRM2 and esidues 105-288 and 27-45 o
FIR RRM1-RRM2–FBP Nbox complex)
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