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Essential role of RBP-J in activation of the K8delayed-early promoter ofKaposi's sarcoma-associated herpesvirus byOR!"#R$%

&an an() &an &uan *epartment of +icrobiolo(y) ,chool of *ental +edicine) niversity of Pennsylvania) Philadelphia) P% ./."0),%Received 1/ +arch 1""23 returned to author for revision 1 June 1""23 accepted 1" ,eptember 1""2%vailable online .8 October 1""2

%bstractK,45 K8 (ene is activated by virally encoded transactivator R$% in delayed-early sta(e of viralreactivation6 $hree R$%-responsive elements7RREs 9ere identi:ed in the promoter6 %mon( them) RRE-;; 9as found to be the most criticalcis-actin( element for R$% transactivation6 ;n thisreport) the mechanism underlyin( R$%-mediated activation of the K8 delayed-early promoter9as investi(ated6 % *uence-speci:c *uencefor RBP-J 7?$?%?%% bet9een thenucleotides @."1 and @."8 relative to the transcription initial site6 Removal or mutation of themotif abolished RBP-J bindin( to the K8promoter and as a conse>uence) R$% failed to bind to and activate the promoter6 %n essentialrole of RBP-J in the transcription of the K8promoter 9as demonstrated by diminishment of the promoter activity in RBP-J-null murineembryonic :broblasts6 $aAen to(ether) R$% activatesthe K8 promoter throu(h an indirect bindin( mechanism) i6e6 bein( recruited to the K8promoter throu(h interaction 9ith RBP-J bound to anRBP-J motif in the promoter6 1""2 Elsevier ;nc6 %ll ri(hts reserved6Key9ordsC Kaposi's sarcoma-associated herpesvirus 7K,453 4uman herpesvirus-8 7445-83 K8 promoter3Replication and transcription activator 7R$%3 RBP-J3?ene eDpression re(ulation

;ntroductionKaposi's sarcoma-associated herpesvirus 7K,45) alsoreferred to as human herpesvirus-8 7445-8) is a (ammaherpesvirus)belon(in( to Rhadinovirus (enus 7han( et al6) .//03+oore et al6) .//26 $his virus has been proven to be associated9ith several mali(nancies includin( Kaposi's sarcoma 7K,7%mbroFiaA et al6) .//!) primary eGusion lymphoma 7PEH7esarman et al6) .//! and multicentric astleman's diseases7+* 7,oulier et al6) .//!6$he open readin( frame K8 of K,45encodes a bIip protein)namely K8 protein or replication-associated protein 7R%P orK-bIip 7Hin et al6) .///3 Ihu et al6) .///6 $hree functions havebeen reportedly associated 9ith this protein6 7i K8#R%P 9as

found to bind to the ori(in of viral lytic *uired for the *

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re>uired for viral lytic replication in the delayed-early sta(e)9hile the latter t9o functions are needed in the immediate-earlysta(e6 $herefore) K8#R%P is an important re(ulatory protein inboth immediate-early and delayed-early sta(es of reactivationof K,456onsistent 9ith the functions of K8#R%P protein in the

immediate-early and delayed-early sta(es) the K8 (ene 9asfound to be eDpressed in both sta(es durin( K,45 reactivation6e previously reported that transcription of the K8 (ene inthese t9o sta(es is controlled by t9o distinct promoters)yieldin( t9o transcripts) an immediate-early mR

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al6) 1""0b6 R$% also acts on RREs in the promoter of K/) butnone of these RREs 9ere found to be bound by R$% directly7eda et al6) 1""16 %n RRE in the OR!L promoter has lo9a=nity for direct bindin( of R$% in an electrophoretic mobilityshift assay 7E+,%) but encompasses stron( bindin( sites forcellular *

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incubated 9ith nuclear eDtract derived from $P%-inducedBBH-. cells6 $he bound materials 9ere 9ashed 9ith.!" m+ Kl-containin( buGer and se>uentially eluted 9ith"" m+) !"" m+ and . + Kl6 $he eluted materials 9ereanalyFed byestern blots 9ith antibodies a(ainst some cellular*

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reco(nition motif ?$?%?%% to a Bam4; reco(nition site7i(6 1%6 $his mutation 9as introduced into a K8 promoterMreporter construct PK8-*E1!" and the eGect of this RBP-Jmotif mutation on R$%-mediated transactivation of thepromoter 9as analyFed usin( a luciferase assay6 Reporterconstructs of 9ild-type K8 promoter 7PK8*E-1!") RBP-J

site mutant 7PK8*E-1!"mJ as 9ell as RRE-;) RRE-;; andRRE-;;; deletion mutants 7PK8*E-1!"S.) S1 and S 9ereintroduced into BH0. and 1/ cells to(ether 9ith an R$%eDpressin(vector 7pR6.-OR!" or empty pR6. vector6$he result sho9ed that 7i in a(reement 9ith previous data)deletion of RRE-;; caused a dramatic reduction of R$%mediatedK8 promoter transactivation by /0V in 1/ cellsand over //V in BH0. cells3 7ii mutation of RBP-Jreco(nition site brou(ht about similar eGects 7/!V reductionin 1/ cells and //V in BH0. cells as seen 9ith RRE-;;deletion mutant 7i(6 1B and 6 $his result indicates that thecentral theme of RRE-;; is the RBP-J reco(nition motif9hich plays a critical role in the K8 promoter for R$%mediatedtransactivation6RRE-;;#RBP-J-mediated R$% transactivation contributes mostto transcription of K8 delayed-early (eneOur data demonstrated that the RBP-J motif resided inRRE-;; is a crucial cis-actin( element for R$% activation ofK8 *E promoter6 4o9ever) RRE-; 9as also reported tocontribute to the K8 promoter activation by R$% and sho9nto be directly bound by puri:ed R$% in vitro 7HuAac et al6)1"".6 urthermore) a functional #EBP bindin( site7desi(nated #EBP-;; 9as found 9ithin the RRE-; andectopic eDpression of #EBP 9as able to enhance R$%transactivation of the K8 promoter in a transient reporter (eneassay as 9ell as K8 (ene eDpression in PEH cells 7an( etal6) 1""6 $hese data su((est that the re(ulation of the K8promoter mi(ht involve multiple mechanisms) includin( direct*

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cotransfected into these cells6 irst) ectopic eDpression ofRBP-J and #EBP alone 7in the absence of R$% did notactivate the K8 *E promoter in either BH0. or 1/ cells7i(6 6 ,econd) cotransfection 9ith RBP-J and R$%brou(ht about small increases in the transcription of the K8promoterMreporter in both cell lines6 $his su((ests that the

physiolo(ical concentration of RBP-J 9as saturated fori(6 .6 ;denti:cation of K8 promoter RRE-;; bindin( proteins by usin( *

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the #EBP eGect 9as very limited 71-fold6 $hese datasu((est that endo(enous #EBP has probably reached asaturated concentration in 1/ cells and is able to activate thepromoter by bindin( to RRE-; and recruitin( R$% to thepromoter6 ;t is 9orthy to note that althou(h the physiolo(icalconcentrations of both RBP-J and #EBP in 1/ cells are

hi(h enou(h for them to bind to their motifs) the contributionof RBP-J to the K8 promoter activity is obviously much(reater than that of #EBP because deletion of RRE-;; ormutation of RBP-J resulted in loss of /0M/!V of total promoter

activity in contrast to the removal of RRE-;) 9hich only costsless than !"V of the activity in 1/ cells 7i(s6 1 and B6;nability of transactivation of the K8 promoter in RBP-J-nullcells and restoration of the promoter activity by ectopiceDpression of RBP-J$o further analyFe the role of RBP-J in activation of the K8promoter by R$%) 9e obtained a mouse embryonic :broblast7+E cell line derived from an RBP-J@#@ mouse 7OAa et al6).//!6 $his RBP-J-null line 7O$.. and its 9ild-typecounterpart O$. 9ere used to analyFe activation of the K8promoter and its deletion mutants by R$%6 Reporter constructsof the K8 promoter as 9ell as its mutant derivatives 9ereintroduced into O$.. and O$. cells alon( 9ith R$%eDpressionvector pR6.-OR!" or empty pR6. plasmid6%s sho9n in i(6 0) R$% 9as able to fully activate the K8promoter 7PK8-*E1!" in 9ild-type O$. cells6 ;n contrast)cotransfection of RBP-J@#@ +E O$.. cells 9ith K8 reporterconstruct and R$%-eDpression vector resulted in very lo9luciferase activity) indicatin( that R$% itself 9as not able toe=ciently activate the K8 promoter in the absence of RBP-J6o-eDpression of R$% and increasin( amounts of RBP-J inO$.. cells resulted in pro(ressively hi(her levels of K8promoter activities in the 9ild-type promoter 7pK8-*E1!" andits RRE-; deletion mutant 7pK8-*E1!"S. 7i(6 0%6 4o9ever)ectopic eDpression of RBP- alone did not activate the K8promoter at all 7i(6 0%6 $he RBP-J-dependent transactivationof the K8 promoter by R$%9as (reatly compromised 9henRRE-;;-deleted 7PK8-*E1!"S1 or RBP-J motif-mutatedreporter constructs 7pK8-*E1!"mJ 9ere used in the assayin RBP-J-null cells) con:rmin( the role of RBP-J motif inRRE-;; in the K8 promoter activity 7i(6 0%6 $he eDpression ofRBP-J in the transfected cells 9as monitored by estern blot9ith an antibody speci:c to RBP-J 7i(6 0B6 e noticed thati(6 06 ontribution of RBP-J to transactivation of the K8 *E promoter6 ildtype7O$. and RBP-J-null 7O$.. mouse embryonic :broblast cell lines

9ere cotransfected 9ith ."" n( of K8 promoter-luciferase reporters and ."" n(R$% eDpression vector 7pR6.-OR!" or empty pR6. or ."" n( of RBP-JeDpression vector or combination of pR6.-OR!" and increasin( amounts7"M1"" n( of RBP-J eDpression vector6 7% Huciferase activities 9ereanalyFed by the dual-luciferase reporter assay6 Relative luciferase activities 9eremeasured as fold activity relative to the basal level of luciferase activity in cellsthat are cotransfected 9ith blanA plasmids) 9ith the error bars representin(standard deviations of the results from t9o independent eDperiments6 7Bestern blottin( sho9in( the levels of endo(enous RBP-J in O$. cells) theabsence of endo(enous RBP-J in O$.. cells and ectopic eDpression of RBP-Jin O$.. cells6i(6 6 EGects of ectopic eDpression of RBP-J and #EBP on transcription

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activity of the K8 *E promoter in BH0. and 1/ cells6 % K8 promoter-luciferasereporter and its RRE deletion mutants 9ere introduced into BH0. and 1/ cells9ith RBP-J or #EBP eDpression vector alone or to(ether 9ith R$%eDpression vector 7pR6.-OR!" or pR6. empty plasmid6 Huciferaseactivities 9ere analyFed by the dual-luciferase reporter assay6 Relative luciferaseactivities 9ere measured as fold activity relative to the basal level of luciferaseactivity in cells that are cotransfected 9ith blanA plasmids p?H and pR6.)9ith the error bars representin( standard deviations of the results from t9o

independent eDperiments6

althou(h ectopic eDpression of RBP-J in O$.. cells partiallyrestored R$%-mediated transcriptional activity of the K8promoter) the levels of the promoter activity 9ere lo9er thanthat in 9ild-type O$. cells6 e thinA that it may be caused bya compromised RBP-J activity 7either in *

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reactivation in the RBP-J-null murine embryonic :broblastcell line6 $he results sho9ed that the reactivation of lytic(ene eDpression) viral *

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promoter present no discrepancy) but an addition to ourAno9led(e about R$% activation of the K8 delayed-earlypromoter6Based on the data and observations from the current andprevious studies) a model for K8 *E promoter activation byR$% can be established as follo9s6 7i R$% activates the K8

*E promoter mainly throu(h RRE-;; by interaction 9ithRBP-J protein6 7ii R$% also binds 9eaAly to RRE-; eitherby direct *

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by *uencin(6R$% eDpression vector 7PR6.-OR!" 9as constructedand described previously 7an( et al6) 1""0a6 RBP-JeDpression vector 7p%+-RBP-J-myc 9as provided by ErleRobertson 7niversity of Pennsylvania and #EBP eDpressionvector 7p,?!-la(-#EBP by ?ary 4ay9ard 7Johns

4opAins niversity6*

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$o transfect human embryonic Aidney 1/ and mouseembryonic :broblast cells) subconWuent cells (ro9n in 10-9ellplates 9ere transfected 9ith ."" n( of p?H- reporter) ." n(pRH-$K reporter and ."" n( pR6.-OR!" usin( ia(enEGectene transfection Ait6 ells 9ere harvested at 08 h posttransfectionfor luciferase assay6

$he dual luciferase reporter assay system 7Prome(a 9asused to eDamine the responsiveness of the promoters to R$%6$ransfected cells 9ere 9ashed once 9ith .\ PB, andsuspended in 0"" [l of .\ passive lysis buGer6 ells 9erefroFen-tha9ed once and centrifu(ed in mirocentrifu(e for. min6 ,upernatants 9ere assayed for :reWy luciferase andRenilla luciferase activities usin( a $*-1"#1" luminometer 9ithdual auto inQector 7$urner *esi(ns6 $he luciferase assays 9erecarried out accordin( to the manufacturer's instruction7Prome(a6%cAno9led(mentse thanA *r6 $asuAu 4onQo 7Kyoto niversity for RBP-J7@#@ mouse embryonic :broblast cell line6 e thanA *rs6 KeHan and Erle Robertson 7niversity of Pennsylvania for RBPJeDpression vector 7p%+-RBP-J-myc and polyclonalantibody to RBP-J) *r6 ?ary 4ay9ard 7Johns 4opAinsniversity for #EBP eDpression vector 7p,?!-la(-#EBP and *r6 *avid HuAac for R$% polyclonal antibody6 ethanA all members of &uan Hab for constructive discussion)su((estion and critical readin( of the manuscript6 $his 9orA9as supported by research (rants from the