HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus

BioMed Cen al Page 1 o 16 (page numbe no o ci a ion pu poses) Molecula Cance Open Access Resea ch HHV-8 encoded LANA-1 al e s he highe o ganiza ion o he cell nucleus Gyö gy S ube 1, Ka in Ma sson1, Emilie Flabe g1, Em ah Ka i2, Laszlo Ma kasz3, Julie A Sheldon2, Geo ge Klein1, Thomas F Schulz2 and Laszlo Szekely*1 Add ess: 1Depa men o Mic obiology, Tumo and Cell Biology (MTC) and Cen e o In eg a i e Recogni ion in he Immune Sys em (IRIS), Ka olinska Ins i u e, S ockholm, Sweden, 2Depa men o Vi ology, Hanno e Medical School, Hanno e , Ge many and 3Depa men o Pedia ics, Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Deb ecen, Hunga y Email: Gyö gy S ube - [email p o ec ed]; Ka in Ma sson - ka ma[email p o ec ed]; Emilie Flabe g - emi [email protected]; Em ah Ka i - [email p o ec ed]; Laszlo Ma kasz - ma[email p o ec ed]; Julie A Sheldon - Sheldon.Ju[email p o ec ed]; Geo ge Klein - [email p o ec ed]; Thomas F Schulz - Schulz.Thomas@mh-hanno e .de; Laszlo Szekely* - [email protected] * Co esponding au ho Abs ac The la ency-associa ed nuclea an igen (LANA-1) o Human He pes Vi us 8 (HHV-8), al e na i ely called Kaposi Sa coma He pes Vi us (KSHV) is cons i u i ely exp essed in all HHV-8 in ec ed cells. LANA-1 accumula es in well-de ined oci ha co-localize wi h he i al episomes. We ha e p e iously shown ha hese oci a e igh ly associa ed wi h he bo de s o he e och oma in [1]. We ha e also shown ha exogenously exp essed LANA-1 causes an ex ensi e e-o ganiza ion o Hoechs 33248 DNA s aining pa e ns o he nuclei in non-HHV-8 in ec ed cells [2]. He e we show ha his e ec includes he elease o he bulk o DNA om he e och oma ic a eas, in bo h human and mouse cells, wi hou a ec ing he o e all le els o he e och oma in associa ed his one H3 lysine 9 i-me hyla ion (3MK9H3). The elease o DNA om he he e och oma ic ch omocen e s in LANA-1 ans ec ed mouse cells co-incides wi h he dispe sion o he ch omocen e associa ed me hylcy osin binding p o ein 2 (MECP2). The localiza ion o 3MK9H3 o he emnan s o he ch omocen e s emains unal e ed. Mo eo e , exogeneously exp essed LANA-1 leads o he eloca ion o he ch omocen e s o he nuclea pe iphe y, indica ing ex ensi e changes in he posi ioning o he ch omosomal domains in he LANA-1 ha bo ing in e phase nucleus. Using a se ies o dele ion mu an s we ha e shown ha he ch oma in ea anging e ec s o LANA-1 equi e he p esence o a sho (57 amino acid) egion ha is loca ed immedia ely ups eam o he in e nal acidic epea s. This sequence lies wi hin he p e iously mapped binding si e o his one me hyl ans e ase SUV39H1. We sugges ha he highly concen a ed LANA-1, ancho ed o he hos genome in he nuclea oci o la en ly in ec ed cells and eplica ed h ough each cell gene a ion, may unc ion as "epigene ic modi ie ". The induc ion o his one modi ica ion in adjacen hos genes may lead o al e ed gene exp ession, he eby con ibu ing o he i al oncogenesis. Published: 13 Ap il 2007 Molecula Cance 2007, 6:28 doi:10.1186/1476-4598-6-28 Recei ed: 14 Feb ua y 2007 Accep ed: 13 Ap il 2007 This a icle is a ailable om: h p://www.molecula -cance .com/con en /6/1/28 © 2007 S ube e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 2 o 16 (page numbe no o ci a ion pu poses) Backg ound Human he pes i us i us 8 (HHV-8) is conside ed as he causa i e agen o Kaposi's sa coma (KS) and is also asso- cia ed wi h p ima y e usion lymphomas (PELs) and mul- icen ic Cas leman's disease (MCD). I is a gammahe pes i us ha shows sequence homology o Eps ein-Ba i us (EBV) and he pes i us saimi i (HVS) ha a e able o ans o m B (EBV) and T cells (HVS), espec i ely. Bo h i uses can cause malignan lympho- mas [3]. HHV-8 encodes a la ge numbe o p o eins ha show s uc u al simila i ies wi h cellula p o eins in ol ed in cellula p oli e a ion, cell cycle egula ion and immune modula ion [4]. A human cyclin D homologue, CYC, ORF72, a bcl-2 homologue, ORF16 [5], an IL-8-like G- p o ein coupled ecep o , GCRP, ORF74 [6] and in e - e on egula o y ac o s, IRFs, ORFK9, ORFK10.5 [4] a e among he genes ha ha e been pi a ed by he i us. The la ency-associa ed nuclea an igen (LANA-1, LNA o LNA-1), encoded by ORF73, is one o ew HHV-8 encoded p o eins ha is highly exp essed in all la en ly in ec ed umo cells [7-9]. This sugges s ha LANA-1 plays a c i ical ole in main enance o la en HHV-8 in ec ion. LANA-1 is a 222–234 kDa phosphop o ein wi h an acidic in e nal epea domain lanked by a ca boxy- e minal domain and an amino- e minal domain [9]. Cons i u i e exp ession o LANA-1 om i s own p omo e in ansgenic mice induced splenic ollicula hype plasia due o an expansion o IgM+ IgD+ B cells and led o inc eased ge minal cen e o ma ion. LANA-1 exp essing B-cell lesions could also p og ess o lymphomas [10]. LANA-1 ac s as a ansc ip ional egula o . I has been shown o bind o p53 and o he e inoblas oma p o ein pRb. This leads o he inac i a ion o p53-dependen p o- mo e s and induc ion o E2F-dependen genes [11,12]. Toge he wi h he cellula oncogene H- as, LANA-1 ans- o ms p ima y a emb yo ib oblas s [13]. I can ansac- i a e he p omo e o he e e se ansc ip ase subuni o he human elome ase holoenzyme [14]. Ac i a ion o el- ome ase is a c i ical s ep in cellula ans o ma ion [15]. LANA-1 is also in ol ed in ansc ip ional ep ession, howe e [16-18]. I can, mo eo e , in e ac wi h he mSin3/HDAC1 co- ep esso complex [17]. I has been also shown o in e ac wi h and inhibi he ATF4/CREB2 ansc ip ion ac o ha in e ac s wi h he basic ansc ip- ion machine y [19]. LANA-1 was also epo ed o bind wo human ch omosome-associa ed cellula p o eins, MeCP2 and DEK [17]. RING3, a homology o he sh ( emale s e ile homeo ic) gene p oduc o D osophila, in e ac s wi h LANA-1 [20]. This esul s in he phospho yla ion o LANA-1. We ha e shown by immuno luo escence ha LANA-1 can e-loca e RING3 in o he e och oma in egions and ha LANA-1 and RING3 co-localize in he nuclea bodies o BCBL-1 cells. Exogenously exp essed LANA-1 inc eased he exp es- sion o RING3 [2]. LANA-1 is associa es wi h cellula ch oma in and s ays on he ch omosomes du ing cell di ision [21]. I main ains he i al genomes du ing cell di ision by e he ing he i al episomes o he ch omosomes [22]. I binds di ec ly o eplica ion o igin ecogni ion complexes (ORCs) ha a e p ima ily associa ed wi h he e minal epea (TR) egion o he HHV-8 genome [23]. Binding o LANA-1 o TR con e s ansc ip ional silencing, on he p omo e o he neighbou ing ly ic gene K1 [24]. LANA-1 is belie ed o play an impo an ole in he supp ession o ly ic i al genes and main enance o i al la ency. The key ly ic eg- ula o p o ein, RTA ac i a es he exp ession o se e al ly ic i al genes by in e ac ing wi h ecombina ion signal sequence-binding p o ein Jkappa (RBP-Jkappa), a an- sc ip ional ep esso and he a ge o he No ch signaling pa hway. Impo an ly, LANA-1 also supp esses RTA ac i - i y by i s di ec binding o RBP-Jkappa [25]. Dis inc egions o he N- e minus o LANA-1 a e espon- sible o nuclea a ge ing and binding o human ch omo- somes [26]. The 1–22 N e minal esidues o LANA-1 bind di ec ly o an acidic pa ch on he co e his one dime s H2A-H2B [27]. LANA-1 shows a cha ac e is ic cellula dis- ibu ion. The HHV-8 episomes and he associa ed LANA- 1 p o ein accumula e in i egula ly shaped bodies in he in e phase nucleus, p e e en ially a he bo de o he e o- ch oma in [1]. I binds o human me aphase ch omo- somes in an appa en ly andom ashion [21,26]. Exogeneously exp essed C e minal pa o LANA-1 p e e - en ially concen a es o pai ed do s a pe icen ome ic and pe i- elome ic egions o a subse o mi o ic ch omo- somes [28]. A sho 15 aa egion in he C e minal pa is esponsible o he associa ion wi h he e och oma in [29]. This ch oma in-binding domain is equi ed o mul- iple LANA-1 unc ions, such as he abili y o bind o and eplica e i al episomes, o modula e ansc ip ion, and o in e ac wi h he membe s o B d ch oma in binding p o- eins B d2/RING3 and B d4s [30-32]. We ha e p e iously shown ha exogenous exp ession o LANA-1 induces a majo e-o ganiza ion o DNA s aining pa e ns. He e we show ha his eo ganiza ion leads o he disappea ance o no mal he e och oma in pa e n in bo h human and mouse cells. In o de o u he cha ac- e ize he e ec o LANA-1 on he e och oma in we ha e compa ed i s dis ibu ion in HHV-8 ca ying cells and in non-in ec ed bu LANA-1 ans ec ed cells in ela ion o di e en he e och oma in ma ke s. Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 3 o 16 (page numbe no o ci a ion pu poses) Ma e ials and me hods Cell cul u e condi ions and cell lines The cells we e cul u ed a 37°C a 5% CO 2 in Isco e's modi ied Dulbecco's cell cul u e medium supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (FBS), 100 U/ml penicillin and 100 U/ml s ep omycin. The cells we e passaged and spli 1:5 e e y ou h day. The cell cul- u es we e egula ly es ed o he absence o mycoplasma in ec ion by Hoechs 33258 s aining. The cell lines used in his s udy we e he ollowing: he HHV-8 in ec ed human body ca i y lymphomas BC-1 and BCBL-1; human b eas ca cinoma MCF-7; human os eosa coma cell line Saos-2, human ce ical ca cinoma HeLa; immo alized mouse ib oblas s NIH3T3 and mouse ib osa coma L (Ame ican Type Cul u e Collec ion (ATCC). T ansien ans ec ion p ocedu e The human o mouse cells we e g own on co e slips in six-well pla es and ans ec ed o 24–48 hou s using a ull-leng h LANA-1 cDNA inse ed in o a pcDNA1 ec o o a LANA-1 dele ion cons uc s [29]. An emp y ec o o a pBabe EBNA-5 cons uc c ea ed by us was used o con- ol ans ec ions. T ans ec ion o cells was made acco d- ing o he manu ac u e 's ins uc ions using FuGene6 (Roche). Immuno luo escence mic oscopy The ans ec ed cells (g own on co e slips) o body ca i y lymphoma cell lines (cy ospinned on o glass slides) we e ixed in me hanol: ace one (1:1) a -20°C o 20 min. The e-hyd a ion o cells was done in PBS o 20 min a oom empe a u e. The ollowing an ibodies we e used in his s udy o immuno luo escence s aining: human an i- LANA KS2 (an ise um, a gi om A ila Juhasz, he De - ma ology Uni o Deb ecen Medical School, Hunga y), abbi polyclonal an i- i-me hyl K9 his one H3 (A gi om D P im Sing); abbi polyclonal IgG an i-mouse MeCP2 ( eac ing wi h bo h human and mu in MeCP2) (Ups a e); FITC-conjuga ed swine an i- abbi (DAKO); hodamine-conjuga ed abbi an i-human (DAKO); FITC- conjuga ed abbi an i-human (DAKO) o Texas ed-con- juga ed ho se an i-mouse (Vec o ) we e used as seconda y an ibodies. The di e en combina ions o p ima y and seconda y an ibodies a e speci ied in espec i e igu es. The con ol ans ec ion o pBabe-EBNA-5 was s ained wi h a mouse monoclonal an i-EBNA-5 (JF186)[33]. Texas ed-conjuga ed ho se an i-mouse (Vec o ) was used as seconda y an ibody. The an ibodies we e dilu ed in blocking bu e (2% BSA, 0.2% Tween-20, 10% glyce ol, 0.05% NaN3 in PBS). The p ima y an ibodies we e incu- ba ed in a humid chambe a oom empe a u e o one Compa ison o exp ession le els o i us encoded and exogeneously in oduced LANA-1 in he nucleus o HHV-8 posi i e BCBL-1 body ca i y lymphoma (le ) and HHV-8 nega i e MCF7 b eas ca cinoma cell (middle)Figu e 1 Compa ison o exp ession le els o i us encoded and exogeneously in oduced LANA-1 in he nucleus o HHV-8 posi i e BCBL-1 body ca i y lymphoma (le ) and HHV-8 nega i e MCF7 b eas ca cinoma cell (middle). Immuno luo escence s aining (g een) using human an i-LANA-1 se um de ec ed by FITC conjuga ed mouse an i-human immunoglobulins. The images a e Z axis p ojec ions o s acks o 10 op ical sec ions, 0.5 mic ome e apa , cap u ed om iden ically s ained and p ocessed nuclei using an au oma ed wide ield luo escence mic oscope. The coun e s aining o BCBL-1 DNA wi h Hoechs 33258 (blue) is shown o easie o ien a ion. The 3D p ojec ion o he plo o he measu ed s aining in ensi y ( igh ) illus a es ha he nuclea oci o la en ly in ec ed cells con ain compa able amoun o LANA-1 o he ansien ly ans ec ed ones. Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 4 o 16 (page numbe no o ci a ion pu poses) hou ollowed by h ee washes wi h PBS. Incuba ion wi h he seconda y an ibodies was done o one hou in humid chambe a oom empe a u e. Double s aining be ween LANA-1 and he di e en ch oma in-associa ed p o eins we e done as ollows: abbi an i- i-me hyl K9 H3 o ab- bi an i-MeCP2, FITC-conjuga ed swine an i- abbi , no - mal abbi , human an i LANA-1 and a las hodamine conjuga ed abbi an i-human. DEK double s aining: mouse an i-DEK, Texas ed conjuga ed ho se an i-mouse, no mal mouse, human an i-LANA1 and FITC conjuga ed abbi an i-human. The DNA was s ained wi h Hoechs 33258. Each incuba ion s ep was ollowed by h ee washes in PBS. The images we e cap u ed wi h one o he ollowing sys- ems: Lei z DM RB wide ield luo escence mic oscope equipped wi h a Hamama su dual mode cooled CCD came a C4880 whe e he images we e eco ded and ana- lysed on a Pen ium PC compu e equipped wi h an AFG VISIONplus-AT ame g abbe boa d using Hipic 4.0.4 (Hamama su), Image-P o Plus (Media Cybe gene ics). Digi al images we e assembled using Adobe PHO- TOSHOP so wa e. Al e na i ely a Zeiss Axiopho mic o- scope was used o econs i u e images om a se ies o op ical sec ions ha we e de-blu ed by emo ing he ou - o - ocus blu using a nea es neighbo de-con olu ion algo i hm de eloped by us. On his sys em he images we e cap u ed wi h a PXL cooled came a (Pho ome ics, Munich, Ge many) and analyzed using ou own image cap u e and analysis p og ams de eloped on ISee g aphi- cal p og amming language unning unde Mand ake LINUX OS on a Pen ium PC compu e [34]. Con ocal images and e y la ge ield mosaics we e cap u ed using ou cus om buil dual mode Ul a iew (combined RS and LCI) sys em (Pe kin Elme ) using imaging au oma ions Quan Cap u e 4.0 and Quan Coun 3 ha we ha e de el- oped using OpenLab Au oma o p og amming en i on- men (Imp o ision). 3D econs i u ion was ca ied ou using Voloci y (Imp o ision) o ImageJ p og ams. Resul s E ec o exogeneously exp essed LANA-1 on nuclea s uc u es o human cells HHV-8 in ec ed cells ha bo LANA-1 in a spa ially s ic ly es ic ed manne . The majo i y o LANA-1 is associa ed wi h well-de ined nuclea a eas ha also con ain i al epi- somes (he e e e ed as LANA bodies). Al hough he e a e mul iple binding si es in he i al e minal epea , we ha e es ima ed ha he amoun o LANA-1 concen a ed in he nuclea oci is o de s o magni ude highe han he one ha can o m di ec con ac wi h he i al DNA. In o de o model he e ec o high LANA-1 concen a ion on he o ganiza ion o ch oma in in he neighbo hood o LANA bodies, we o e exp essed LANA-1 in ansien ly ans- ec ed MCF7, HeLA o Saos-2 cells. The le el o exp ession was de e mined by measu ing he luo escence signal in ensi ies on iden ically p ocessed, immunos ained slides, using manual, semi-au oma ed o ully au oma ed wide- ield o spinning disc con ocal luo escence mic os- copy. The measu emen da a demons a ed ha he ocal exp ession le els o LANA-1 in he LANA bodies we e compa able o he le els eached in he ansien ly ans- ec ed cells (Figu e 1). Exp ession o LANA-1 in compa a- ble quan i ies ha occu in he LANA bodies has led o p o ound ea angemen o nuclea s uc u es in he an- sien ly ans ec ed cells. In human cells his is mos p om- inen ly demons a ed by he e ec on pe inucleola he e och oma in. Two majo , dis inc ype o ch oma in al e a ions we e obse able. In a ac ion o ans ec ed cells LANA-1 o e exp ession led o he almos homogene- ous elimina ion o ch oma in s aining pa e n (Figu e 2 middle panel) whe eas in o he cells a no el condensed ch oma in pa e n appea ed, ha was somewha simila o he mo phology o p ema u e ch omosome condensa- ion obse able in mi osis/in e phase cell hyb ids (Figu e 2 bo om panel). Impo an ly bo h ype o ch oma in change had a majo e ec on he he e och oma in. LANA-1 (g een) dissol es DNA (blue) om pe inucleola he e och oma in in ans ec ed MCF7 cellsFigu e 2 LANA-1 (g een) dissol es DNA (blue) om pe inucleola he e och oma in in ans ec ed MCF7 cells. Inc easing amoun o ans ec ed LANA-1 (compa e op o he middle o bo om panels) leads o he elimina ion o he DNA s ain- ing (blue) in he pe inucleola he e och oma ic ings. Adja- cen non- ans ec ed cells se e as con ols. Righ panel shows magni ied pic u es o ch oma in o ganiza ion o he selec ed nuclea a eas selec ed by ed bo de boxes in he le panel. The middle and bo om panels ep esen he wo dis inc ypes o ch oma in e ec s: The smoo hing ou o he ch oma in s aining (middle panel) e sus induc ion o newly condensed ch oma in co ds (bo om panel). Impo an ly bo h changes lead o he elimina ion o pe inucleola he e o- ch oma in. Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 5 o 16 (page numbe no o ci a ion pu poses) Dissolu ion o DNA om pe inucleola he e och oma in is no accompanied by he elease o ime hyla ed lysine 9 his one H3 (3MK9H3) – g een immuno luo escence s aining in LANA-1 ans ec ed cells ( ed)Figu e 3 Dissolu ion o DNA om pe inucleola he e och oma in is no accompanied by he elease o ime hyla ed lysine 9 his one H3 (3MK9H3) – g een immuno luo escence s aining in LANA-1 ans ec ed cells ( ed). 3MK9H3 s aining clea ly iden i ies pe i- nucleola a eas (whi e a ows) wi h diminished he e och oma ic DNA s aining in he ans ec ed cells. Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 6 o 16 (page numbe no o ci a ion pu poses) Inc easing le els o LANA-1 led o he disappea ance o he e och oma in om he pe inucleola a eas as de ined by Hoechs 33258 s aining (compa e he op panel o Fig- u e 2 o he middle o bo om panel). Impo an ly his e ec was no associa ed by a simila elease o he he e o- ch oma in ma ke ime hyla ed lysine 9 on his one H3 (3MK9H3) om he pe inucleola a eas. On he con a y 3MK9H3 posi i e he e och oma in emnan s appea ed o be collapsed in o smalle sphe ical s uc u es (Figu e 3). Measu ing he amoun o 3MK9H3 in he nuclei o MCF7 cells, 48 hou s a e ans ec ion, using au oma ed Ex ended Field Lase Con ocal Mic oscopy (EFLCM), we ound no signi ican di e ence be ween he o al amoun LANA-1 exp ession does no e ec 3MK9H3 (g een) le els as measu ed using ex ended ield lase scanning mic oscopy (EFLCM) ha au oma ically cap u ed 300 adjacen ields as a mosaic o Z p ojec ed images o 12 op ical sec ions eachFigu e 4 LANA-1 exp ession does no e ec 3MK9H3 (g een) le els as measu ed using ex ended ield lase scanning mic oscopy (EFLCM) ha au oma ically cap u ed 300 adjacen ields as a mosaic o Z p ojec ed images o 12 op ical sec ions each. The o al luo escence in ensi y measu emen o 3MK9H3, DNA (blue) and LANA-1 ( ed) o he indi idual nuclei is plo ed in he o de o inc easing amoun o LANA-1. The amoun o 3MK9H3 s aining is also compa ed on popula ion le els o LANA-1 posi i e and nega i e nuclei on a box cha . Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 7 o 16 (page numbe no o ci a ion pu poses) o 3MK9H3 in he un ans ec ed and LANA-1 ans ec ed cells (Figu e 4). E ec o exogenously exp essed LANA-1 on nuclea s uc u es o mouse cells Mo phological analysis o he e och oma ic s uc u es is cumbe some in human cells because o he a he di use bo de be ween he euch oma in and he e och oma in a eas. The disc imina ion be ween he wo ypes o ch o- ma in is much easie in mouse cells whe e he pe icen o- me ic al a-sa elli e epea s a e o ganized in e y well de ined he e och oma ic ch omocen e s. We ha e p e i- ously shown ha LANA-1 has e ained i s abili y o a ge mouse he e och oma in in BCBL-1/Sp2 human/mouse synka yon hyb ids [1]. In o de o es he e ec o LANA- 1 on mouse ch omocen e s we ha e ans ec ed A9 and L cells as well as NIH3T3 ib oblas s wi h LANA-1. All ee lines showed he same e ec . Inc easing amoun o LANA- 1 has led o he disappea ance o ch omocen e s by Hoechs 33258 s aining and o ma ion o condensed ch oma in a he nuclea pe iphe y o in he pe inucleola a eas (Figu e 5). In e es ingly he disappea ance o he bulk o he DNA om he ch omocen e s, as illus a ed on single na ow con ocal sec ions o ans ec ed and con ol nuclei (Figu e 6) was no ollowed by he disappea ance o 3MK9H3 s aining (Figu e 6). As in human cells, LANA- 1 ans ec ed mouse cells con ained simila amoun s o 3MK9H3 as non- ans ec ed cells and bo h he numbe and he s aining in ensi y o indi idual 3MK9H3 oci was unal e ed. Impo an ly, howe e , he localiza ion o 3MK9H3 oci was d ama ically changed (Figu e 7). Whe eas in he un ans ec ed cells he 3MK9H3 oci we e e enly dis ibu ed h oughou he en i e nucleus, hey we e almos exclusi ely localized o he nuclea pe iphe y in he ans ec ed cells (Figu e 8). High esolu ion op ical sec ioning and 3D econs i u ion o he con ocal image se ies showed ha he eloca ion o he oci was an ea ly e en ha has p eceded he elease o Hoechs s ained ch oma in om he ch omocen e s (Figu e 9) [see Addi- ional iles 1 and 2]. The elease o Hoechs s ained DNA om he ch omocen e s was accompanied wi h a majo ea angemen in he s aining pa e n o an o he he e o- ch oma in binding ac o , he me hyl cy osine binding p o ein 2 (MECP2) in mouse L-cells. Inc easing exp es- sion o LANA-1 led o he g adual dissolu ion o MECP2 oci ha we e s ingen ly associa ed wi h he ch omocen - e s in un ans ec ed cells. In ans ec ed cells, MECP2 was eleased om he oci and appea ed in he a eas o he newly o med condensed ch oma in bundles bu showed no co-localiza ion wi h LANA-1 i sel (Figu e 10). This lack o co-localiza ion was also consis en wi h he absence o co-localiza ion be ween LANA-1 and MECP2 in HHV-8 ca ying BCBL-1 cells (Figu e 11). Mapping he LANA-1 egion equi ed o he ch oma in e ec s using dele ion mu an s We ha e es ed a se ies o C- e minal dele ion mu an s ha did o did no con ain he cen al acidic epea E ec o LANA-1 on mouse pe icen ome ic he e och oma in o ganized as ch omocen e sFigu e 5 E ec o LANA-1 on mouse pe icen ome ic he e och oma in o ganized as ch omocen e s. Inc easing amoun o LANA-1 (g een) leads o g adual disappea ance o ch omocen e s in he ans ec ed nuclei o mu ine L-cells. DNA s ained wi h Hoechs 33258 (blue). Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 8 o 16 (page numbe no o ci a ion pu poses) High esolu ion compa ison o LANA-1 ( ed) posi i e L-cell nucleus wi h an adjacen non- ans ec ed cell in a single op ical sec ion ha slices bo h nuclei in he middle le elFigu e 6 High esolu ion compa ison o LANA-1 ( ed) posi i e L-cell nucleus wi h an adjacen non- ans ec ed cell in a single op ical sec ion ha slices bo h nuclei in he middle le el. The in ensi y plo is eco ded along he whi e line and demons a e a massi e elease o he bulk DNA (blue) om he ch omocen e s in he ans ec ed cell wi hou e ec ing he 3MK9H3 (g een) le els in he emnan s o he ch omocen e s (whi e s aple on he igh side o he line plo ). Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 9 o 16 (page numbe no o ci a ion pu poses) egions o hei e ec on he ch oma in o ganiza ion in human and mouse cells. The C e minal unca ion had no e ec on LANA-1 induced ch oma in ea angemen , while dele ions o he cen al acidic epea elimina ed his e ec (summa ized in Figu e 12). Dele ion mu an s lack- ing he cen al epea s (del a mu an s) e ained he capac- i y o a ge he su ace o he e och oma in bo h in human and mouse cells, bu had no e ec on he o gani- za ion o ch oma in i sel (Figu e 13). To iden i y he egion ha was in ol ed in he ch oma in eo ganizing o LANA-1 mo e p ecisely, we es ed mu an s ha e ained he immedia e neighbo ing sequences o he cen al epea s. We ound ha a mu an (del 332–972) ha lacked he in e nal epea s bu e ained he immedia e ups eam egion ha p ecedes he DE epea s was ac i e in ea anging he ch oma in (Figu e 14). The compiled da a showed ha his 57 amino acids long egion (aa. 275– 332) was equi ed o he ch oma in e ec s. Impo an ly his a ea is o e lapping wi h he p e iously mapped bind- ing si e o he his one me hyl ans e ase SUV39H1 [35]. Discussion HHV8 ca ying cells can ha bo up o a ew dozen i al episomes ha localize in disc e e nuclea compa men s delinea ed by LANA-1. LANA-1 as mul i unc ional i ally encoded p o ein, in ol ed in he main enance o he i al episomes, egula ion o i al la ency, ansc ip ional egu- la ion o i al and cellula genes and impai men o cell cycle checkpoin s [36]. Se e al o hese unc ions may play a ole in he HHV8 induced malignan ans o ma- ion o Kaposi sa coma and body ca i y lymphoma cells. In his pape we ha e p esen ed addi ional e idence ha LANA-1 may also ha e undamen al e ec s on he o gan- iza ion o he in e phase nucleus. They a e mos clea ly seen in mouse cell nuclei whe e he pe icen ome ic he e- och oma in o ms easily ecognizable ch omocen e s. The bulk o he he e och oma in associa ed DNA is eleased om he ch omocen e s wi hou a ec ing hei 3MK9H3 con en . This sugges s indi ec ly, ha a la ge pa o he DNA ha is associa ed wi h he ch omocen e s con- ains nucleosomes ha a e no ime hyla ed on he 9 h lysine o his one H3, a modi ica ion ha is conside ed o be he hallma k o he e och oma in o ganiza ion. Ou da a sugges ha ch oma in wi h 3MK9H3 modi ied Al hough single op ical sec ions (le ) o LANA-1- ans ec ed nuclei may sugges ex ensi e dec ease in 3MK9H3 s aining, econs i u ion o he summa ized s aining signal om he en i e s ack o 15 images ( igh ) shows no de ec able al e a ion in he o al le els o 3MK9H3 in mouse L cell nucleiFigu e 7 Al hough single op ical sec ions (le ) o LANA-1- ans ec ed nuclei may sugges ex ensi e dec ease in 3MK9H3 s aining, econs i u ion o he summa ized s aining signal om he en i e s ack o 15 images ( igh ) shows no de ec able al e a ion in he o al le els o 3MK9H3 in mouse L cell nuclei. (LANA-1 – ed, 3MK9H3 – g een, DNA – blue). Publish wi h BioMed Cen al and e e y scien is can ead you wo k ee o cha ge "BioMed Cen al will be he mos signi ican de elopmen o dissemina ing he esul s o biomedical esea ch in ou li e ime." Si Paul Nu se, Cance Resea ch UK You esea ch pape s will be: a ailable ee o cha ge o he en i e biomedical communi y pee e iewed and published immedia ely upon accep ance ci ed in PubMed and a chi ed on PubMed Cen al you s — you keep he copy igh Submi you manusc ip he e: h p://www.biomedcen al.com/in o/publishing_ad .asp BioMedcen al Molecula Cance 2007, 6:28 h p://www.molecula -cance .com/con en /6/1/28 Page 16 o 16 (page numbe no o ci a ion pu poses) RTA: a no el mechanism o es ablishmen o la ency. J Vi ol 2005, 79:7453-7465. 26. Piolo T, T amie M, Coppey M, Nicolas JC, Ma echal V: Close bu dis inc egions o human he pes i us 8 la ency-associa ed nuclea an igen 1 a e esponsible o nuclea a ge ing and binding o human mi o ic ch omosomes. J Vi ol 2001, 75:3948-3959. 27. Ba be a AJ, Chodapa ambil JV, Kelley-Cla ke B, Luge K, Kaye KM: Kaposi's sa coma-associa ed he pes i us LANA hi ches a ide on he ch omosome. Cell Cycle 2006, 5:1048-1052. 28. Kelley-Cla ke B, Balles as ME, Koma su T, Kaye KM: Kaposi's sa - coma he pes i us C- e minal LANA concen a es a pe i- cen ome ic and pe i- elome ic egions o a subse o mi o ic ch omosomes. Vi ology 2006. 29. Viejo-Bo bolla A, Ka i E, Sheldon JA, Na han K, Ma sson K, Szekely L, Schulz TF: A Domain in he C- e minal egion o la ency- associa ed nuclea an igen 1 o Kaposi's sa coma-associa ed He pes i us a ec s ansc ip ional ac i a ion and binding o nuclea he e och oma in. J Vi ol 2003, 77:7093-7100. 30. You J, S ini asan V, Denis GV, Ha ing on WJ J , Balles as ME, Kaye KM, Howley PM: Kaposi's sa coma-associa ed he pes i us la ency-associa ed nuclea an igen in e ac s wi h b omodo- main p o ein B d4 on hos mi o ic ch omosomes. J Vi ol 2006, 80:8909-8919. 31. 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