ABFI contributes to the chromatin organization of Saccharomyces cerevisiae ARS1 B-domain

Biocl e t Bic

Biochimica et BiophysicaActa 1219 (19~

s to the chromatin or l cerevisiae A R S 1 ] Patrizia Venditti a Giovanna Costanzo

a Rod(

a Centro di Studio per gli J li Acidi Nuclei 9ndazione 'Istituto Pasteur-Fondazione Cenci Bolognetti ', c / o Dipari Jartimento di G ~ienza', Rome, It~ Sapienze

on o f SaccharoJ Lin [ a, Giorgio Camillon Italy gia Molecolare, Universit~ degli .~

Received 20 April 1~

tract

?he involvement of the ABFI transcription factor in organizing 'gamzmg the chromatin structure of the Saccharomyces cerevi~ been previously postulated. We studied the ARS1 chromatin sl structure both on the chromosom~ and on plasmids cart has been ~ted ABFI binding sites, using a recently developed no-backglround technique for nucleosome eosome mapping, coupled wit mutated micrococcal •ococcal nuclease in vivo footprinting. We show that ABFI pr~ protein acts as a boundary element of chromatin structur invasion by nucleosomes toward the essential A-domain. Keywords: vords: Nucleosomepositioning; In vivo footprinting; Nystatin

1. Introduction Nearly all the DNA in a eukaryotic chromosome is generically associated with histones fistones in the form of nucleosomes. Therefore, understandin ding the structure and function of eukaryotic chromosomes rests on analysis of the first level of the chromatin org~anization: that of the nucleosome. Functionally crucial chromosom omal regions are charac~mosomal terized by defined chromatin structures (reviewed in [1-3]). Centromeric and telomeric regions show strong nuclease protections surrounded by precisely positioned nucleosomes [4,5]. Gene regulatory ,ry sequences and replication origins are areas of specific nuclease hypersensitivity and exact nucleosome positioningg [3]. Precisely positioned nucleosomes can assume a critical [tical role in transcription and replication. Three main mechanisms that :hat are not mutually exclusive have been shown to operate in positioning of nucleosomes in vivo [3]: (a) histone-DNA intei contexts and (c) three-dimensional

structures. ' Boundary' refers to a chromati physically limits a random rar arrangement DNA [6]. Examples include in( both the alten aries and of flanking sequences se in yeast pla DNA-binding proteins like 1 GRF2 [9] and y~ sor [10]. The general propert aerties of the yeast A replication are describe escribed in [11]. Low reso of positioned nucleoson :leosomes on the region ha [12]. Deletion analysis has shown that A~ vided into three domains: domaii A, B and C [13, number of proteins that th bind to the AR~ been characterized [14-16]. [14A single-strant ing protein has been identified i on the b~ interactions with the T-rich T-I strand of the AF sus [17,18]. More recently, recen a multiprotein ( Origin Recognition Complex) CoJ that specific cellular origins off DNA replication has beeJ purified from the yeast S. cerevisiae [19]. e I footprinting the ORC pl ction spanning from positJ [28]. A strong correlation gin recognition by this ac

P. Venditti et al. / Biochimica et Biophysica A

ries have independently ng protein, ABFI, which if RTCRYNsACG ([39] if is located in either the A domain A, present in a e promoters for a variety site acts as an upstream and references therein). ions or deletions in the 1 B-domain has produced conflicting results, linkerning analysis of the ARS1 sequence [21] has recently led that ARS function requires an intact ABFI-binding Interestingly, the same authors showed that the posieffect of an intact ABF1 binding site can be replaced ther transcription factor binding sites. enomic footprinting has shown protection of the I-binding site [22]. l this paper we report data on the effect of the ABFI dn on the chromatin organization of ARS1. The inement of the ABFI transcription factor in organizing ',hromatin structure of the S. cerevisiae ARS1 region )een previously postulated. Along with high resolution acoccal nuclease (MNase) footprinting, two new techniques ~s for chromatin analysis were used: (a) digestion of chromatin aaatin directly inside viable spheroplasts [23]; (b) a positive ive assay for determination of the nucleosomal borders [24]. We show that in the ARS1 region the ABFI protein .,in plays a role as a boundary effector: its presence limitss the multiple nucleosome positioning on the B-domain., preventing the nucleosomal invasion of the adjacent A domain.

Y. requi

ere grown at 30°C in Yt aedia [25].

2.2. J

I chemicals

M were Prom tatin

mclease and T4 polynuc from Boehringer; Taq pol ,ase from Seikagaku, Toky ; radiochemicals from Ame

2.3. ]

prmation

Y~

mation was performed aci ethod described in [26].

lithiu 2.4. nucle

treatment with nystatin an

Tt procedure is described in expo] ~wing cells (0.1-0.5 A6001 with 27], then washed once witl7 pellet spended in nystatin buffer I 1.5 mM CaCI2, 20 mM Tris-HCl (pH 8.0) and 50 p,g/ml nystatin ystatin). Spheroplasts in i were treated at 37°C for fol 15 min with MNa The enzymatic concentrations (see Results). Resu stopped by addition of 10% 1 SDS, 50 mM El the reaction volume). Proteinase K was sample and the DNA pu )urified according to [ 2.5. Plasmids

Plasmid DNAs used were yRpl7 (New labs) and yRpl7-SB (this (thi work), yRpl7-SB from yRpl7 by introducl ltroduction of a dodecamer .~CGGATCCGCG) in the ker sequence (CGCGGt site of the ARS1 region ton and by mutating tl 755(T ~ G), 757(G C 765(T ~ C) and G ~ C), the ARS1 sequence (nu numbered according t tion and mutagenesis were we obtained by reve

2. Materials and methods 2.1. Yeast strains and culture media

The yeast strain used in this is work was W303-1a (Mata, ade 2-1, ura 3-1, his 3-11,15, trrp 1-1, leu 2-3,112, canl-100) (from R. Rothstein and kindlyy provided by R. Stemglanz). TRP1 coding region

EcoRI

Ec RI

858-868 56O 00

-

map p ~ i t i o n

ctional domains functional

B

A

C

oligoe uaed

Fig. 1. Diagram of the S. cerevisiae ARS1-TR

38] are shown using the numberin

P. Venditti et al. / Biochimica et Biophysica 1

ad mutagenic oligos as (yRpl7-SB) the original GCTAA-#770 sequence

is s CTC cate quen

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Thes yRp~ onto

9y #750-AAATTGCCTC ACGGTA-#770 (underlin( ons; the interruptions repo the site of the BamHI lil ons alter the TRP1 gene re dd resulting in plasmid in~ nedium not supplied with t

2.6.

of labeled primers

ARS1 1 2 3 4 56

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oligonucleotides used as ~ns corresponded (primer primer #2) to positions 63, o positions 812-831 (prin data of [28] (see also Fig. g using [32 P]ATP and T4 1 armed according to stand; )n products were purified ,phoresis.

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10 /xg of genomic DNA D were reacted w polymerase and 75 000,cpm of end-labeled ( (specific activity 1-2 /xCi/pmol). /~ The sat cled 30 times through the following steps min, 55°C for 2 min, and an 72°C for 2.5 min. products were phenol eextracted, ethanol pr solved in formamidee an( and dyes and analyzed ing polyacrylamide gel.

700-2.8. Analysis of microc micrococcal nuclease bor amplification (MBLA>

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Monomeric DNA, de defined as the limit dl by nucleosome protectk rotection on DNA toward micrococcal nuclease, ~ was prepared accord cedure of [24]; basicallyy, nystatin treated spt subjected to MNase dig(~stion (20 U / m l or at 37°C for 15 min and 1the reaction stopped EGTA to 5 mM; after proteinase K treat SDS, samples were p~henol extracted thJ ethanol precipitated. TI The samples were r

i ~ ~

-

Fig. 2. MNase analysis of th the B domain chromosom ddC sequencing lane of yRp yRpl7 with primer #3. Pri~ products pun iod fro, from nystatin-permeahilized treatment with 2, 5, 10 and 20 U / m l of micrococcal nu or of DNA purified from sph( spheroplasts, untreated (lane sample (lane 7) was the he nrime~ primer extension product of 1 with 0.2 U / m l of nuclease; purit ion pattern. Sequence is indexed dicate, respectively protection o~ spheroplast chromatin compared ed areas is shown on the fight.

P. Venditti et al. / Biochimica et Biophysica/

MNase footprints ARS1based plasn 101112

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v D n 1 7 - _-O S.BR of yRpl7 naked DNA digested in digested in vitro with 0.1 and 0.~ creasing amounts of MNase (2, tie amounts of MNase as above. L

~. Venditti et al. / Biochimica et Biophysica A

t run in 1.5% agarose gel ladder. Monomeric DNA tase, labeled at low spen on a denaturing polyaally nicked monomeric monomeric DNAs were nultiple cycles of primer h 5'-labeled oligonucleoting the borders of the omer-length MNase digestion products (the in vivo,~cted nucleosomal DNA) that contain the indicated primers.

- j

E,~ ident (1) f e a a c p (2) f r

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In vitro nucleosome reconstitution he reconstitution procedure was the salt dilution (1.0 IaC1 to 100 mM NaC1) protocol [30], utilizing a 30:1 tr ratio of nucleosome core particles from chicken lrocyte to acceptor DNA (plasmid yRpl7 or yRpl7at a DNA concentration of 10 /zg/ml [31].

3.2. ARSJ

Partial purification of ABFI protein and evaluation utant sequence binding affinity

he ABFI protein was purified according to [32]. For The band shift experiments [33] the ultrogel-heparin 175 mM (NH 4)2SO4 fraction was used. The binding affinity for the mt sequence was determined in band shift competition mutant riments where a labeled fragment containing the ABF1 experiments ing site of the S. cerevisiae L2A gene [32] was bindin enged by increased amounts of either w.t. or mutant challenged sed were produced by PCR fragments. The fragments used amplification of a region between ~¢een position 195 and 830 of yRp17 or yRp17-SB DNAs.

3. Results High resolution footprinting ng experiments using MNase as a probe for DNA-protein interactions were carried out to investigate the molecular organization of the S. cerevisiae ARS1 region B-domain in (Fig. 1) and the rules governing nucleosome positioning tg in this DNA tract.

ides 767 to 748 (numberiJ ; the in vitro defined ABI y using the yeast purified be protected in vivo [2~ itection implies a high de ABFI recognition sequenc~ s 730 to around 560, corres protected region as descri chnique on isolated min 121). ,~gion upstream of position csis is precluded by insuffi

11

(3) a d 11

. . . . . . . . . . .

three following protected

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TI ~e localized around posit ry close ~,rlg. r.s map very cJO~ to the ABFI prc (Fig. suggesting that the binding bim of this transcr might influence the chromatin chrc organization The ABFI function in the 1 ARS1 region re undetermined: however, the disruption of i on a centromeric ARSl-based ARS1 plasmid abc tion [21]. We investigat~ Lgated the possibility th~ work as a chromatin org :ganizer acting as a p ary by limiting the multiple mu alternative pc nucleosome wich covers the ARS1 B-domai~ of the A & potential nucleosome invasion in

1<>1 1~>1

purification of nucleosomal monomer do DNA

3.1. In vivo high resolution~ MNase footprinting of the chromosomal copy of the ARS1 $1 B domain Yeast spheroplasts were permeabilized with nystatin and exposed to different amounts ants of MNase. The cleavages effected by MNase in the B-domain were mapped by extension of primer # 3 (Fig.• 2, lanes 3 to 6). A defined th cleav c ' l ~ v ~ a ~ i n t c * n ~ i t i ~ di'f-f~.rprofile was clearly visible with ences relative to the reference sampl significant Taq polymerase pausing c tions used, as shown by the elonga

micrococcAI nuclnese limit digestion

purify full-length monomer DNA strands

.~ . . . . . . . .~----.# _ _

multiple-round • of specific prime GEl. ANALYSIS A

ication of Micrococcal Border by Lil

~e borders by linear amplification arrows indicate specific primers

P. Venditti et al. / Biochimica et Biophysica/

hat the invasion of the overing the C-domain is 36].

nvestigate ABFI function aal mutation of the ABFI b ~17-SB plasmid, see Mater

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!or detail) which was used, in parallel with yRpl7, for ods for yeast cells transformation. Gel shift experiments (see Materials and methods) :nce was unable to efficiently showed that the mutant sequence bind a partially purified ABFII protein fraction (not shown), Determination of plasmid stabilit~ tability for both yRpl7 and yRpl7-SB has been performed: led: a slight decrease (about 10%) in stability for mutantt DNA, relative to the wild type, has been observed (datat not shown),

3.3. In vivo high resolution MNase footprinting of the yRpl 7 (wild type) and yRp17-SB '-SB (mutant) episomal copies of the ARS1 B-domain Cells were subjected to n ystatin and MNase treatment and the purified DNA was used as a template for

~tension of primer # 3 (F ested with MNase (lanes 'Rp17 chromatin digested me (lanes 3-5). The AB] Rpl7 chromatin samples [ 745. As evaluated by sca ot shown) the protection (3 n the chromosome, implyin ling sites on the average cells. A more complex cl ons and enhancements is e gel, starting from posit heterogeneous nucleosom ,~rnative positions on the pl the chromosomal copy, as see below). rison with the yRpl7-SB r two c :an be reached: (i) in the m inting (as compared to the the ! positi 15) is completely lost an enhax ;e sites is obtained at the location; (ii) a difference from the wild locati digestion pattern is detec detectable in the mutant. MNase accessil chromatin seems to be more n ABFI bindi fun that the lack of a functional influence the chromatin organization in the sequences.

multi nakec comr amou visib] cleoti metri as co panc3 copie revea uppel sugg~ with tion MBL Fr

3.4. Comparison of nut nucleosomal arrange~ and yRp17-SB by MBLA analysis To further substantiat stantiate the result, we appl technique to the chromatin chrom~ of both yRpl7 containing cells. This no-background t~ scheme in Fig. 4) is in fact capable of complex MNase footprii printing pattern due to lapping nucleosome pos ~ositions by revealing individual positions. In addition to the ac some mapping, this assay ass provides inforrr relative abundances of the tl existing nucleoso Fig. 5A, shows the 1;~rimer # 1 extensie the nucleosomal DNA monomer n of the wik (lane 5) as compared to those of the mutant 6). The patterns obtained obtain in lanes 5 and

Fig. 5. MBLA on yRpl7 and yRpl7-SB -SB episomal DNAs. (A) 5' end-labeled MNase generated DNA monomer.,strands from yRpl7 (lane (lane 10) containing cells. Lanes 2-3 and 8-9: sequencing lanes from primer #1 (A and C) of yRpl7 and yRp yRpl7-SB, respectively. Lanq #1 extension on yRpl7 and yRpl7-SB, SB, respectively (to evaluate Taq polymerase pausing). Lanes 5 and 6: pri primer #1 elongation prodl purified from cells containing yRpl77 and yRpl7-SB, respectively. Numbers in parenthesis refer to map posi ~ositions according to Ref. [ arrow refer to the nucleosomal borders. rs. Two graphic representations of analyzed sequences (yRpl7, left) and (yi yRpl7-SB, right) are also., densitometric analysis of band intensit ;ity from panel A, lane 5. Lower: densitometric evaluation of band intensity. :y from lane 6, panel A. ( ~' primer n 7-8: sequencing lanes from oligo # 2~.(A and (C) of vRn17 and vRnl 7-SR DNA~ re~net~tivelv I ~ane~ q and 6: # 2 extension prod~ yRpl7-SB, respectively (Taq polymerase pausi urified from cells containing yRpl respectively. Numbers in parenthesis refer to w refer to the nucleosomal borde unique graphic representation is reported beca A tract. (D) Upper: densitometric intensity from lane 4 in panel C. Lower: densit • Numbers on densitograms refer 5 panel C) and map positions is i

P. Venditti et al. / Biochimica et Biophysica ;

altiplicity of nucleosomal I mutant plasmids implies ire positions; (ii) most of

the L mut~ diffe

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the mutant plasmid. It more than 50% of the ,~w positions (see lane 6 he A-domain; the corretype plasmid are comsee lane 5). The corre(panels B and D) allow ~ferences in the nucleosohe MBLA assay was repeated on the same monomeric ts using primer # 2 (Fig. 5C), in order to define the site putative nucleosome borders and double-check nformation obtained. Comparison of lanes 4 and 5 in 5C confirms the data reported in panel A; in particular listribution of signals is coherent with an overall shift e nucleosome positions towards the A-domain. Panel rows the densitometric scannings for a quantitative ration. With the MBLA technique we are unable to nucleosome borders inside or in proximity (10-15 bp) te selected primers sequence because the presence of abeled oligo obscures the elongation products nearby. ig. 6 summarizes these data and displays the positions adeosome on the wild type and mutant sequences as ~ i n e d by MBLA analysis. The preferential positions determined

D

of the histor octarr the nl mutal right (histo In ber c comp positi the of th. positi some

n the wild type sequence a] # 1, 2, 3 and 4; in parti 2, the most abundant, ck lapped on the chromosom~ nucleosome shift occurs' map and the new position #5, 6 and 7). (i) on the episomal DNAs, nucleosome positions a] e chromosomal copy; (ii) tt litatively and quantitativel the mutant sequences; (iii ein reduces the number o: oiding the potential invasi omain.

3.5. l on yt

sis of in vitro reconstitutet p17-SB DNAs

ro plasmids appear to share B~ te investigated the influenc some ~' on nucleosome positio sequence 'per se" nstituted in vitro on the , somes were reconstitute by MBLA mutant plasmids and analyzed al conclude that: (i) in the absence of accessor two DNAs allow nucle(:osome formation on quences (compare lanes 3 and 4 in Fig. 7), v are again c difference; (ii) multiple positions 1 of the borders mapping in the region betwe 740 and 800; (iii) the more heterogeneol obtained in vitro sugge,sts that the DNA s observed in does not possess the selectivity sek

4. Discussion

1

Nucleosome positioni ositioning has been sugget the most active element dements in the regulation tion [3,36]. Several repo :ports point to the prec of nucleosomes in manyy eukaryotic genes [ gate the principles of nucleosome setting studied the molecular art ular arrangement of the nu( on the B-domain of yeas ,east S. cerevisiae A R S the potential role of the ABFI protein in p nucleosome.

e

4.1. The nucleosome location loc in the chromG ARS1 B-domain region

o i~

The molecular organi anization of the ARS1 copy was defined by M]Nase in vivo footpri g r n m t h ; ¢ ~ v n P r ; m ~nent nt x ~ conclude that: (i) i we ~osome positioning is well rding to the low resolutio~ )rotection to MNase cleava nding site (box #1).

P. Venditti et al. / Biochimica et Biophysica

)some positioning

vere found that bind the • nucleosome [3]. Their .d he range of nucleosome oundary mechanism was two sequences related to at no direct evidence for has been provided yet. In ARS1 region the ABFI binding site maps very close to nucleosomal position defined in Fig. 2. Therefore, we analyzed the potential role of the ABFI protein as a adary for nucleosome positioning. At this purpose, a ation of the ABFI binding sequence was generated in yRpl7 plasmid, in order to investigate the chromatin mization in the absence of bound ABFI.

a

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ncreased number of overl~ e wild type and mutant se moda =tamers, with both qualitati • Being that the ABFI bi~ tative ', that we mutated in the Alq the o: ~matin organization sugge varial hysical barrier, by limitin funct vicinity. In particular, At occu, a of the A domain, known t cleos =tion [36]. tal fe ositioning is determined by N1 ds. At the DNA level, rotat trans] e positioning through repe caus~ ments with helical periodic able can be represented by spe latioi ~s (possibly acting as sit~ tion he rotational signal is intri bindi nponent [37] we evaluate to th A sequence 'per se' to dire abilit absence of any additional 1 posit: osonl tion experiments (Fig. 7) 1 "lude that the wildd type and mutant DNA se~ clude exhibit appreciable differences diffe in nucleoson It appears that the ARS1 ARS B-domain contain tional signal leading to t( heterogeneous bt positioning in vitro and in vivo. In vivo ,~ strong translational element, elen further reduci~ of nucleosome positions on the wild type A for the role of ABFI in the organization o presented in Fig. 8. copy some

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The data reported in this paper supports in controlling nucleosol tcleosome positioning: the agreement with a previ, >revlous report showing ABFI with other DNA DN.~ binding proteins ARS1 replication efficiency efficie at a normal le~ that it functions primaril )rimarily at the level of chr zation [21]. Ordered chromatin structure might ha~ keep the essential ARS1 ARS A domain free o (and therefore more accessible acc to the repli~ cry). Our data indicate that th one way of prc domain from nucleoson :leosome invasion is base(

Fig. 7. A n a l y s i s o f M B L A of nueleosomes after in vi L a n e s 1 - 2 and 5 - 6 : C and T s e q u e n c i n g lanes f r o m o respectively. L a n e s 3 and 4~ pfi enerated m o n o m e r obtained from y R p l 7 and y R p l 7 - S B D N A s , re~, ar borders. The single a r r o w st rns. N u m b e r s identify the b a n d s r~

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The ARS1-TRP1 regi ion of S.cere TI Fig. 8. Proposed model for the role of ABF I in chromatin organization, anization of the ARS1-TRP1 region. Ellipses refer to nucleosome pos represent ',sent the ABFI protein; striped circle, the A domain binding factor. factor. FFunctional domains of the ARS1 regJion are shown below. ence.~., on the B - d o m a i n , o f a b i n d i n g site for the abundant and essential A B F I protein.

Acknowledgements This w o r k w a s supported ~d by P r o g r a m m i Finalizzati B i o t e c n o l o g i e and B i o s t r u m eentazione n t a z.,ione i o r and Ingegneria GeErnesto Di Mauro, Sabrina netica (C.N.R.). W e thank Venditti, M i c a e l a Caserta and td K e l l y P. W i l l i a m s for helpful discussion and critical readin ~ading o f the manuscript. The w o r k of L i u d m i l l a R u b b i inn constructing the y R p l 7 - S B mutant and the technical assistance o f A r c a n g e l o Di Francesco are also a c k n o w l e d g e d .

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