Gene expression in murine erythroleukemia cells

J. Mol. Biol. (1984) 172, 417-436

Gene Expression in Murine Erythroleukemia Cells T r a n s c r i p t i o n a l C o n t r o l a n d C h r o m a t i n S t r u c t u r e o f the a ~ - G i o b i n G e n e MICHAEL SllEFFERY, PAUL A. MARKS AND RICHARD A. RIFK1ND

De Wilt Wallace Research Laboratory Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, N.Y. 1002I, U.S.A. (Received 25 April 1983, and in revised form 27 August I983) The transcriptional activation and chromatin structure of the ~l-globin gene was ana/)~ed during induced erythroid differentation in murine erythroleukemia cells (MELC). In unindueed MELC, a low level of at-globin, coding-strand-specific transcription is deteetable. Hexamethylene bisacetamide (HMBA)-mediated MELC differentiation is associated with a l0 to 20-fold increase in the rate of ulglobin gene transcription. In induced MELC, ul-globin gene transcription initiated predominantly near the c~p site, occurs only off the coding strand, and might terminate, or attenuate, in a region 50 to 250 base-pairs 3' of the polyadenyl~tion site. Before transcriptional activation of the gene, chromatin surrounding the gene displays overlapping DNase I and $1 nuclease sensitive sites, which map to a region 100 to 200 base-pairs 5' of the cap site. After induction, the nuclease sensitivity of these pre-established, overlapping sites increases. In addition, induction generates novel, non-overlapping DNase I and $1 nuclease sensitive sites, which map to regions centered 300 base-pairs 5', and al)proximately coincident with the cap site, respectively. We compared the timecourse of aj-globin transcriptional activation to the chromatin structure changes. A twofold increase in gene transcription is detected within two cell cycles (approximately 24 hours) of exposure of cells synchronized in the Gjearly S-phase to inducer. Transcription rates continue to increase for at least 48 hours in MELC cultured with HMBA {the latest time assayed). Chromatin structure changes appear nearly complete after two cell cycles.

1. I n t r o d u c t i o n

Murine hematopoietic precursor cells transformed by Friend virus complex can generate erythroid cell lines, murine e r y t h r o l e u k e m i a cells, which appear to be blocked in their differentiation at a relatively late stage in erythropoiesis (for a review, see Marks & Rifkind, 1978). This block in erythroid differentiation is phenotypieally reversible in vitro. Expression of differentiated characteristics occurs after cells are treated with a n u m b e r of agents, t e r m e d inducers (Friend et al., 1971; Reuben et al., 1976). Virtually all cells in susceptible M E L C t t Abbreviations used: MELC, murine erythroleukemia cells; Me280, dimethy|sulfoxide; HMBA hexamethylene bisacetamide; kb, 103 bases or base-pairs; bp, base-pairs. 417 0022-28'36/84]040417-20 $(}3.00/0 © 1984 Academic Press Inc. (London) Ltd.

4Is

M. SHEFFERY. l'. A. .MARK,~ AND R. A. RIFKIND

t)opulations t reate(l with potent inducers initiate a program of differentiation that inelutles the expression of erythroeyte-spe('ifie genes and a loss of the capacity for (.ell division (Gusella el td., 1976: Fil)ach el td., 1977). Transfi)rmed cell lines, stleh as MEIAL which can be induced to express a differentiation program involving well-characterized genes, such as the globin genes, offer a d w m t a g e s tbr studying molecular events involved in regulating gene cxpressi(m. Since molecular features of uninduced MEI,C can be defined, and events initiate(l hy inducers can I)e analyzed as differentiation l)roceeds, l)otential temporal or ol)ligatory/non-obligatory relationships between molecular events a.~s(,eiated with gene expression might l)e established readily in this in vitro differentiating s.vstem. In atltlition. MEI,C can be synchronized with respect to the cell vyele (Gambari et ai., 1978), and the relationship of the molecular changes required for differenliation-sl)ecific gene expression to cell division cycle events can I)e examined. Recently. several m(~le('ular events associated with glohin gene exl)ression in MEI,(' have been descril)ed. GIobin gene expression is regulated to a large degree at the level ()f transcripti¢)n when ME1,C are cultured in the presence of dinl¢,lhylsulf(~xide or hexamethylene bisa('etamide (Hofer et al., 1982; Profous.lu('helka et ttl., 1983). Transcril)tional regulation of the fl'""i-globin gene has been the most extensively stu(tied. Using a nuclear RNA chain elongation assay, a l0 to 20-fold increase in fl""i-globin transcription is observed after Me2SO or HMBA induction of MEI,(Y (Hofer & l)arnell. 1981: Hofer et al.. 1982). Transcription initiates at or ('lose to the (.ap site. occurs predominantly off the coding strand, and continues 1.5 kl) i~evond the l)oly(A) a(idition site before termination (Hofer et al.. 1982). In addition we (Sheffery et al.. 1982) and others (Hofer et al., 1982) have shown thai increased transcription is accompanied I)y the estahlishment of a I)Nase 1 sensitive region 5' tt) the fl'""i-globin gene. The program of gene expression during induced differentiation of MELC includes the co-ordinate(1 expression of the a-globin and fl'""i-glol)in genes. The results reported here focus on molecular events associated with the expression of the ~l-globin gene during HM l~A-induced ME1,C differentiation. We have defined several characteristics of al-glohin transcription in induced MEI,C. We have determined changes in chromatin structure that oc('ur during HMBA-mediated MELC al-globin gene expression. We have also determined the teml)oral relationship between changes in chromatin structure and increased u~-glol)in gene transcription during induced 5IELC differentiation.

2. Materials a n d M e t h o d s (a) ('~,ll cMhlre ~lnd synch ron izrdion

.MEI,C line 1)819 was maintained a.s described (Ohta el al.. 1976). Exl)eriments were routinely initiated by inoculating exponentially growing cells at l05 cells/ml in tile presence or absence of 5 m,~l-HMBA. (:ells synchronized with res|)eet to the G t/early S-phase of the cell (.ycle were obtained by centriftlgal elutriation (Gaml)ari el td., 1978). Cell cycle i)osition wa.~ determined by analyzing samples f~f ceils for i)XA content I)y flow microltuorimetry [Gambari e/¢d.. 1978).

~tt-GLOBIN GENE EXI'ICESSION IN INDUCED MELTS

419

(b) Preparation of cloned DNA fragments DNA prepared from a recombinant phage (Nishioka & Leder. 1979) containing the at-globin gene was digested with Sacl, and the 3-1 kb fragment was purified from agarose gels by clectroelution (Smith. 1980). After cleavage with appropriate restriction endonucleases, subfragments were purified from polyacrylanlide gels by electroelution (Smith, 1980). Fragments A, B, C and l) (see Fig. l) w4-re cloned into appropriate restriction endonuclease digests of l)lasmid I)UC9 which, like the *ll3 I)hage vectors (Messing et al.. 1981), vontains its cloning sites clustered in a defined region of a portion of the laeZ gene. After transformation of Escherichia coli strain JM83, plasmids containing inserts were identified as white colonies on ampicillin-containing X-gal plates. Both I)XA strands of fragments A to D were also cloned into the single-strand phage M l3mp8 or Ml3ml)9, as described (Messing et al.. 1981). Coding and non-coding strands were determined either by comparison of the 5' to 3' polarity of restr,i,etion sites in ~l-globin and vector l)NAs, or hy I)NA sequence analysis using the dideoxy method (Sanger et al., 1977), followed by coml)arison of sequence data with the known ,~t-globin gene coding arr(l 5' flanking region I)XA sequence (Nishioka & Ledcr. 1979). Frr%,ments E 1 and E 2 were cloned as follows. A derivative of pBR322 containing non-globin information inserted at the BamHl-Hindll[ sites was provided by Dr Allen Oliff. The insert contains a single Sacl site. After cleavage with Sacl and Pvull. the fragment containing the pBR322 origin of rel)lieation and Amp gene was recovered by electroelution, and ligated to fragments E 1 and E2. After transformation, insert-containing colonies were identified by colony hybridization to a radiolabeled Sacl fi'agment corrtaining tire "~q-globin gene. Non-globin sequences contained ira the parent vector were removed by 8acl/BomHl digestion, followed by electroelution of the c~-globin containing fl'agment. Fragment aZ was cloned by treating the 3.1 kb SacI fragment with phage T4 DNA polymerase and ligating the blunt-ended molecule to phosphorylated H i n d l l I linkers (Bahl & Wu, 1978). After cleavage with H i n d l l l and P~'tI, the fl'agment Z was l)urified I)y electroelution, and inserted into a Hindlll/P.stl digest of pUC9. Insert-containing l)lasmids were screened on aml)icillincontaining X-gal plates, as described above. (c) RNA chain elongation in isolated nuclei and dot hybridization.~ RNA chain elongation in isolated nuclei was l)erformed as described (Hofcr & Darnell, 1981), except that unincorl)orated nucleotide was removed by precipitating labeled, purified nuclear RNA with trmhloroacetm acid as described (Groudine et al., 1981). Reaction mixes, containing 250 pCi of [32p]UTP]5 × 107 nuclei, were incubated for 5 rain at 37°C. R.NA purification and alkali breakage was performed as described (Hofer & Darnell, 1981 ). Dots of cloned DNA fragments were bound to 0.45 l~m nitrocellulose filters (Kafatos el el.. 1979) using a Minifold (lot apparatus (Schleieher and Schuell). Approximately 5 pg of I)NA was applied per dot. Equal amounts of acid-precipitable counts were used in each hybridization reaction. Hybridization, RNase A and Tt digestion, and washing of filters were performed as described (Hofer & Darnell, 1981). (d) DNase I digeslion of nuclei, DNA isolation, and blot hybridizations Nuclei (5 x 107]ml) were prepared, digested with l)Nase I, lysed, and DNA was purified as described (Sheffery el al., 1982). Purified DNA was incubated with restriction endonucleases under the conditions recommended by the supplier (New England Biolabs). Elcctrophoresis, transfer of DNA to nitrocellulose, hybridization, and filter washes were performed as described (Stalder el al., 1980; Sheffery el al., 1982).

(e) S I nuclease digestion of nuclei Nuclei (5 x 107/ml) were prepared as described (Sheffery el al., 1982), except that after washing, nuclei were resuspended ill 30 mM-sodium acetate (pH 4.5), 50 mM-NaCI, 2 raM-

42n

M. S H E F F E R V . P. A. MARKS A;~;i) R. A. R I F K I N I )

ZnS()+. Vari.us linlOlllliS Of S t nu(qease (l]oehringer Mannheinl: in lO0 In)i-sodilinl acetate (Ill{ 4,5). (I-I inM-ZnN(),l) werl+ adtletl to sillnliles (if nuclei and reiletions were incubated at ;17:( ' fiw 30 niin. (.']iilnlling (if nil('lei in ,Hi nuclease digestion liuffer was nliniinize(] liv gentle agitlition. Mit,roxrolfiv t,.'.~aliliilatioil showt, d Ill) detecial)le ilii(']l, ilr lysis (hiring iilcli|)ati(lil. A f t r r ililzextion, the lltt (if tim ri,actiinl mixture was adjusted to 7.5, and ntl(.lei were lysed lll~d I)NA wits llrl,llared tls dl,scril)ed (SiletTery el al., 198°).

3. Results T h e ili()llS(, :~l-glot)in geile is c o n t a i n e d (ill il" 3"l kll Sacl r e s t r i c t i o n en(tonuelease rrilgmelil ( N i s l l i l i k a & i,eder, i979). T o liliilivze tile lli(lie('lliill" clianges associated w i t h lilt" illtluce(I expression (if tile l l - g l o h i n gelle, u'e preliare(l I ) N A clones fronl the 3.1 k h ,%',r.] f r a g m e n t t h a i span the e n t i r e f r a g l n e n t . F i g u r e ] s h o w s tile (h,.-~ignatiolls or lhe cloned friignieni,~ and tlleir ]oeation relative t,o the ~ l - g i o b i n ~elle. T w o ['rll~lllents (~IZ ~n(i ~1,4) fire ]oimte(] 5' of tile l l - g ] o i ) i l l Call site ali(| ('(llllilill no l l - g ] o l i i l l stru(_'tUl'il] seilUeliOe illfOrliltltiol] (tile 3' end of ~ I A is 10 bp 5' (it" tile ~l-gi()hin ~elll,, (;ill) site). |*~rllglnelits ~1 B, (',, |) anii E i eontah~ 0;i-gloi)in strtletllrill si,(lUel~t~c~ i n f o r n l a t i ( m and are h o m o l o g o u s to p o r t i o n s o f 0~i-giobill mllXA. [~'i'li~mtmt Ill?, 2 (.(in~liil'iSes tim 3' Ilalf o f ['i'agnleilt 0~iE l, a n d I)egins a l i l l r ( i x i m a t e t y 50 II D) b e y o n d the p u t a t i v e p o l y ( A ) a d d i t i o n site of' the ul-globin

g~,ll~' (Nishioka & I,e(h.r. 1979). F r a g m e n t ~i!"]2 theretTire struelural suqui;nee infornlation, in addition to this set of ])XA t'rliglnlmts, h o t h I)NA strands f r o m regions designated I~relmred ill the singh,-strand I)NA (.Ioning ve(.tor, phage 1(,t81- st,e Fig. 1 ).

contains no ul-globin cloned, double-stralld aiA, B, C and l) were M13 (Messing et al.,

(a) 7'ramvcriptional regulation of the ots-globin ,qene Thl, t:hmlJd ~l-t41ol)in gcne, f r a g m e n t s (Fig. I) trans(~l'il)lioilal r e g u l a t i o n or the gelle, i?or these ('/ilture(I ill the l)resen('e or allsence o [ ' 5 m M - H M i I A ('ells wci'~; washed, illi('lei were lii'el)al'e(] and inctihated A

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Pill. 1. (Jlollq~.t 0%-ghJbin gent'. ['ragmcnts. Th,' lueation of the 0~l-ghJtiin t~ene is shown diagranlatieally +in +i ,~;tlr| restrieti+nl I'llfJollll(;](;li~#~ J'Ftt~llllfll{ i:onlliilling t|iu gene (Nishioka & Loller, 1979). The ~OliO iS rt, lln.si+nie(I ht.ginniilg ill the cap site (CAP) an(I ending +it the putative pol)'lA) addition site (l'(tlNli%)). l il,.n ll(ixt+s I't~|irt't;ll{ t=.XI}llS, Iilled |itixt,s iiltroils, (!lolled ('ragnll+litS I)lt~l)lll'e,iI aero~s the +'~'l!~'ll rpligllll.lll art, indicated IO" h+l,ler designations atiox'e the gelle. Nilnlbt.rs in l)arentheses indicate tllliIF+llilllliil~ h'ligt|l.~ 0n kb)
a ~ - ( I I , ( ) B I N ( ' E N E lgXI~I¢ESNI()N IN I N I ) U C E I ) MEI,('

421

chain elongation mixture containing la2p]UTP to lal)el, in ~:ilro, those nascent RNA (.hains alrea(ly initiated in vivo (Hot'er & l)arnell, 1981). The ial)eled nuc.lear RNA was l)rel)ared (Groudine ¢;1 ai., 1981) and hybridized (Hole( & l)arnell, 1981) I.o cloned ~-globin I)NA fl'agments (Fig. 1) that ha c! I)een denatured (for doul)lesl.rau(l cloned |'ragments; Hofer et al., 1982) and immobilized as dots on ifitroc(,llul()se filters (Kai'atos ¢.,1 al., 1979). The results (Fig. :2) allow ('haracterization oI" several aspects of ~l-glol)in gene transcril)tion. The autora(liographic signals obtained after hybridizing in. vih'o elongated nuclear RNAs prepared from induce(l ME1,C (Fig. 2(a), lane 2) to clone(l I)NA fragments containing al-giohin strut.rural infi)rmation (~1 B, C, l) and El) sllow a marke(l in(.rease in al-globin-sl)ecific hyl)ridization when compared to nuclear RNAs l)rel)are(l fi'oln uninduced (:ells (Fig. 2(a), lane 1). This indicates that increase(I c(1-glol)in gone expression is regulated to a large (lcgree at the level of de t~o~,o transcril)tion. Hyhridization of iabele(l nuclear transcril)ts to clone(! a l-glohin gent t)'agments was quantitated I)y densitometry of autoradiogral)hs or hy liquid so.in(ilia(ion (;ounting of in(livi(lual clots. I~oth methods show that (:ulture of MEI,C, with 5 mM-HMBA fbr 48 hours results in a 10 to 2()-t'ol(l increase in hyl)ri(lizati(m to ul-glol)in gone coding sequences (data not shown). It has I)ecn (lemonstratcd that fl'""J-gloi)in gene transcription also increases al)l)roximately 10 to 2()-tld(I during induced differentiation (Hofer et al., 1982). For the l)url)ose of comparison, we h~ve confirme(l this observation with cloned I)NA t)'agments h(ml()log(~iis to specific regions of the fl'""J-globill gene (Fig. 2(a), ~tncs 3 and 4). In order to determine whether newly synthesized el-glol)in gene nuclear trans(,ripts reflect mRNA precursor tbrmation, single-strand I)NA clones r()nlaining either coding or non-(,'oding stran(ls of the fragments [)repare(I f'r'om the ~l-globin gene were used. The results (Fig. 2(b), right) show that, after induction, hybridization of" nut:lear transcripts elongated in vitro occurs almost ex('lusively to clones containing the co(ling strand. Before induction, nuclear elongation lrans(.ripts also hybridize to al-globin coding strand I)NA (Fig. 2(b), left). ('~,nl)arison of hyl)ridization to coding uersus non-coding I)NA strands shows that the level of hybridization is low, but above background. This result suggests that a 2-gh)bin specific nuclear transcril)ts are synthesized in uninduced 5'1ELC, but to a gr(,atly reduced extent when compared to induced cells (Fig. 2(b), right). In contrast to the marked inducer-mediated increase in transcril)tion of sequen(.cs homologous to al-globin structural inlbrmation, we expected, and ti)und, little in(.reased hyl)ridization of nuclear elongation transcripts to sequences Ul)strcam from the cap site (compare hybridization to alA in lanes I and 2 as well as hybridization to the appropriate plasmid control designated "I)UC", in the same lanes). Quantitation showed consistent, small (2-fold) increases in hybridization to both double and single-strand |)NA clones containing, fragment alA. This finding is most evident by coml)arison of hybridization to single-strand I)NA clones. After induction, increased hybridization to ulA occurs l)redominantly to the clone containing the a~A fragment associated with the a I-glol)in gene coding strand (Fig. 2(b)). Thus, while most transcription appears to begin very close to the cap site, the nuclear elongation assay consistently detects low levels of upstream transcripts during active expression o|" the al-globin gene.

4"2"2

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I"I(;. ~. Tra,~c.rilJthm n(.tivntio, of lh** ~i-~h~hin gP1n'. (n) ('h~l,'d ~ I ni.l/}'"Lglohhl ~.nP Frngment~ (d,'~i~n.'~t.d a.~ in l*'i~. I, ~md n.~ i . Fig. I of Hof'.r ~: I ) n r . . l l . I.qSI. re,~l.~(,tiv.l.v) w~'r. immohilized i . the mHnt.*r,'d lan..~ as dot.~ o . . i t r o ( * . l l u l . s e tilters, as imlienh.*d i . the Figure. nnd h vhridiz.d to H.NA i~rPpnr'(.d Fr.m 5 m i . l~ul.~*-htb,*h'd rnwl.nr (.hai. ,.hm~nlion t rnu.~(,riplion r~*n(*lion~. An equivnh,nt n u m h . r . f . . . n t . ~ w~,.~ n d d . d to .n('h tilh*r nnd ~ult~rndi.~r~,ph.~ ~l' wn.~hed tilt.r.~ nr~' M.~wn. Nu(,h.i (.,× I07) f~r . m d . n r e h . i n e l . . ~ n l i o n trnn,~(,ripthm rvn~'th~n.~ w(.r," prt*l.m*d From ~,xl.m.nthd pOln, lati(,n~ of MI*~L(' eulturt.I in the .h.~.n,,,. 0") ~l' t h . i~l','~el)u~• oF i l . h . ' ( ' r (~ m.~-H.~IBA) For 4,~ h (indi~.~t.d .! tt.* t,~l~ of tl.~ l"i~un*). Otl.~r DNA d.t,.~ wer. u.~c*d .x hybridization (~mtr'(,l~. A (*I)NA .I.m* thnt hybridiz..~ to mwh*ar HNA t**'~m.~('rilm~pr.~,.nt in h - t h .nindu(*,*d and h . h w . d MI':IX' i.~ d~,si~m,t~.*d "('O.I/'" (D~rman ~,1 a/.. I!.~1). A I)NA -I.n..~l.'eifi(' t'(,r t h . m(.~x~* immnn()~lohulin nll,hn gem.** a ~ . m ' not expressed durirux .~1EI.(~ ditt'~*r,.*.li n t l . . , in deni~;mted **1~:(". 'l'h(. pin.mid v~.*hieh" u,~ed to ~:(~n.~tt'~,(*t.,~ub.lone.~ ~(t-A. B, (' and I) i.~ d..~ignnted " p t ' ( " ' . TI.. plasmid w.hieh' u.~t.d to eou.~tru(,t •~ubclon..~ ~(~E,. ~ I"~, and .~ul.:lor.*s #A. B. C, I'; ~,nd I: is ,le,~ig.atc*d "pIIP.". (h) ,";ingh,-str~nd I)NA ~dor.*,~ ~(~rrt, s l . m d i . ~ , to th(: (.oding ((~) or n~m-(:~ding [N(~) strand (~t" region.~ designnl~.l A. B, C nml I) (.~*'. Fig. I) werP immobilized as dot.~ on nitr,..elh,l.~e tilt er,s. I*'ilters w . r . hyl~ridize.d to 5 rain p u l ~ label.d nuch*~=r . h a i . el.ngatior~ trans*~ript~ (.~e~, abovL*) l~rel.tred From . x l . m e n t i n l l.~p.lntion,~ (~t" 51EI.C eull..r*~d i . tl,. nb.~en~*. (CONTI~.OI.) or pr,~*~er.*e ( I N I)L!(~'I']I)) .t' 5 m.~- II ~1BA t'(~r 4~ h. T h . r..~ul|i.g autoradio~raph is shown. (~:) Nm'lei (r~x I(}v) for m . d e n r (~hni. el(mgnlion lnm,~eripti(,n

~ t - ( ; I , ( ) B I N (~I.;NE E X I ' I / E S , S I O N

IN INDU('ED

MELC

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It has been (lemonstrate(I in the case of the fl"'"J-globin gene (Hofer et al., 1982) that nuclear chain elongation transcripts terminate more than 1300 b I) 3' of the fl,,,,,i 1)oly(A ) addition site. To (letermine if c~l-globin gene nuclear" elongation transcril)ts extend heyon(I sequences tbund in the mature mRNA, we compared autova(liogval)hie siglmis ol)tained by hyl)ridizing nuclear chain elongatioll RNA l)vel)are(l fl'om cells culture(] in the presence or absence of HMBA to subclone ~1E2, whi(.h begins 50 l)I) downstream fl'om the putative pol3:(A) additi(m site. The results (Fig. 2(a), lanes 1 and 2) show little increased hyt)ridizatioll to this small (190 hi)) 3' flanking sequence |)'agment when compared to anotl~er small f'vagment, the 170 b 1) ul B fragment, containing c~l-globin structural inlbrmation. (The al)l)rOl)riate l)iasmid control for subclone ulE 2 is designated "I)BR", lanes 3 and 4.) Quantitation of hyl)ridization I)y liquid scintillation counting of exl)evimems similar to lhat shown in F i g u r e 2 shows a 1.3-fold increase in hyl)ridizati(m of fl'agment a l e 2 after induction compared to a greater than tenli)ld increase in hyl)ridization to fragment ~1 B (normalized for hyl)ridization to plasmid eotltrols). This restzlt suggests that ~x-globin gene transcripticm may terminate or atteuuate relatively (:lose to the 3' end of al-gi()bil~ mR.NA, and contrasts with results obtained f'oi' fl'""! described abovc (Hofer ~: 1)arnell, 1981; Hofcv et ~d.. 1982; Fig. 2(a) lanes 2 and 4: coral)are hybridizatiotl of z~ E~ to /J,,,,,i ti'agment fiE, whi(:h ends 800 I)I) 3' of the fl'""J poly(A) site, and flF, which ends 1300 I)l) 3' of the l)oly(A) site). (I)) Time-course of imt~tction of c~rglobin ffene trana'criptiou The time-('ourse of" the tvanscril)tional activation of the ~t-globirl gelm is also shown in Figure 2(c). MELC populations highly enriched in the G l/early S phase of' the (:ell oyele were obtained by centrifugal elutriation (Gambari et al., 1978). The synchronized l)opulation of cells was culture~ with 5 mM-HMBA for one or two (;ell cycles (approx. 12 and 24 h. respectively). In the same experiment, exponentially growing, nol~-synchronizcd cells were cultured with 5 m.~I-HMBA for 36 ov 48 hours. As a cozltrol, an exponentially growing population was cultured without in(lucev tbr 48 hours. After culture, nuclear chain elongation reactions were performed, and the resulting labeled l~NA was hybridized to cloned fragments of the ul-globin gene (Fig. 2(e), lanes I to 5). For comparison, labeled RNA obtained fl'om cultures incubated ~or 48 hours with or without

reactiotls were prepared fron~ synchronous or exponential populations of MELC cultured in the absence or the presence o|' inducer (.5 mM-HM BA) |'or various periods of time. Synchronous populations (enriched in G I/early ,S-phase) were cultured with inducer for I cell cycle (ICC), or o cell cycles (2CC) as monitored by analysis of" k~ell I)NA content by flow mierofluorimetry (see Materials and Methods). I,;xponential populations of cells were cultured without HM BA for 48 h (e) or with inducer for 36 or 48 h (indicated at the top of the Figure). Hybridization controls are designated as in (a). (The relativel.y low intensity of the signal fl'om the (3031 dot at I cell cycle is an artifact due to an error in I)NA loading. Several other experiments (noL shown) demonstrate t h a t the intensity of the signal from the COM dot is approxim~Ltely the same in control and 1 cell cycle samples. The results for b3"hridizati,m of nuclear transcripts Le c~t.globin cloned fragments are not affected.) Cloned ~j (left) and /trnaj (right) globin gene fragments were immobilized on nitrocellulose filters and are designated as described in (a).

424

M. SHI,IFFERY. i'. A. MARKS AND R. A. RIFKINI)

inducer was also hyi)ridized to cloned fragments of the fl"'J-giobin gene (Fig. 2(c), lanes 6 and 7). The results show that the amount of labeled RNA hybridized to the ~t-globin gene fragments increased approxinmtely twofold after one to two cell cycles of exposure to HMBA (Fig. 2(c), lanes 2 and 3). When nonsynchronized cells cultured with HMBA for' 36 and 48 hours are examined, the increase in labeled RNA hybridized to f~'agments of the al-globin gene is l)rogressively greater (Fig. 2(a), lanes 4 and 5). At 48 hours, the amount of hybridization to plasmid I)NA controls also increases (Fig. 2(c), lanes 5 and 7). The amount of hybridization to plasmid controls is somewhat variable (compare dots labeled pBR in Fig. 2(a), lanes 3 and 4 to dots labeled pBR ira Fig. 2(c), lanes 6 and 7). and is detected only on filter's that also hybridize relatively large amounts of labeled RNA (an equivalent number of counts is originally applied to each filter). Nevertheless, hybridization to plasmid controls is usually relatively low, and is sui)tracted for quantitative analyses. Quantitative results, obtained by densitometric scanning of representative clots show increases of approximately two- to threefold after one and two cell cycles, approximately tenfold by 36 hours, and approximately 20-fold by 48 hours (data not shown). These results are comparable to those observed for the time-course of the HS! BA-mediated increase in fl'"~Lglobin gene transcription in MELC (Salditt-Georgieff et al., 1984), and suggest that the rate of :¢l-glohin gene transcription in 3IELC increases progressivel.v over the initial 48 hours of culture with HMBA. (c) DNase 1 sensitive sites near the arglobin ffene

We have previously shown that specific sites in chromatin containing the ~l-globin gene become sensitive to digestion I)y DNase I when M E LC are cultured in the presence of HMBA (Sheffery et al., 1982). \Ve examined the chromatin structL, re of the ul-glol)in gene in greater detail using cloned al-globin gene fragments (Fig. l). Fragment ~IZ abuts the Sacl restriction endonuclease site upstream (5') of the al-globin gene, and is derived entirely from al-globin gene 5' flanking sequences (Fig. I). Fragment alZ hyt)ri(iizes with high preference, but not exclusively, to the 3"! kh SacI fragment contaiuing the al-giobin gene. Other ,~,'acl fragments, which hybridize weakly to alZ, hybridize strongly to an a-giobin cl)NA prol)e (Sheffcry et al., 1982). Thus, Sac] fragments that weakly hybridize tire ulZ probe probal)ly do so because of a low level of 5' flanking sequence homology between al-globin and closely related genes, such as 0¢2, and other' u-like globin sequences known to be dispersed throughout the mouse genome (I,eder et al., 198l). Because of its strong specificity for the a~-globin gene, we used fragment arZ as an indirect end-labeling probe (Wu, 1980) to map DNase I sensitive sites near the gene. Cells were cultured in the presence or absence of 5 m,~t-HMBA for 48 hours, I)y which time both al-globin and fl'""J-giobin genes are being actively transcribed (Fig. 2). DNA prepared from DNase ] digested nuclei was digested with Sael, sized by agarose gel electrophoresis, blotted to nitrocellulose filters (Southern, 1975), and hybridized to fragment alZ, which had been radiolabeled in vitro by nick translation (Weinstock el al., 1978). In induced cells, I)Na.~e I (0.4 to 1.2 pg/ml) digestion of nuclei generates two DNA fragments

al-(-'L()B1N GENE EXPRESSION

IN INDUCED

UNINDUCED

DNose(Fg/m[)

0 i

0-2

. l " :

.~ :' . .;.: .'.

•

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.,

1,2

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.

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: ~ , ' , ::~'~":G;::~::.!,.', ~:~'-','./' , ~ , " - ¢ ' 5

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2

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~NDUCED

0,40,60,81,0 .,

MELC

3

4

5

6

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7

8

9 10

11

12

3.1 kb{u I)

"

~£~,%;~.~

13 14

UNINDUCED

Sac --41

~

1-8 kb

1.7 kb

-

~

',

1.8 kb INDUCED

Fro. 3. Sensitivity to DNase [ of chromatin containing tile ql-globin gene. Nuclei (Sx 10~[ml} prepared from uninduced M EI,C (left) or MEI,C induced by culture ill the presence of 5 ml~I-HMBA (right) were incubated with amou~lts of DNase I indicated above tile ¿alles. Lane numbers arc indicated below each hmc. DXA was purified, digested with Sac]. sized by gel electrophoresis, blotted (Southern, 1075), and hybridized to fragment at-Z (see Fig. 1) that bad |)een radiolabeled in vitro (Weinstock el al., 1978). The resulting autoradiograph is shown. The 3.1 kb Sacl restriction endonuciease fragment cont:dning the avglohin gene is indicated at tile right. A DNase 1 generated subfl'agment ()f approximately /.Skb. detected in unindueed cells, and 2 DNase I generated subfragmeats of 1.8 nnd 1.7 kb detected ill induced cells are indicated at tile right. The locations of DNase ] cleavage sites, mapped on t h e Sac] restriction endonuclease fragment containing the u~-gtobin gene, are indicated diagramaticalty by vertical arrows at the bottom of the Figure. Open arrows: DNase I cleavage site detected in uninduced cells. Filled arrows: DNase I cleavage sites detected after induction. Distances of cleavage sites from the [eft-hand Sac] site are also indicated. The al-globin gene is represented as in Fig. l.

42~i

M. S H E F F E R Y ,

i ). A. M A R K S

A N D R. A. R I F K I N D

from the 3.1 kb fragment containing the ~l-globin gene, which migrate at 1.8 and 1.7 ki) (Fig. 3). Since the ~I-Z probe abuts the Sacl restriction endonuclease site upstream of the 0¢l-globin gene, the two DNase I sensitive sites can be mapped to regions centered 200 and 300 bp 5' of the ~l-giobin cap site (Fig. 3, bottom). Another faintly detected fragment of approximately 4 kb is also generated from induced cell ehromatin (see Sheffery et al., 1982). This fi'agment is presumably generated from another a-like globin gene activated during MELC diffbrentiation. An al-globin specific DNase I generated subfragment is also detected in cells cultured without HMBA (Fig. 3). At least two features distinguish this subfragment from those detected after cells are induced by HMBA. First, the sul)fragment detected in uninduced cells migrates as a single diffuse band, not as a doui)let (even at higher levels of digestion; see below). Second, approximately two to three times more DNase I (0-8 to ].2/zg/mi) is required to generate the subfragment observed in I)NA prepared from uninduced MELC compared to amounts required to produce the subfragments detected after induction. The mobility of the subfragment detected in cells cultured without inducer was used to ma I) the DNase l sensitive site to a region centered approximately 200 bp upstream from tim al-giobin cap site. This DNase I site spans approximately 100 bp and partially overlaps the pair of DNase I sensitive sites detected after induction. However, it does not extend as far 5' of the al-globin gene as the l)Nase 1 sensitive region detected in induced cells (Fig. 3, bottom). (d) S I uttclease sensitivity of chromatin containing the arglobin gene It has been demonstrated that the single-strand specific nuclease, $1, can make site-specific, double-strand cuts in chromatin. The regions of chromatin cleaved b V S1 nuclease are often included in, or are adjacent to, regions that are also sensitive to digestion by DNase I (Larsen & Weintraub, 1982). We examined the sensitivity of chromatin containing the al-g[obin gene to $1 nuclease in MELC cultured in the presence or absence of 5 mM-HMBA for 48 hours. Samples of nuclei were digested with increasing amounts of $1 nuclease. DNA was prepared, digested with Sacl, and analyzed by the Southern blot technique with the al-Z probe. The results (Fig. 4) show that when cells are induced for 48 hours, S, nuclcase digestion of nuclei generates two al-globin specific DNA fragments that migrate as diffuse bands centered at approximately 1.8 and 2.¢~.~b. These mobil|ties were used to map the Sl-sensitive sites. One is found approximately 200 bp 5' to the al cap site, and the other virtually coincides with the a~-globin cap (Fig. 4, bottom). Tim $1 site centered 200 bp upstream of the al-globin gene is located within one of the DNase I sensitive regions detected in cells actively expressing the ~l-globin gene (see Fig. 3, bottom). The second $1 nuclease site, centered near the cap site, does not overlap either of the two DNase I sensitive sites described above. This latter $1 nuclease sensitive region, although not marked by sensitivity to cleavage by DNasel, is known to include DNA sequences that could form energetically stable stem-loop structures (Nishioka & Leder, 1979). Again, a faintly detected fragment of approximately 4 kb is generated in induced cell nuclei, presumably from another activated a-like globin gene.

GENE EXPRESSION

~-GLOI~[N

UNINOUCED

IN INDUCED

MELC

427

INDUCED

S! {U x I 0 - ~ ) 0

]

5

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0

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Fro. 4. S 1 nuclease sensitivity of chromatin containing the =l-globin gene. Nuclei (5 x |O~Jml) from uninduced (left.) or induced (right) MELC were incubated with the amount of S 1 nuclease indicated shove each lane. Lane numbers are indicated below each lane. DNA was prepared, blotted, and hyhridized with the =]-Z probe (Fig. I) as described in Fig. 3. The 3. I kb Sac[ fragment containing tile a:-globin gene, and subfragments of 2-0 kb and 1.8 kb generated by Sj nuclease are indicated at the right of the resulting autoradiograph. Locations of S] nuciease cleavage sites are shown diagramatical]y ~Lt the hol~tom of the Figure by vertical arrows. Open arrow: S~ nuclease cleavage site detected in uninduced cells. Filled arrows: Si nuclease cleavage sites detected after induction. The distance of the cleavage sites from the left-hand Sacl site is also indicated. The ;¢t-globin gene is represented as in Fig. I.

S: nuclea~se digestion of nuclei prepared yields only a 1.8 kb subfragment (Fig, 4), shows that sixibld more $1 nuclease is subfragment. Comparison of S, and DNase I

from cells cultured without HMBA and comparison with induced cells required to initially generate this cleavage sites in uninduced cells (see

42s

M. S H E F F E R Y .

P. A. M A R K S A N D R. A. R I F K I N I )

Fig. 3) shnws that these two nuelease sensitive regions partially overlap. Thus, in polmhltions of unin(tuced 31EI,C, a region centered 150 to 200 bp upstream of the u~-globin gene cap site is already sensitive to both Sa nuelease and I)Nase l. ('ulture in the presence of inducer leads to an increase in the sensitivity of this n,gion to both nueleases, and to the appearanee of two additional, nonow,rlapping, nuclease sel,sitive sites in regions of chromatin both upstream (the novel l)Nase 1 site ; Fig. 3) an(t downstream (the novel $1 nuclease site; Fig. 4) fi'cml the nuch,ase sensitive region cleaved ira uninduced cells.

(e) Direct comparison of DNase I and St nuclease cleavage ,site,s, and digestion of naked DNA Sin(.e the l)Nase I an(i St nuclease sensitive sites are clustered ira a small region (approx. 3t)(I I)p), an additional experiment was performed to verit3, the mapping results ((lescril)e(l above), which indicate distinct l)Xase I and $1 nuclease sensitive sites near the transcriptionally active ~l-glol)in gene (Figs 3 and 4). Nuclei l)relmre(l fi'om cells cultured for' 48 hours in the l)resenee or al)senee of inducer were digested with l)Nase 1 or Sr nuelease. I)NA was purified, and digested with Nacl. Sami)les corresponding to l)Xase I digests of chromatin fi'om unin(hwe(l ('ells. and i)Xase I or Sl nuclease digests of chromatin fi'om induced cells were subjeete(I to eleetrophoresis. Samples were blotted an(l hyl)ridized to lhe :~IZ probe. The results (Fig. 5Ca)) confirm thai $1 nuclease digestion of ehromatin fr()m induce(l (:ells generates a sul)fi'a,~,'ne~li ~'~fal)l)roximately 2.0 kl), which is not (lete(.ted as a product of I)Nase I (ligestior, of induced cell chromatin (Fig. 5Ca). compare lanes 8 and 9 with lanes 10 t. 14). The results also confirm that even crier extensive digestion hy l)Nase l, chromatin containing the g l g l ° b i n gene in uninduccd (:ells yields only a single diffuse subfragnlent centere(I near 1"8 kb (Fig. 5Ca), hums 2 to 6), whereas a broader region of chromatin is accessible to cleavage by l)Nase I in induce(l cells (Fig. 5Ca), lanes l0 to 14). l,esser exl)osures of the nuclease sensitive region detected after indu(.tion show a doublet of l)Nase I sensitive sites (see Fig. 3, hums 8 to 14). We also verified that the I)Xase I sensitive sites we detect are clue predominantly to features (5 chromatin stru('ture rather than to preferential cleavage by I)Nase 1 of specific nueleotide sequences near the ~1-globin gene. For' these experiments, the purified, cloned 3.1 kb I)NA fi'agment containing the al-giobin gene was digested with increasing anmunts of l)Nase I, subjected to electrophoresis, blotted, and hybridized to alZ. T h e results (Fig. 50))) show t h a t I)Nase | does preferentially (:leave naked DNA containing the al-globin gene near some of the same sites cleaved in chromatin (Fig. 5(b), subfragments labeled 1.8 and 1.7 kb). However, other sites, which are also preibrentially cleaved by DNase I in naked I)NA (Fig. 5(b), subfragments labeled !-6 and 1.4 kb), are detected in greatly reduced a m o u n t s in l)Nasc I digests of chromatin when compared to the fragments described above (see Figs 3 and 5Ca)). In addition, the subfragments generated by l)Nase I cleavage of naked DNA are only faintly detectable above a high background of nearly random cleavage. It thus seems likely t h a t the DNase ] sensitive sites we detect in the chromatin of uninduced and induced MELC are

~ts-GL()BIN GENE

v

UNINDUCED

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INDUCED

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,

,

.

.

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tip

-

6.4

~ t l =

. . . .

.

:,

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12 13 14 15

(b)

l"lf'. 5. I)irect conll)arison of l)Nase 1 and St nuclease cleavage sites, aml l)Nase I digestion of naked a t-glohin I)NA. (at Nuclei (5 x 10~/ml) prepared from unindueed (left) or induced (right) MELC were incubated without (indicated by a zero above a lane) or with increasing atnounts of DNase I (indicated by an arrow above lanes) or with the amount of S t nuclease indicated above appropriate lanes (hum numbers are indicated a t the bottom of the Figure). DNA was prepared, blotted, and hybridized with the a s Z probe (Fig. It, as described in Fig. 3. Al)proximately twice as much DNA was added to lanes 8 and 9 compared to hums 2 to 6 and l0 to 14. Sizes (in kb) or marker DNA fi'agments (3l) in lanes l, 7 and 15 are indicated at the left of the Figure. The 3.1 kb SacI fragment containing the at-globin gene, S t nuclease generated subfragment of 2.0 kh and I-8 kb, and DNase 1 generated subfragments of 1.8 kb and 1"7 kb are indicated at the right. (b) A purified, protein-fl'ee (naked) DNA fl'agment containing the ~l-globin gene (the 3.1 kb Sacl fragment) was incubated without or with increasing amounts of DNase I (indicated at the top of the Figure as in (at). Samples were subjected to eleetrophoresis, blotted and hybridized to the atZ probe (Fig. It. The 3.1 kb SacI fragment containing the ctt-globin gene is indicated at the right of the Figure, as are the sizes (in kb) of several discrete subfragments detected above the background of DNase I digestion products.

~:m

M. S H E F F E R Y .

!'. A. M A R K S A N D R. A. R I I ; K I N 1 )

due. in large measure, to features of chromatin strueture established near the gene. (f) 7'ime-co~tr.~e of appearance of D.\'~t.~e 1 ,s'ensitiee sites near the ~l-globin gene We investigated the time-course of appearance of inducer-mediated DNase I sensitive sites in ehromatin containing the ~-glol)in gene. For these experiments, we determine the time of appearance of th ,~ induetion-speeifie 1.7 kb DNase I generated subfragment detected with the ~I-Z probe to minimize problems of interpretation t|lat might arise due to the DXase ] sensitive site present in I CC DNase(,~o/r~l)

36 HR

2 CC

0"4

0

0"2 0 " 4 0 " 6 0 " 8

I'0

0 0-20.40.60-81.0

1"2

•

+

i

,--

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FIG. 6. Timing of chromatin structure change.s near the ~-g]obhl gene. Synchronous I)opulations of MELC (enriehed in (;I/early S-phase) were cultured with 5 m.~I-HMBA fi~r ! or 2 cell eyeles, and an exp¢mential population was cultured with inducer for 36 h (culture periods are indicated t)y iCE:, 2(2C, and 36 h, re.speetively). Nuclei (5 x lO~/ml) were prepared and ineul)ated with the amount a~f DNase I indicated above each lane. Lane numbers are indicated beJow each lane. DNA was prepared, digested with Sac|, hybridized t() the ' ~ l Z ' p r o b e (Fig. I) as described for Fig. 3, and subjected to autoradiography. The 3. t kh S~,c| fragment containing the =l-glohin gene and the 1.8 kb and 1.7 kb I)Nase I generated subfragments are indicated at the righ¢ of the Figure.

~-GLOBIN

GENE EXPRESSION

IN INDUCED

MELC

431

uninduced cells (see Fig. ~t,~. Populations of MELC synchronized in the G1]early S-phase were cultured with 5 m.~-HMBA for one and two cell cycles. In addition, we examined an exponentially growing, non-synchronous population cultured with HMBA for 36 hours. After culture for these times, nuclei were prepared and digested with DNase I. Purified *,')NA was digested with SacI, and Southern blots were analyzed with the labeled al-Z probe. After exposure of synchronized cells to 5 mM-HMBA for one ceil cycle (Fig. 6, lane l), the 1.7 kb induction-specific subfragment is detectable. After two cell cycles, (Fig. 6, lanes 2 to 7), this fragment is readily apparent, and the cleavage pattern is indistinguishable from the pattern of DNA fragments generated from chromatin of an exponentially growing population of cells exposed to inducer for 36 (Fig. 6, lanes 8 to 14) or 48 hours (Fig. 3). Thus, inducer-mediated generation of the 1.7 kb subfragment appears to be established early during induction, and is readily detected within two cell cycles. These findings with respect t~ gl-globin are similar to results obtained by analyzing the appearance of a DNase I sensitive region 5' to the fl,,,i giobin cap site relative to the increase in fl'""i-globin gene transcription (SaldittGeorgieff et al., 1984). These results suggest that the establishment of inducermediated, 5' DNase I sensitive sites associated with a 1 and fl'""i-globin gene activation occur early during inducer mediated differentiation of MELC. 4. Discussion

The present study provides further evidence that complex alterations in the cilromatin structure of specific genes are associated with their transcriptional activation during differentiation (Groudine & Weintraub, 1981; McGhee et al., 1,081; Sherman & Beckendorf, 1982; Weintraub et al., 1981; Wu & Gilbert, 1981; Sheffery et al., 1982; Hofer et al., 1982). In this paper, we have described the

DNose I

UNINDUCED

,NOUCEOn $1

INDUCED

of co

It°cgCc' g

UNINDUCED ~

~

to gc gc fcg fcfffggog fctgoggg

Flo. 7. Sumnmry of chromatin and I)NA sequence tbatures near the al-globin gene. The location of DNase I (see Fig. 3) and S I nuclease (see Fig. 4) sensitive sites in ehromatin engaged in active (INDUCED) or inactive (UNINDUCED) transcription of the a : g l o b i n globin gene are indicated by vertical arrows. Open arrows: DNase I (above the line) and $1 (below the line) sites present before active gene transcription. Filled arrows: DNase I and S 1 nuclease sites present during active at-globin gene transcription. The u:globin gene is designated as in Fig. 1. Inset: The nucleotide sequence of the region indicated by the small open box to the right of the a : g l o b i n gene is shown in a potential stemloop configuration. A 9 base-pair direct repeat sequence is also indicated. DNA sequence features similar to those indicated may have important effects on transcription through this region (see the text).

43"

M. SHEFFERY. P. A. MARKS AND R. A. RIFKIND

changes in ehromatin structure associated with HM BA-mediated expression of the ",l-gloi)in genes in ME1,C. In addition, we have defined several features of the ~l-globin gene transcriptional domain (Fig. 7). We found that inducer-mediated increased c~l-giobin gene expression in MELC is regulated, in part, by a 10 to 20-fold increase in transcription (Fig. 2). We assume that ~,-giobin gene transcripts might also contribute, in part, to the signal detected fl'om ~l-giobin coding region fragments. To quantitate this contribution, cloned fragments specific for ~2-globin gene transcripts will have to be prepared. By using cloned I)NA fragments spanning the ~l-globin gene (Fig. 1), we have also been able to investigate specific structural features of the ~l-globin gene transcription domain. Although most a l-giohin transcripts appear to initiate near the cap site, we detected consistent, twofold increases in hybridization to cloned I)NA fragments prepared from the region designated al A, whose 3' end is 10 bp Ul)st~ream from the cap site (Fig. 2). Thus, the nuclear chain elongation assay dete(:ts a h)w level of Ul)stream transcripts that. might normally occur during active expression of the al-globin gene. Comparison of hybridization to single-strand DNA clones containing coding or non-coding strands of fragments ulA, B, C and D shows that transcription oe(:urs ahnost exclusively off the coding strand (Fig. 2(b)). Although this result is anti(.ipatcd, it distinguishes the hybridization pattern of the al-globin and ~m,~i_ globin genes, since it has been shown that in. vitro elongated nuclear transcripts l~repared from uninduced MELC hybridize to both I)NA strands of a small region near the 5' end of the ~""i-giobin gene (Salditt-Georgieff el al., 1984). The source of the sequen(:es hybridizing to the ~o~i non-coding strand remains unclear. Although we do detect low levels of al-globin transcripts in uninduced MELC, they appear to be coding strand specific, and distributed uniformly across the gene (see below). The nuclear (:hain elongation assay detects reduced levels of transcription in a region 3' of the putative polyadenylation site of the al-globin gene (Fig. 2). The 1)NA sequence 3' of the polyadenylation signal in ai (Nishioka & Leder, 1979) contains a 9 b 1) direct repeat (A-G-C-C-A-A-A-G-A-A-G-C-C-A-A-A-G-A), and several regions of hyphenated dyad symmetry that could form energetically stable slem-ioop structures. One possible stem-loop structure is depicted in Figure 7 (inset). Other such structure.s are possible, including ones that bring the 9 bp direct repeat into a loop. DNA sequences containing potential stem-loop structures have I)een shown to be important in regulating transcription termination in both prokaryotes (Rosenberg & Court, 1979) and eukaryotes (Birchmeier et al., 1982; Hay et al., 1982). For example, in sea urchin histone gene I)NA sequences, potential stem-loop structures have been shown to be necessary, though not sufficient, signals for correct transcription termination (Birchmeier et al., 1982). ]n the simian virus SV40, regions of DNA sequence containing hyphenated dyad symmetry have been shown to be involved in attenuating transcription, appal;ently by mechanisms analogous to those occurring .in prokaryotes (Hay et al., 1982). Our results suggest that DNA sequences in fragment alE~ might be near, or include, termination or attenuation signals for ai-glohin gene transcripts. We speculate that some of the DNA sequence features

~I-GLOBIN GENE EXPRESSION IN INDUCED MELC

433

shown in Figure 7 (inset) play a role in attenuation or termination of ~l-globin gene transcription. Our analysis of the time-course of al-globin transcriptional activation shows twofold increases in transcription within one to two cell cycles (Fig. 2(c)). The major increases in transcription occur somewhat later; by 36 hours, transcription is approximately 60~o of the rate detected at our latest assay time, 48 hours. These results show that transcription of the avglobin gene increases to its maximal rate over a period of at least 36 to 48 hours. The transcriptional activation of the flm~J-globingene follows a similar time-course (Salditt-Georgieff et al., 1984). Experiments analyzing the time-course of development of inductionspecific DNase I sensitive sites (Fig. 6) suggest that the establishment of the chromatin structure associated with the actively transcribed state is nearly complete within two cell cycles (approx. 24 h). One possible interpretation of these results is that the establishment of chromatin structures associated with the active transcription of the al-globin gene occurs slightly before the transcription peaks, and may thus be independent of the increased transcription of these genes. This interpretation would be consistent with reports showing that DNase I sensitive sites need not be directly linked to gene transcription (Groudine & Weintraub, 1982; Weintraub et al., 1982; Nasmyth, 1982). However, while our results show that the chromatin structure associated with active al-globin gene transcription is established early during induced differentiation of MELC, transcription rates, although low, are clearly increased from uninduced levels even after one cell cycle. Thus, we have not been able to definitively show the independence of the active chromatin structure from any increase in transcription. [t has been established that compounds such as dexamethasome (Scher et al., 1978; Lo et al., 1978; Tsiftsoglou et al., 1979; Chen et al., 1982) can block induced differentiation of MELC. In addition, inducer-resistant variants of MELC, which show restricted responses to specific inducers, are available (Ohta et al., 1976; Marks et al., 1983). It may thus be possible to manipulate the MELC system so as to determine the relationships between the establishment of chromatin structures associated with the actively transcribed state in MELC and transcription itself. The low level of al-globin gene transcripts detected in uninduced MELC occurs in the absence of the non-overlapping pattern of DNase I and $1 nuclease sensitivity associated with the actively transcribed state (Figs 3, 4 and 5). Overlapping DNase I and $1 nuclease sensitive sites are detected in uninduced MELC, and it is possible that these sites play a role in maintaining or permitting the low level of al-globin gene transcription detected in uninduced cells. However, alternative functions for these sites must be considered. First, previous studies have demonstrated that DNase I sensitive sites can "mark" genes before their active expression (Keene et ai., 1981; Sledziewski & Young, 1982). Thus, the DNase I and Sl nuclease sensitive sites we detect in uninduced MELC may represent pre-established sites which normally mark the transcription unit before its activation. Although the original stimulus generating them is no longer present, these sites could have been stably propagated (Groudine & Weintraub, 1982) and may represent a normal event in erythroid differentiation that has been |9

4:14

M. SHEFFEI~Y, I'. A. MAI,~KS AND R. A. RIFKINI)

"'frozen" by ~ransl'ormation of MF, I,C. S(~eon(t, the ['a(;t that these sites are deteeted in h¢)th unindu['ed and i~l(tuced cells suggests that the nucleases may he reeognizing a e()mmon structure, important in utht, r cellular ft,netions, sueh as an active origin of replication or a eelluhtr enhancer se(luenee. By analogy with results obtained i~1 polyt)ma or SV40 systems, these regions might also t)e nuelease sensitive sites (Herl)omel et (tl., 1981: Shakhov et cal.. 1982). Our analysis of the ehromatin sirtl(!tUl'e o|" the :¢l-glohin gem~ after transcriptional aelivation of the gene has revealed a relatively enm[)lex pattern of nuelease sensitive sites. As (lescri~)e(t above, l~efore induetion overlapping I)Xase I and $1 nuetease sensitive sites are detected 5' of the al-glot)in gene ('~p site (Figs 3. 4 and 5). After induction, the nuelease sensitivity of these sites i~crr, ases a n d new. non-c)verlapl)ing 1)Nase I and S 1 nuelease sites develop. The detection of multiple $1 nuclease and I)Nase I sensitive sites 5' (ff active genes using indirect end-labeling probes has been described (see. for examl)le, I,arsen & Weintrauh, 1982). The visualizati()n of multiple IJr¢)(luets (rather than only the most probeproximal fi'agment) suggests that at early stages of digestion, only one potentially accessible site is cleaved in most nuclei. The intensity el" the resulting subfragments woulti thus he related to the ]lrohahility of cleavage at a given accessible site. In MEIA' the in(tuetion-si~e('ific DNase I sensitive region maps 3(tO 1)t) 5' of the :t~-glot)in cap site (150 [)l) upstream of the I)Nase ] site detected in unindueed cells), a~(l the induction-specific Sl sensitive site mat)s to ¢t region coincident with the cap site (200 bp downstream fi'om the $1 nuctease site detected in uninduced (.ells: see Fig. 7). The detection of new S 1 nuclease and l)Nase I sensitive sites after euiture in the presence of in(tueer suggests t h a t while s(Jme ~'hanges in chromatin structure may have alrea(ty been estat)lished ne~r the oci-globin gene in MEI~C, additional changes might ])e required f~)r activation o|" ~l-glot)in t rans('ription. Interestingly. the ])Xase l and S 1 sites associated with the actively transcribed gene do not overlap, suggesting that, at least in some cases, different struetures can he recognized l~y these two nucleases (Figs :~1. 4. 5 and 7). Presumat)ly, 81 nuciease sensitivitv indicates that l)NA in this region is single-stranded, perhaps in a stem-loop configuration. A potentially stable stem-loop structure has already I)een noted in tim I)NA sequence near the ul-globin cap site (Nishioka & Leder, I979) and our mapping indicates t h a t this DXA sequen¢~e th]ls in the region cleaved by S 1 nuclease after inducer-mediated transeri}~tional a(.tivation. Potential stem-loop structures may thus play important roles in both the ii~itiation and termination o|" transcripl~ion (l~arsen & Weintr~ul), t 982; Birchmeier et al., 1982; see Fig. 7, inset), although the presence of nucle¢~tide sequences ealml)le of forming such structures is apparently not suificient rot' either f'unetion. The experiments descril~e(l here provide a haste tYamework ft)r understanding the regulation of ~¢l-glohin gene expression during induced (|ifferentiation of *IE[,C. ]t remains to be established how (and it) the structures we detect in MELC are established during normal routine crythroid diffL'rentiation, and how they are activated and maintained during gene expression.

~1-(II.()BIN GI,:NE EXI'RESS]ON IN INI)UCE1) MELC

435

These st.dies wer(~' supp~)rted, i~l part, by grants from the National Cancer Institute (I)() 1 (~A-3-7t~8 a . d CA-08748), the Ameri(:an Cancer Society (CH-681)), and the Bristol Myers (]an(.(.r Grant Program. REFERENCES

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Edited by P. Chambon