Vol. 11, No. 9MOLECULAR AND CELLULAR BIOLOGY, Sept. 1991, p. 4324-43320270-7306/91/094324-09$02.00/0Copyright C 1991, American Society for Microbiology

Derepression of Mouse r-Major-Globin Gene Transcriptionduring Erythroid Differentiation

KAY MACLEOD AND MARK PLUMBt*

CRC Beatson Laboratories, Beatson Institute for Cancer Research, Garscube Estate,Bearsden, Glasgow G61 IBD, United Kingdom

Received 10 January 1991/Accepted 7 June 1991

Functional analysis of the mouse ,I-major-globin gene promoter has revealed a negative regulatory element(-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activityis induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1(-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directedmutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity inuninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters duringMEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activityin uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-bindingactivity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating theeffects of the repressor element. The BBl-binding factor also competes with the CCAAT-binding protein forbinding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity duringdifferentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor alsodown-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding proteinto the proximal CCAAT motif.

The interactions between transcription factors bound atpromoter, enhancer, or locus control region (LCR) se-quences determine globin gene expression during develop-ment and differentiation of erythroid cells (1-12, 14-18, 20,23, 24, 26-29, 32, 34-39, 41, 44-51). These regulatory ele-ments reside in developmentally regulated and tissue-spe-cific nuclear DNase I-hypersensitive sites (DHSs) (4, 23, 24,26, 37, 39, 47) and are accessible for binding transcriptionfactors in vivo (23, 24, 26, 39). The characterization ofpromoter sequences in the absence of enhancer or LCRelements has defined transcription factor binding sites whichare required for the modulation of both promoter activityand promoter function (5, 6, 11, 12, 14, 18, 23, 32, 38, 44) andwhich may mediate putative promoter-enhancer-LCR inter-actions (1, 2, 7, 8, 10, 16, 28, 29, 35, 48, 50).The promoter DHSs of globin and other erythroid-specific

genes in erythroid cell nuclei extend some 150 to 250 bpupstream of the transcription initiation site (4, 23, 24, 26, 39)and contain conserved sequences such as the ATA (- 30 bp),CCAAT (-70 bp), and CACC motifs which are essential forpromoter function (6, 11, 32). Additional proximal promoter(-1 to -100 bp) transcription factor binding sites, such asGATA-1 (36) (also called NF-E1, GF1, EF1, or Eryfl [17,36, 38, 49]) and Spl (26), have also been described, and it isthe interactions between these proximal promoter nucleo-protein complexes which presumably favor the formation ofan active transcription initiation complex.

Studies of the transcription factor binding sites in thedistal region of the globin gene promoter DHS (-100 to -250bp) suggest that these sites modulate the activity of theproximal promoter (5, 12, 14, 18, 38). For example, thehuman adult 3-globin gene distal promoter contains two

* Corresponding author.t Present address: Institute for Cancer Studies, St. James's

University Hospital, Leeds LS9 7TF, United Kingdom.

GATA-1 (-120 and -190 bp) binding sites and the sequenceAAGCCAGTG (-155 bp, which we call the BB1 sequence),which are involved in the induction of transcription duringterminal erythroid differentiation (12). Similarly, the chickenadult P-globin gene promoter contains stage selector ele-ments which bind proteins whose abundance changes duringdevelopment and differentiation (14, 18, 23).The enhanson compositions of globin gene promoters and

enhancers are very similar (12, 14, 17, 18, 26, 36-39, 41, 47,49), suggesting that the differential regulation of globin geneexpression during development and differentiation is a con-sequence of different combinatorial arrangements of a lim-ited number of transcription factor binding sites. Further-more, the developmental expression of the chicken adult andembryonic and the human adult and fetal ,-globin genes ismodulated by the competition between promoters for eitherenhancer or LCR regulatory elements (2, 3, 7, 16, 18, 35).The ability of each promoter to compete changes duringdevelopment as the availability of critical transcription fac-tors changes (15, 23, 24).

Saturation and linker scanning mutagenesis of the mouse3-major-globin gene proximal promoter sequences revealed

that intact TATA, CCAAT, and CACC motifs and a directlyrepeated sequence (,BDRE) are required for promoter func-tion (6, 11, 32, 44). We have now extended this analysis toinclude a GATA-1 binding site at -60 bp. However, thenuclear DHS in mouse erythroleukemia (MEL) cells extendsover some 250 bp (4), and little is known about the functionof these distal sequences.

In this study, we have used in vitro protein binding assaysand site-directed mutagenesis to further characterize themouse ,B-major-globin gene promoter. We have defined amodular negative regulatory element in the distal promoter(-100 to -250 bp) which is derepressed during MEL celldifferentiation. Sites for binding of three proteins (nuclearfactor 1 [NF1], GATA-1, and BB1) are required for repres-sor function, and only BB1 DNA-binding activity decreases

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V-MAJOR-GLOBIN GENE TRANSCRIPTION 4325

during terminal differentiation. The BB1-binding factor alsobinds the CCAAT motif and may repress transcription bydisplacing the CCAAT-binding protein (CBP). The modula-tion of the BB1-binding factor and its interaction withproximal and distal promoter sequences imply that it iscentral to the regulation of promoter activity during eryth-ropoiesis.

MATERIALS AND METHODS

Cell lines and cell culture. Adherent MEL cells (F4-12B2),mouse fibroblasts (STO) (38), and mouse macrophages(J774.2) (40) were grown in special liquid medium (GIBCO-BRL) supplemented with 10% fetal calf serum (GIBCO-BRL) and 4 mM L-glutamine. F9 murine embryonal carci-noma cells (13) were grown in Dulbecco's modified Eagle'smedium, 10% fetal calf serum, 4 mM L-glutamine, and 1 mMsodium pyruvate.Nuclear protein extraction. Nuclei were prepared and

extracted with 0.35 M NaCl. The eluate was precipitatedwith ammonium sulfate (0.32 g/ml), redissolved, and dia-lyzed against storage buffer (50 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES;pH 7.9], 5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol,20% glycerol) as described previously (38).

Construction of wild-type and mutant plasmids. pOGH,containing a promoterless human growth hormone (hGH)reporter gene, and pTKGH (pOGH driven by the herpessimplex virus type 2 thymidine kinase promoter) (42) wereobtained from Biogenesis Ltd.ppWT was generated by cloning a 370-bp BamHI-HindIII

restriction fragment of the wild-type mouse ,B-major-globingene promoter (-346 to +24 bp) into the BamHI-HindIII siteof pOGH.Mutant constructs were generated by the polymerase

chain reaction technique (22), using the wild-type pBWTconstruct as a template. The oligonucleotide primers used(Fig. 1B) introduced 3- to 4-bp substitutions which abolishprotein binding to the particular site. Amplified mutantpromoter fragments were then restricted and cloned into theBamHI-HindIII sites of pOGH, and mutants were screenedby sequencing.DNase I footprint protection assay. Restriction fragments

were 5' end labelled and isolated after secondary restrictionas described previously (38). Markers were the G+A chem-ical sequencing reaction (31) of the probe.DNase I footprint protection assays were performed in a

final volume of 100 ,ul of storage buffer in the presence of 1pug of poly(dI-dC), 2 ng of end-labelled probe, and up to 98 ,udof nuclear extract in the presence or absence of double-stranded oligonucleotide competitor (100 ng) (38).

Gel shift assays. Blunt-end 5'-end-labelled double-strandedoligonucleotides were prepared and used in gel shift assays(38). Labelled oligonucleotide (100 pg) was incubated on icefor 1 h with protein extracts (O to 10 ,ul) in the presence of 6,ug of poly(dI-dC) and in the presence or absence of compet-itor oligonucleotides (100 ng) in a final volume of 20 pul ofstorage buffer. Samples were electrophoresed in a 5% poly-acrylamide gel in 0.2x Tris-borate-EDTA buffer for 2 h at4°C at 150 V, and the gel was dried for autoradiography.

Transfections and hGH assays. Exponentially growing F4-12B2 MEL cells (106 in 10 ml) were transfected by thecalcium phosphate precipitation technique (19, 38). Fortransient expression studies, 20 p.g of test plasmid, 10 pug ofreference plasmid pHSV3GAL (21), and 10 pug of carrierplasmid pIC20H (30) were used. Sixteen hours later, 10 ml of

fresh medium was added. Cells and medium were harvestedafter 48 h, the medium was assayed for hGH, and cell lysateswere assayed for 3-galactosidase activity (21).

Stable transfectants were generated by transfecting cellswith 40 p.g of test plasmid and 0.5 pLg of HOMER 6 plasmidcontaining the neomycin resistance gene (43). Sixteen hourslater, 10 ml of fresh medium was applied. Stable transfec-tants were selected by treatment with geneticin (80 pug/ml;GIBCO-BRL) for 2 to 3 weeks, and colonies (more than 100)were pooled. Transfectants were induced to differentiatewith 4 mM N,N'-hexamethylenebisacetamide (HMBA) for 5days. Medium was collected at intervals and assayed forhGH activity.

Levels of hGH in the culture medium (100 pll) wereassayed by using the Allegro dual-growth-hormone-specificmonoclonal antibody system as recommended by the sup-plier (Biogenesis Ltd.). In transient transfections, hGH geneexpression was calculated following the deduction of back-ground counts (cells transfected with pOGH) and correctedfor transfection efficiency by using the ,B-galactosidase ac-tivity assayed in cell lysates (21).RNA analysis. A 515-base single-stranded uniformly

[ca-32P]dCTP-labelled probe, complementary to ppWT se-quences from position -346 bp relative to the mouse ,-ma-jor-globin gene promoter to position +145 bp relative to thehGH reporter gene (42), was synthesized by using a 17-baseoligonucleotide primer (GGTTAGTGCCCCCGTCC) com-plementary to hGH gene sequences between + 145 and + 128bp.DNA probe (2.5 x 105 cpm) was hybridized to 10 pLg of

total cellular RNA (prepared by the RNAzol technique;Biogenesis Ltd.) overnight at 50°C in the presence of 0.25 MNaCl, 0.03 M sodium acetate (pH 4.6), 1 mM ZnSO4, and200 pug of sonicated salmon sperm DNA per ml. Hybridswere then digested with 100 U of Si nuclease (GIBCO) for 1h at 37°C, and nucleic acids were isolated and resolved bydenaturing 6% polyacrylamide gel electrophoresis and auto-radiography.

RESULTS

Characterization of protein binding sites in the mouseP-major-globin gene promoter. The nuclear DHS of themouse ,-major-globin gene promoter in MEL cells spans a250-bp (-1 to -250 bp) sequence (4). A GATA-1 proteinbinding site at -212 bp (38) and conserved motifs such as theCCAAT, CACC, ,BDRE, and TATA motifs, which wereidentified in functional assays (6, 11, 32, 44), have beendescribed. Using in vitro protein binding assays and com-petitor oligonucleotides (Fig. 1B), we have identified threeadditional protein binding sites (Fig. 1A).

Footprint analysis of the distal (-160 to -250 bp) region ofthe promoter with MEL cell nuclear extract reveals twomajor protein binding sites (Fig. 2A). Competition experi-ments confirm that the erythroid GATA-1 nuclear proteinbinds to the GATAAG sequence (-215 bp) (38). Binding isabolished by competitors containing either the mousea-globin gene promoter GATA-1 binding site (aG2 [38]) orthe homologous sequence (G215) but not by competitorscontaining an NF1 recognition sequence (adenovirus originof replication NF1 binding site [Ad-NFl]) or CCAAT (BCT)motif. Interestingly, a competitor containing a potentialGATA-1 binding site at -60 bp (G60; Fig. 1 and 2) doescompete for binding, suggesting there is a -60 bp GATA-1binding site present in the mouse P-major-globin gene pro-

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4326 MACLEOD AND PLUMB

A. Mouse 1-major globin gene promoter.

-260 -240 -220ATTGAGCAAA TGCGTTCGOC AAAAAGGATG CTTTAGAGAC AGTGTTCTCT GCACAGATAA GGACAAACAT

NF1 GATA-1

-200 -180 -160 -140TATTCAGAGG GAGTCCCAGA GCTGAGACGT CCTAAGCCAG TGAGTGGCAC AGCATGTCCA GGGAGAAATA

BB1

-120 -100 -80TCGCTTCGTC CTCACCGAAG CCTGATTCCG TAGAGCCACA CCCTGGTAAG GGCCAATCTG CTCACACAGG

-60 -40 -20 -1ATAGAGAGGG CAGGAGCCAG GGCAGAGCAT ATAAGGTGAG GTAGGATCAG TTGCTCCTAC

GATA-1

B. Competitor and mutant primer oligonuclootides:

BNF1 : gatcGCAAATGCGTTCGCCAAAAAGGABNF1M : GCAAATGCGTTCGCCggttAGGATGCT

G215 gatcCTCTGCACAGATAAGGACAAACAG21 5M TGTTCTCTGCACAGcTccGGACAAACATTA

BB1 gatcCTGAGACGTCCTAAGCCAGTGAGBB1M GAGCTGAGACGTCCTcctaaAGTGAGT

BCC CCTGATTCCGTAGAGCCACACCCTGGTBCCM CCTGATTCCGTAGAGCtgCAggaaGGT

BCT gatcCtggtAAGGGCCAATCTGCTCACBCTM gatcCTGGTAAGaGCCAATCTGCTCAC

G60 gatcCACACAGGATAGAGAGGGCAGGAG60M TGCTCACACAGctgcagGAGGGCAGGA

aG2aP3aAd-NFl:

gatcCGGGCAACTGATAAGGATTCCCAgatcCAAACCAGCCAATGAGAACTGCTCCAgatcCTTATTTTGGCTTGAAGCCAATATG

FIG. 1. (A) Sequence (-1 to -270 bp) of the mouse ,-major-globin gene promoter. Protein binding sites and conserved consensussequences are underlined. (B) Sequences of the competitor oligonucleotides derived from the protein binding sites (BNF1, -250 bp; G215,-215 bp; BB1, -165 bp; and G60, -60 bp) and conserved consensus (BCC, -90 bp; and BCT, -75 bp) sequences. Primer oligonucleotides(BNF1M, G215M, BB1M, BCCM, and G60M) were used to mutate the corresponding sites. aG2 and aP3a are the mouse a-globin promoterGATA-1 and CCAAT binding sites, respectively (38); Ad-NF1 is the adenovirus origin of replication NF1 binding site (33).

moter, as has been described for the chicken ,B-hatching-globin gene promoter (36).The second major protein binding site (-250 bp, Fig. 2A)

contains a GCCAA sequence (Fig. 1A) which resembleseither one half of the NF1 inverted repeat consensus recog-nition sequence (TTGGC(N)4CCAA [25]) or a CCAAT mo-tif. Neither the P-major-globin gene CCAAT (BCT) nor theGATA-1 binding site sequence (G215, G60, or aG2) com-petes for binding. Binding can be competed for by thehomologous sequence (BNF1) or Ad-NF1, suggesting thatthe binding factor is a member of the NF1 family of tran-scription factors.The sequence AAGCCAGT (-160 bp; Fig. 1A) is also

found in the distal human P-globin gene promoter where, insynergy with two GATA-1 binding sites, it has been impli-cated in the up-regulation of transcription during MEL celldifferentiation (12). Although the mouse sequence (BB1) isnot apparently bound in our DNase I footprint protectionassays (data not shown), gel shift assays with labelled BB1probe and MEL nuclear extracts revealed specific binding(Fig. 3A), as has also been shown for the human ,-globingene BB1 sequence (12). Binding to BB1 cannot be com-peted for by competitor BNF1, Ad-NF1, or the mouseax-globin gene CCAAT motif (aP3a) but can be competed foreither by the homologous BB1 sequence or by the mouse,-major-globin gene CCAAT motif (BCT) (Fig. 3A), con-

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V-MAJOR-GLOBIN GENE TRANSCRIPTION 4327

A. pBWT B.pBNFlM pBG215M

U.

XZV- in,O< ci u,m m XCo t

ce0 laz cL J040 00 U - 4 m 0a a Competitor

NFI

GATA 1

w w - op WV __

- i - I - _. -

t~~~~I4pl* tl Gi-n_ - _ _ * _.

_- U _- U,C~ L. _- C LL _O Z N 0 z NO - m C U - m O Competitor

NF1

-- 255 bp

- - 245 bp

- - 220 bp

- - 210 bp

G ATAl

--170 bp

FIG. 2. (A) Footprint analysis of the mouse P-major-globin gene distal promoter sequences. Plasmid ppWT was 5' end labelled at position-100 bp of the noncoding strand. Probe (5 ng) was incubated with 0 (Con.) or 90 iLl of MEL cell nuclear extract in the presence or absence(-) of competitor oligonucleotide (100 ng; Fig. 1B and Materials and Methods), as shown. Markers are the G+A chemical sequencing reaction(31). The NF1 and GATA-1 binding sites are marked. (B) Footprint analysis of mutant plasmids ppNFlM and p,G215M. Labelling andfootprint reaction conditions were as described above. As shown, mutagenesis abolishes NF1 binding in p3NFlM and GATA-1 binding inpPG215M.

firming that the BB1-binding factor is a member of theCCAAT box-binding factors (12). The BB1 probe is specif-ically bound with MEL, mouse brain, or liver nuclearextracts but is only weakly bound with those from HMBA-induced MEL, J774.2, or F9 cells (Fig. 3B), suggesting thatthe BB1 DNA-binding activity is differentially expressed indifferent tissues and is down-regulated during MEL cellterminal differentiation.To determine the relationship between the BB1 binding

factor and the mouse ,-major-globin gene CCAAT box-binding factor, we compared the gel shift analyses of themouse --globin CCAAT motif (BCT; Fig. 3C) and the BB1sequence (BB1; Fig. 3A). MEL nuclear extract binding toBCT yields two nucleoprotein complexes (BCT1 and BCT2)which are specifically competed for by BCT competitor butnot by BNF1 or Ad-NF1, suggesting that neither complexcontains NFl. Furthermore, aP3a competitor (containing themouse P-globin gene CCAAT box) results in loss of theBCT1 (but not BCT2) complex, suggesting that the sameCBP (9, 38) can bind both the murine a- and ,B-globin geneCCAAT motifs. However, only one nucleoprotein complex(equivalent to BCT1) has been observed in gel shift experi-ments with the mouse a-globin gene CCAAT sequence (38).

Since the tissue distributions and electrophoretic mobili-ties of the BCT2- and BB1-binding factors are very similar(Fig. 3B and D) and the BB1 and BCT sequences cross-

compete (Fig. 3A and 4), it is likely that the same proteinbinds the mouse P-major-globin gene GGCCAAT and BB1sequence motifs but cannot bind (or has a low affinity for) themouse a-globin gene AGCCAAT motif.

Mutagenesis of the mouse ,B-major-globin gene promoterGGCCAAT motif to the mouse a-globin gene AGCCAATsequence results in the up-regulation of promoter activity(32). We therefore compared the MEL nuclear proteinbinding with binding of the wild-type GGCCAAT sequence(BCT) and the AGCCAAT point mutation (BCTM) in gelshift assays (Fig. 4). Whereas the BCT probe yields the twoBCT1 and BB1/BCT2 nucleoprotein complexes, the BCTMprobe gives a strong BCT1 complex and only a weakBB1/BCT2 complex. In both cases, the BCT1 complex isdifferentially abolished by the aP3a or BCTM competitor andthe BB1/BCT2 complex is differentially abolished by theBB1 competitor. However, BCT2 complex formation withthe BCTM probe appears to be increased when the BCT1complex is differentially abolished by either the aP3a or BCTcompetitor, suggesting that the two factors are competing forbinding. Furthermore, the finding that BCT competes onlyweakly for either the BCT1 or BCT2 complex formed withthe BCTM probe indicates that the binding factors have a

higher affinity for the AGCCAAT sequence. The finding thatthe GGCCAAT-to-AGCCAAT point mutation results in theup-regulation of promoter function in vivo and a decrease ofBB1/BCT2 protein binding in vitro suggests that BB1/BCT2binding to the wild-type GGCCAAT motif represses thepromoter whereas CBP/BCT1 binding activates the pro-moter.Mutagenesis of distal promoter elements results in up-

regulation of expression in transient transfection assays. Todetermine the functional significance of the BNF1, BB1, andtwo GATA-1 protein binding sites, we used site-directed

- 255 bp

- 245 bp

- 220 bp

- 210 bp

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4328 MACLEOD AND PLUMB

Comrnpet t or

U-- z

- L-L COm z u a-

- Co m < m

W WWWBBt

I

BB1pro be

Compet i tor

--Z v U GL

_ C m

+ c

--

Wu ~wi - BB1

BBIprobe

N

IJ c

U1

B CT BCTM Probe

0uom

2

-., < m CL e

C

co_ CD m cn m _ : co m CO COmP.

- BCT1- BCT2

FIG. 4. Gel shift analysis of MEL cell nuclear extracts bound toBCT or BCTM probe. 5'-End-labelled BCT or BCTM probe (100 pg)was incubated with uninduced MEL cell crude nuclear extract in thepresence or absence of cold competitor (Comp.) oligonucleotide(100 ng) as shown. The BCT1 and BCT2 complexes are shown.Sequences of the oligonucleotides used are shown in Fig. 1B.

BCT1

- 8CTl -BCT2

BCT

OCT

propbe

FIG. 3. Gel shift analysis of nuclear extracts using double-stranded oligonucleotide probes. 5'-End-labelled BB1 (A) or BCT(C) probe (100 pg) was incubated with uninduced MEL nuclearextract in the presence or absence (-) of competitor oligonucleotide(100 ng) as shown. BB1 (B) or BCT (D) probe was incubated withnuclear extracts from uninduced (MEL-) or HMBA-induced(MEL') MEL cells or from brain, liver, J774.2, or F9 cells in theabsence of competitor. The specific shifted nucleoprotein com-plexes (BB1, BCT1, and BCT2) are shown.

mutagenesis to introduce 3- to 4-bp substitutions in theprotein binding sites (p,NF1M, p3BB1M, p,BG215M, andpPG60M, respectively), using the primer sequences shownin Fig. 1B. Footprint (Fig. 2B) and gel shift (data not shown)analyses confirmed that the sequences no longer bind proteinin vitro. The CACC motif was also mutated (pj3CCM) as a

control (32). The ability of the wild-type and mutant promot-ers to drive the hGH reporter gene was then determined intransiently transfected MEL cells. The levels of hGH pro-duced were determined, standardized for transfection effi-ciency, and expressed relative to that obtained with the hGHplasmid driven by the herpes simplex virus thymidine kinasepromoter (pTKGH).Compared with the wild-type promoter (p,WT), the mu-

tant promoters fall into two classes (Fig. 5). The proximalGATA-1 (pPG60M) and CACC (p,CCM) promoter mutantsare comparatively inactive, confirming and extending previ-ous results (6, 11, 32, 44). In contrast, the distal NF1(pPNFlM), GATA-1 (pPi215M), and BB1 (pPBBlM) pro-moter mutants are four- to eightfold more active than ppWT.This finding indicates that the distal sequences are acting as

a negative regulatory element modulating the function of the

proximal promoter. The proximal promoter sites (CCAAT,CACC, PDRE, TATA, and GATA-1), on the other hand,appear to be essential for promoter function.

Mutagenesis of the distal promoter element results in re-

duced inducibility during differentiation. To determine theeffects of the mutations on activity of the promoter duringterminal differentiation, stable MEL cell transfectants weregenerated. Southern blot analysis showed that pools ofstable transfectants (greater than 100 colonies) containedequivalent numbers of copies of integrated plasmid (-50copies per cell; data not shown). Similarly, the relative levelsof hGH gene expression driven by the various promoterconstructs in the stable transfectants were comparable tothose observed in the transient transfection studies (Fig. 5).Comparison of the relative hGH gene expression in stable

transfectants before or after HMBA-induced terminal differ-entiation is shown in Fig. 6. While HMBA-induced differen-tiation resulted in a 14-fold increase in hGH gene expressionfrom the wild-type promoter construct (pPWT), a reducedbut significant 4- to 6-fold increase was observed with thedistal promoter mutants (p,NF1M, pP215M, and pPBB1M),whereas the pTKGH control gave only a 2.2-fold increase.The two proximal promoter mutants (pPCCM and p,60M)remained inactive during differentiation. Total cellular RNAprepared from the MEL cell stable transfectants containingthe p,WT, p,NF1M, p,215M, and pPBBlM constructs wasthen analyzed by the S1 nuclease protection assay. A515-base probe, which is complementary to the ppWTsequences between position -346 bp of the mouse p-major-globin gene promoter and position + 145 bp of the hGH gene,was hybridized to total cellular RNA and assayed. As shownin Fig. 7, two major bands were detected. The 515-base bandcorresponds to the uncleaved probe, and the 169-base frag-ment corresponds to initiation at the mouse f3-major-globingene promoter cap site (145 bases of hGH gene sequencesand 24 bases of mouse ,-major-globin gene sequences),which was not observed when the probe was hybridized toyeast tRNA (data not shown). Furthermore, quantitation ofthe S1 nuclease 169-base digestion products indicates thatcorrect initiation in the various stable transfectants beforeand after the induction of terminal differentiation correlateswith the levels of hGH gene expression assayed in the same

cells (Fig. 6 and 7).The repressive effect of the wild-type distal promoter

A

C.

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p-MAJOR-GLOBIN GENE TRANSCRIPTION 4329

Relative Growh Hormone ctivity

-346 -250 -200 -150 -100 -50

DHS

(W 1(AA) (~~T~ATA

(w~)CAA)~~T~ATA

C"IATA~) &TATA)TTA

("I CT~) (a) 6~6T TT

1 2 3 4

Pon

pBNF1M

pBG215

p$BlIM

pBCCM

pOG60M

HSV-TK

7

D uninduced stable (n=5)

tranient (n=8)

h

-'Vt

FIG. 5. Ability of the wild-type (ppWT) and protein binding site mutant (pPNF1M, pPG2155M, pPBB1M, poCCM, and pPG60M) mouse3-major-globin gene promoters to drive hGH gene expression, assayed in transient or stable transfections in MEL cells. hGH gene expression

in the transient experiments is corrected for transfection efficiency; values were subtracted from those obtained with the promoterless pOGHplasmid and expressed relative to that obtained with pTKGH. Values are the means + standard errors of at least five experiments. In stabletransfectants, hGH gene expression was estimated over a 24-h period and expressed relative to the value for pTKGH.

element is therefore relieved during differentiation, and thiseffect correlates with the observed decrease in BB1/BCT2binding activity. Furthermore, the four- to sixfold up-regu-lation of gene expression observed with the distal promotermutants during differentiation also correlates with a decreasein BB1/BCT2 binding, suggesting that a decrease in BB1/BCT2 binding to the GGCCAAT motif and a correspondingincrease in CBP/BCT1 binding during differentiation activatepromoter activity.

DISCUSSION

In this study, we have characterized a negative regulatoryelement in the distal half (-100 to -250 bp) of the mouseP-major-globin gene promoter. The negative regulatory ele-ment represses transcription in MEL cells and is dere-pressed during terminal erythroid differentiation. Mutagen-esis experiments indicate that at least three in vitro proteinbinding sites in this sequence (NF1, GATA-1, and BB1) arenecessary for repression. As BB1 DNA binding decreasesduring differentiation and levels ofNF1 and GATA-1 bindingdo not change significantly, the BB1 binding factor appearsto play a central role in modulating P-major-globin genetranscription during terminal differentiation.

Protein binding studies confirm that the BB1 motif(AAGCCAGT) is bound by a nuclear factor which is relatedto the CBPs (12) since it also binds the mouse P-major-globingene GGCCAAT motif. However, the mouse ,-major globingene GGCCAAT sequence is also bound by a factor (BCT1)which is related to the mouse a-globin gene promoterAGCCAAT-binding factor, which binds only CBP/BCT1 (9,38) and whose abundance does not alter significantly duringterminal erythroid differentiation (37a). Mutagenesis of themouse P-major-globin gene GGCCAAT motif to either AGCCAAT or GACCAAT results in the up-regulation of pro-moter activity (32). Furthermore, a truncated mouse n-ma-jor-globin gene promoter (-106 to +26 bp) which lacks thedistal repressor element but contains the GGCCAAT,CACC, PDRE, and TATA motifs responds to terminalerythroid differentiation (11). This finding suggests that theBBl/BCT2-binding factor may have a dual role in repressingmouse a-major globin gene promoter activity in uninducedMEL cells: (i) together with GATA-1 and NF1 in the distalpromoter region, it is required for repressor activity; and (ii)in the proximal promoter, it competes with CBP/BCT1 forbinding to the GGCCAAT motif, thus reducing promoteractivity. We therefore suggest that during terminal differen-tiation, as BB1/BCT2 binding activity decreases, the repres-

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-346 -250 -200 -150 -100 -50

DHS

~~ (~~~~ ~~j~ATTATAT

6TwI i;) AwAT

(GATAI)CBB I (CTAXTAT~A

Relative Growth Hormone Activity (pHSY-TK=2)

5 10 15 20 25 30

paeT

pBNFlY

pBG215M

pBBlM

pSCCM

pBG601

HSV-TK

(1=14)

(1=4.5)

(1=5.9)

.Th(1=4.3)

F'

D uninduced (n=5)

* induced (o=5)

FIG. 6. hGH activity assayed in stable transfectants containing the ,-major-globin gene promoter/hGH constructs. The cells wereincubated in the presence (solid bars) or absence (open bars) of HMBA for 5 days. The fold increase (I values) of hGH gene activity due toMEL cell differentiation was estimated relative to that in uninduced cells (also see Fig. 4) and expressed relative to the value for pTKGHuninduced transfectants.

-4

FIG. 7. Si nuclease analysis of MEL cell stable transfectantRNA. The 515-base probe was hybridized to total cellular RNAprepared from poWT (lanes 1 and 2), pPG215M (lanes 3 and 4),p,BlM (lanes 5 and 6), and p,NFlM (lanes 7 and 8) stable MEL celltransfectants before (lane 1, 3, 5, and 7) or after (lanes 2, 4, 6, and8) the induction of terminal differentiation. Hybrids were digestedwith S1 nuclease and resolved by denaturing gel electrophoresis (seeMaterials and Methods). Lane M, molecular weight markers ([y_32p]ATP-labelled HaeIII-digested 4X174 DNA); lane p, undigestedprobe.

sor element is deactivated, CBP/BCT1 binds to the GGCCAAT motif, and transcription is activated. A CCAATdisplacement protein which binds the human -y-globinCCAAT motif has been identified; it binds a sequence thathas been implicated in the Greek form of hereditary persis-tence of fetal hemoglobin (45).

Repression of the chicken ,B-adult-globin gene promoteralso involves the competition for protein binding to aCACCC motif (CON) which overlaps a palindromic proteinbinding sequence (PAL). PAL is a potent repressor ofpromoter activity, whereas the highest levels of the activatorCON are observed in actively transcribing erythrocytes (14,23). Similarly, a transcription factor (NF-E4) which ispresent only in mature definitive chicken erythroid cells andwhich binds sites in the chicken 3-adult-globin gene pro-moter and 3' enhancer has been identified (18), indicatingthat activator and repressor transcription factors are modu-lated during chicken erythroid differentiation. A silencerelement has been described in the distal promoter sequences(-177 to -392 bp) of the human e-globin gene (5), but its roleduring erythroid differentiation has yet to be determined.

Globin gene enhancer and LCR sequences evidently mod-ulate promoter activity during erythroid differentiation anddevelopment (7, 15, 27, 28, 41) and interact with transcrip-tion factors, some of which either are tissue specific, such as

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V-MAJOR-GLOBIN GENE TRANSCRIPTION 4331

GATA-1 (17, 27, 36, 38, 47, 49, 51), or are modulated duringdifferentiation (14, 18, 23). Promoter competition for enhanc-er/LCR sequences during development has been reportedbetween members of the human or chicken 13-globin multi-gene families (2, 3, 8, 16, 29, 48). It has been suggested thatthe interactions between nucleoprotein complexes at thepromoter and enhancer/LCR regulatory elements are medi-ated by specific protein-protein interactions which loop outintervening sequences (3, 8, 18, 20) and which ultimatelycontrol globin gene expression.The mouse ,-major-globin gene promoter is only one of

many cis-regulatory elements involved in transcriptionalregulation of this gene, and we cannot exclude the possibilitythat both activation and repression regulatory mechanismsare involved. An enhancer element has been identified 3' tothe mouse ,B-major-globin gene, and there is evidence thatthe distal promoter repressor element is required for maxi-mal enhancer function during terminal erythroid differentia-tion (29a). The cis-regulatory elements involved in regulationof the mouse ,B-globin genes are currently being defined,characterized, and used to reconstitute a regulatory systemwhich will increasingly resemble that operating in vivo.

ACKNOWLEDGMENTSThis work was supported by the Cancer Research Campaign.

K.M. was supported by a Medical Research Council researchstudentship.We thank P. Harrison and B. Ozanne for help in preparing the

manuscript.REFERENCES

1. Antoniou, M., E. deBoer, G. Habets, and F. Grosveld. 1988. Thehuman 3-globin gene contains multiple regulatory regions: iden-tification of one promoter and two downstream enhancers.EMBO J. 7:377-384.

2. Antoniou, M. and F. Grosveld. 1990. ,B-Globin dominant controlregion interacts differently with distal and proximal promoterelements. Genes Dev. 4:1007-1013.

3. Behringer, R. R., T. M. Ryan, R. D. Palmiter, R. L. Brinster,and T. M. Townes. 1990. Human y- to ,B-globin gene switching intransgenic mice. Genes Dev. 4:380-389.

4. Benezra, R., C. R. Cantor, and R. Axel. 1986. Nucleosomes arephased along the mouse P-major globin gene in erythroid andnonerythroid cells. Cell 44:697-704.

5. Cao, S. X., P. D. Gutman, P. G. Dave, and A. N. Schechter.1989. Identification of a transcriptional silencer in the 5'-flankingregion of the human e-globin gene. Proc. Natl. Acad. Sci. USA86:5306-5309.

6. Charnay, P., P. Mellon, and T. Maniatis. 1985. Linker scanningmutagenesis of the 5' flanking region of the mouse 13-majorglobin gene: sequence requirements for transcription in eryth-roid and nonerythroid cells. Mol. Cell. Biol. 5:1498-1511.

7. Choi, 0. R., and J. D. Engel. 1986. A 3' enhancer is required fortemporal and tissue-specific transcriptional activation of thechicken adult 1-globin gene. Nature (London) 323:731-734.

8. Choi, 0. R. B., and J. D. Engel. 1988. Developmental regulationof 13-globin gene switching. Cell 55:17-26.

9. Cohen, R. B., M. Sheffery, and C. G. Kim. 1986. Partialpurification of a nuclear protein that binds to the CCAAT box ofthe mouse al-globin gene. Mol. Cell. Biol. 6:821-832.

10. Collis, P., M. Antoniou, and M. Grosveld. 1990. Definition of theminimal requirements within the human 1-globin gene and thedominant control region for high level expression. EMBO J.9:233-240.

11. Cowie, A., and R. M. Myers. 1988. DNA sequences involved intranscriptional regulation of the mouse 1-globin promoter inmurine erythroleukemia cells. Mol. Cell. Biol. 8:3122-3128.

12. deBoer, E., M. Antoniou, V. Mignotte, L. Wall, and F. Grosveld.1988. The human ,B-globin promoter; nuclear protein factors anderythroid specific induction of transcription. EMBO J. 7:4203-4212.

13. deGroot, R. P., J. Schoorlemmer, S. T. vanGensen, and W.Kruijer. 1990. Differential expression of jun and fos genesduring differentiation of mouse F9 embryonal carcinoma cells.Nucleic Acids Res. 18:3195-3202.

14. Emerson, B. M., J. Nickol, and T. C. Fong. 1989. Erythroid-specific activation and derepression of the chick ,B-globin pro-moter in vitro. Cell 57:1189-1200.

15. Emerson, B. M., J. Nickol, P. D. Jackson, and G. Felsenfeld.1987. Analysis of the tissue-specific enhancer at the 3' end of thechicken adult 1-globin gene. Proc. Natl. Acad. Sci. USA84:4786-4790.

16. Enver, T., N. Raich, A. J. Ebens, T. Papayannopoulou, F.Costantini, and G. Stamatoyannopoulos. 1990. Developmentalregulation of fetal-to-adult globin gene switching in transgenicmice. Nature (London) 344:309-313.

17. Evans, T., and G. Felsenfeld. 1989. The erythroid-specific tran-scription factor Eryfl: a new zinc finger protein. Cell 58:877-885.

18. Gallarda, J. L., K. P. Foley, Z. Yang, and J. D. Engel. 1989. The1-globin stage selector element factor is erythroid-specific pro-moter/enhancer binding protein NF-E4. Genes Dev. 3:1845-1859.

19. Graham, F. L., and A. J. van der Eb. 1973. A new technique forthe assay of infectivity of human adenovirus 5 DNA. Virology52:456-467.

20. Grosveld, F., G. B. Van Assendelft, D. R. Greaves, and G.Kollias. 1987. Position independent, high-level expression of thehuman 13-globin gene in transgenic mice. Cell 51:975-985.

21. Hall, C. V., P. Ellen Jacob, G. M. Ringold, and F. Lee. 1983.Expression and regulation of Escherichia coli lac gene fusions inmammalian cells. J. Mol. Appl. Genet. 2:101-109.

22. Higuchi, R., B. Krummel, and R. K. Saiki. 1988. A generalmethod of in vitro preparation and specific mutagenesis ofDNAfragments: study of protein and DNA interactions. NucleicAcids Res. 16:7357-7373.

23. Jackson, P. D., T. Evans, J. Nickol, and G. Felsenfeld. 1989.Developmental modulation of protein binding to 1-globin generegulatory sites within chicken erythrocyte nuclei. Genes Dev.3:1860-1873.

24. Jackson, P. D., and G. Felsenfeld. 1985. A method for mappingintranuclear protein-DNA interactions and its application to anuclease hypersensitive site. Proc. Natl. Acad. Sci. USA 82:2296-2300.

25. Jones, K. A., J. T. Kadonaga, P. J. Rosenfeld, T. J. Kelly, and R.Tjian. 1987. A cellular DNA binding protein that activateseukaryotic transcription and DNA replication. Cell 48:79-89.

26. Kemper, B., P. D. Jackson, and G. Felsenfeld. 1987. Protein-binding sites within the 5' DNase I-hypersensitive region of thechicken aD_globin gene. Mol. Cell. Biol. 7:2059-2069.

27. Knezetic, J. A., and G. Felsenfeld. 1989. Identification andcharacterization of a chicken a-globin enhancer. Mol. Cell.Biol. 9:893-901.

28. Kollias, G., J. Hurst, E. deBoer, and F. Grosveld. 1987. A tissueand developmental specific enhancer is located downstreamfrom the human 1-globin gene. Nucleic Acids Res. 15:5739-5747.

29. Kollias, G., N. Wrighton, J. Hurst, and F. Grosveld. 1986.Regulated expression of human A-y- 1-, and hybrid -y1-globingenes in transgenic mice: manipulation of the developmentalexpression patterns. Cell 46:89-94.

29a.Macleod, K., and M. Plumb. Unpublished data.30. Marsh, J. L., M. Erfle, and E. J. Wykes. 1984. The pIC plasmid

and phage vectors with versatile cloning sites for recombinantselection by insertional inactivation. Gene 32:481-485.

31. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeledDNA with base-specific chemical cleavage. Methods Enzymol.65:499-560.

32. Myers, R. M., K. Tilly, and T. Maniatis. 1986. Fine structuregenetic analysis of a 1-globin promoter. Science 232:613-618.

33. Nagata, K., R. A. Guggenheimer, and J. Hurwitz. 1989. Specificbinding of a cellular DNA replication protein to the origin ofreplication of adenovirus DNA. Proc. Natl. Acad. Sci. USA80:6177-6181.

VOL . 1 l, 1991

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.65.

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34. Ney, P. A., B. P. Sorrentino, K. T. McDonagh, and A. W.Nienhuis. 1990. Tandem AP-1 binding sites within the humanP-globin dominant control region function as an inducible en-hancer in erythroid cells. Genes Dev. 4:993-1006.

35. Nickol, J. M., and G. Felsenfeld. 1988. Bidirectional control ofthe chicken 1- and e-globin genes by a shared enhancer. Proc.Natl. Acad. Sci. USA 85:2548-2552.

36. Perkins, N. D., R. H. Nicolas, M. A. Plumb, and G. H. Goodwin.1989. The purification of an erythroid protein which binds toenhancer and promoter elements of hemoglobin genes. NucleicAcids Res. 17:1299-1314.

37. Philipsen, S., D. Talbot, P. Fraser, and F. Grosveld. 1990. The1-globin dominant control region: hypersensitive site 2. EMBOJ. 9:2159-2167.

37a.Plumb, M. Unpublished data.38. Plumb, M., J. Frampton, H. Wainwright, M. Walker, K. Ma-

cleod, G. H. Goodwin, and P. R. Harrison. 1989. GATAAG; acis-control region binding an erythroid-specific nuclear factorwith a role in globin and non-globin gene expression. NucleicAcids Res. 17:73-92.

39. Plumb, M., V. V. Lobanenkov, R. H. Nicolas, C. A. Wright, S.Zavou, and G. H. Goodwin. 1986. Characterisation of chickenerythroid nuclear proteins which bind to the nuclease hypersen-sitive regions upstream of the PA_ and 3H-globin genes. NucleicAcids Res. 14:7675-7693.

40. Ralph, P., and I. Nakoinz. 1975. Direct effects of immunopoten-tiators on monocytic, myelomonocytic, and histiocylic or mac-rophage tumour cells in culture. Cancer Res. 37:546-550.

41. Reitman, M., and G. Felsenfeld. 1988. Mutational analysis of thechicken ,B-globin enhancer reveals two positive-acting domains.Proc. NatI. Acad. Sci. USA 85:6267-6271.

42. Selden, R. F., K. B. Howie, M. E. Rowe, H. M. Goodman, andD. D. Moore. 1986. Human growth hormone as a reporter gene

in regulation studies employing transient gene expression. Mol.Cell. Biol. 6:3173-3179.

43. Spandidos, D., and N. Wilkie. 1984. Malignant transformation ofearly passage rodent cells by a single mutated oncogene. Nature(London) 310:469-474.

44. Stuve, L. L., and R. M. Myers. 1990. A directly repeatedsequence in the 1-globin promoter regulates transcription inmurine erythroleukemia cells. Mol. Cell. Biol. 10:972-981.

45. Superti-Furga, G., A. Barberis, G. Schaffner, and M. Busslinger.1988. The -117 mutation in Greek HPFH affects binding ofthree nuclear factors to the CCAAT region of the -y-globin gene.EMBO J. 7:3099-3107.

46. Talbot, D., P. Collis, M. Antoniou, M. Vidal, F. Grosveld, andD. R. Greaves. 1989. A dominant control region from the human,B-globin locus conferring integration site-independent geneexpression. Nature (London) 338:352-355.

47. Talbot, D., S. Philipsen, P. Fraser, and F. Grosveld. 1990.Detailed analysis of the site 3 region of the human f-globindominant control region. EMBO J. 9:2169-2177.

48. Trudel, M., and F. Costantini. 1987. A 3' enhancer contributesto the stage-specific expression of the human 13-globin gene.Genes Dev. 1:954-961.

49. Tsai, S. F., D. I. K. Martin, L. I. Zon, A. D. D'Andrea, G.Wong, and S. H. Orkin. 1989. Cloning of cDNA for the majorDNA-binding protein of the erythroid lineage through expres-sion in mammalian cells. Nature (London) 339:446-451.

50. Van Assendelft, G., 0. Hanscombe, F. Grosveld, and D. R.Greaves. 1989. The f-globin dominant control region activateshomologous and heterologous promoters in a tissue-specificmanner. Cell 56:969-977.

51. Wall, L., E. deBoer, and F. Grosveld. 1988. The human ,B-globingene 3' enhancer contains multiple binding sites for an eryth-roid-specific protein. Genes Dev. 2:1089-1100.

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