Proc. Nad. Acad. Sci. USAVol. 86, pp. 2554-2558, April 1989Biochemistry

An erythroid-specific, developmental-stage-independent enhancerfar upstream of the human ",f-like globin" genes

(chloramnphenicol acetyltransferase assays/K562 cells/MEL cells)

DOROTHY Y. H. TUAN*, WILLIAM B. SOLOMON*t, IRVING M. LONDON*t, AND DAVID P. LEEt§

*Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology and tDepartment of Biology, Massachusetts Institute ofTechnology, Cambridge MA 02139

Contributed by Irving M. London, December 16, 1988

ABSTRACT We have identified an erythroid-specific en-hancer element far upstream of the human "l,-like globin"genes, at 10.2-11.0 kilobases 5' ofthe embryonic e-globin gene,and thus at 53-54 kilobases 5' of the adult .8-globin gene. It iscapable of enhancing the expression of a cis-linked test gene byup to 300-fold. This enhancer element is apparently develop-mental-stage-independent, as it is functional at the embryonicand the adult developmental stages in erythroid cells that areexpressing the respective f-like globin genes. The enhancer andglobin promoter sequences work in synergy and are capable ofconferring on 'a cis-linked gene the high transcriptional effi-ciency (enhancer function), erythroid specificity (enhancer andpromoter functions), and developmental-stage specificity (pro-moter function) that are characteristic of the in vivo transcrip-tion of the fl-like globin genes in erythroid cells.

DNA-mediated gene transfer experiments have shown thatthe human ,B-globin gene with its immediate 5' and 3' flankingsequences can be expressed in a tissue-specific and devel-opmental-stage-specific manner (1-5). The transcriptionalefficiency of such introduced f-globin genes is, however,generally low. Efficient transcription may require anotherlevel of cis control by sequence elements that reside outsideof the globin structural genes and their immediate flankingsequences.Using DNase I-hypersensitivity mapping to identify the cis

control elements situated both near and far from the human"p-like globin" gene cluster (5' e Gy-Ay-p 3'), we have foundminor DNase I-hypersensitive sites located in the promoterregions near actively transcribed globin genes and majorDNase I-hypersensitive sites located very far upstream anddownstream ofthe ,-like globin gene domain (6, 7). The 5' and3' major hypersensitive sites appear to be erythroid specificand developmentally stable; i.e., they remain hypersensitivethroughout various stages of erythroid cell development. Themore proximal minor hypersensitive sites near the 5' end ofglobin genes appear only at specific developmental stages. Theabove characteristics of the major and minor sites have led us(7), as well as others (8), to postulate that transcriptionalactivation of the human P-like globin genes may consist of atleast two separate but synergistic activation steps mediated bydistant cis regulatory sequences (major sites) and sequencesmuch closer to the globin genes (minor sites).The functional characteristics of the far upstream regula-

tory sequences (i.e., tissue specificity and stimulation ofgeneexpression over long distances) are the properties of tran-scriptional enhancer sequences (9-11). We report here that a0.8-kilobase (kb) DNA fragment directly underlying one ofthe far upstream major DNase I-hypersensitive sites, at-10.2 to -11.0 kb 5' of the embryonic e-globin gene andtherefore between -53 and -54 kb 5' of the adult p-globin

gene, possesses enhancer activity. When coupled to eitherthe viral simian virus 40 (SV40) promoter or to the e-globinpromoter, it stimulates the expression of a linked chloram-phenicol acetyltransferase (CAT) gene (12) by up to 300-fold.Coupled to the globin promoter, this enhancer sequenceshows strict erythroid specificity. Furthermore, the enhancerelement appears to be functional in erythroid cells at embry-onic and adult developmental stages, a finding consistentwith the developmental-stage stability of its DNase I hyper-sensitivity and its proposed crucial role in globin geneactivation throughout erythroid development.

MATERIALS AND METHODSConstruction of the Recombinant CAT Plasmids. CAT plas-

mids containing the SV40 promoter were obtained by addingBamHI linkers to the 1.9-kb HindIII fragment containingDNase I hypersensitive site II (HSII) and splicing the fragmentor its subfragments derived by Bgl II cleavage (Fig. lA) intothe Bgl II site 5' of the CAT gene in pAlOCAT2 (12). The1.9-kb HindIII fragment was excised from a 5'E II phage clone(13). The enhancerless pep plasmid was constructed by splic-ing a 200-base-pair DNA fragment containing the E-globinpromoter bracketed by aBamHI site and a Pvu II site (14) intopAlOCAT2 plasmid, from which the SV40 early promoter hadbeen deleted by Bgl II and Stu I double digestions. pep HSIIor pep HSII was constructed by triple ligations of the BamHI-linkered 1.9-kb enhancer fragment, the 200-base-pair BamHI-Pvu II E-globin promoter fragment, and pAlOCAT2 fromwhich the SV40 promoter had been deleted as above by Bgl II-Stu I digestions. pep HSII (0.8) was obtained by removing the1.1-kb 3' subfragment from the 1.9-kb enhancer fragment inpep HSII with complete Bgl II and partial BamHI digestionand recircularization of the remaining large fragment. Inoculaof pSV2CAT and pAlOCAT2 (12) were obtained from N.Rosenthal (Children's Hospital, Boston, MA).

Cell Cultures. Adherent K562 cells (M. Fordis, NationalInstitutes of Health) were maintained in RPMI 1640 mediumsupplemented with 10% (vol/vol) fetal calf serum (FlowLaboratories or HyClone) and 20 ,M hemin (Calbiochem).HeLa S-3 cells (from the Massachusetts Institute of Tech-nology cell culture center) and mouse 3T3 F442A cells (15)(J. J. Chen, Massachusetts Institute of Technology) weremaintained in Dulbecco's modified Eagle's medium plus 10%calf serum. THP-1 cells (16) (M. Fenton, Boston University)were maintained in RPMI 1640 and 10% fetal calf serum.MEL cells (J. Banerji and M. Baron, Harvard University)

Abbreviations: SV40, simian virus 40; CAT, chloramphenicol ace-tyltransferase; HSI, -II, -III, and -IV, DNase I hypersensitive sitesI, II, III, and IV.tPresent address: Department of Medicine and Morse Institute ofMolecular Biology, State University of New York Health ScienceCenter Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203.§Present address: University of Minnesota Medical School, Minne-apolis, MN 55455.

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were maintained in Dulbecco's modified Eagle's mediumplus 10% fetal calf serum.

Transfections. Adherent K562, HeLa, MEL, and mouse 3T3cells were transfected by the calcium phosphate precipitationmethod (17). The suspension THP-1 cells were transfected byeither the DEAE-dextran method (18) or electroporation (19).Recombinant plasmid DNA (10-20 pg), which produces linearresponses in CAT enzymatic assays, was used to transfectequal numbers of host cells by either the calcium phosphateprecipitation or the DEAE-dextran method. The cells wereglycerol shocked 4-20 hr after transfection. Forty-eight hoursafter the glycerol shock, the cells were harvested, and extractsfrom 2 x 106 HeLa or 3T3 cells or 2 x 107 of other cells wereprepared (12). Since HeLa and 3T3 cells take up DNA morereadily than other cells, 10 times fewer of these cells were usedfor extracts. Thirty micrograms of recombinant plasmid DNAwas electroporated into THP-1 cells suspended in RPMI 1640without fetal calf serum by electric pulses at 300 V in a BRLCell-Porator apparatus.CAT Assays. Cell extracts obtained from transfected cells

were first heated at 650C for 10 min. This step is required forK562 and MEL extracts to avoid the decrease in CATenzymatic activity that we have observed (20) and thatprobably results from degradation of the CAT enzyme bytemperature-sensitive proteases, which may be relativelyabundant in these cells. The CAT assays were then carriedout as described (20).

RESULTSEnhancer Function Is Located in a 0.8-kb DNA Fragment

Directly Underlying HSII at 10.2-11.0 kb 5' of the E-GlobinGene. We have previously shown by transient CAT assaysthat in 20 kb ofDNA contiguous to the 5' end of the E-globingene (see Fig. 1A) only HSII at -10 to -11 kb, spanned by

A1 kb

HS1V HSX[ HSIIH IH H H HBg H

3.3 1.9 1.0 2.3 1.9

0.8 1.1

BpA1OCAT2

Bg Ba____1

pSV2CAT 1f~nT B

HSIIpA HSII _., fa

1.9 HSIIpA HS]I Ba

1.9

pA HSII (0.8) l; Ba0.8

pA HSII (0.8) I0.8

-

~ ~ BapA HSII (1.1) i lCAT I1.1

a 1.9-kb HindIII DNA fragment, possesses significant en-hancer function, whereas DNA fragments spanning HSI,HSIII, and HSIV as well as the neighboring DNA fragments,which do not span any DNase I-hypersensitive sites, do notexhibit such enhancer function (ref. 20; D. Oh and D.Y.H.T.,unpublished observations). In the 1.9-kb DNA fragmentexhibiting enhancer activity, HSII is located at the 5' end ofthe fragment (Fig. 1A). By subdividing the 1.9-kb fragment,we show here that the enhancer function does not reside inthe 3' subfragment, which spans no detectable hypersensitivesite, and that it resides in the 0.8-kb 5' subfragment thatdirectly underlies HSII. The recombinant CAT constructscontaining the enhancer fragments linked in cis to the CATgene and the SV40 promoter are shown in Fig. 1B. They weretransfected into K562 cells, which possess properties char-acteristic of human erythroid cells (21).

In Table 1, the relative CAT enzymatic activities arepresented. The CAT plasmids pA HSII (1.1) or pA HSII (1.1)containing the 1.1-kb 3' subfragment, which does not spanHSII, and the enhancerless reference plasmid pA1OCAT2 donot noticeably enhance the transcription of the CAT gene.Both the 1.9-kb and the truncated 0.8-kb DNA fragments, ingenomic orientation, in pA HSII and pA HSII (0.8), respec-tively, enhance CAT activity by -30-fold. The same frag-ments in reverse genomic orientation also enhance thetranscription of the CAT gene but at a level 2-3 times greaterthan that with the fragments in genomic orientation. Thisorientation effect suggests the possibility that one aspect ofenhancer function in vivo may also involve interaction of theenhancer with more upstream elements.The above functional assays thus define enhancer activity

to within a 0.8-kb DNA fragment directly underlying HSIIbetween a Bgl II site at -10.2 kb and a HindIII site at -11.0kb 5' of the cap site (13, 14) of the e-globin gene (Fig. 1A). The

HSII H

3.3

Ba Pv 6HI

Cp,p [.___ Bap(.p :ICAT Ia

HSIIBPEp HS]I 4 a

1.9

P-pH~I BgHSX BapEp HSII .1AIt

1.9

- HSIaHSHp p HSII(0.8) Lam B

0.8

FIG. 1. Partial restriction maps of recombinant CAT plasmids containing the enhancer element in combination with either the SV40 or theE-globin promoter. (A) The partial restriction map of DNA spanning four erythroid-specific major DNase I-hypersensitive sites 5' of the E-globingene. Restriction sites used in plasmid construction are shown. H, HindIll; Pv, Pvu II; Ba, BamHI; Bg, Bgl Il. Numbers below the map denotesizes (in kb) of the various HindIll fragments or subfragments. Vertical arrows denote locations of the erythroid-specific hypersensitive sites (HSI-HSIV). The filled box represents the E-globin gene. (B) Recombinant CAT plasmids containing the SV40 promoter. The hatched box 5' of the CATgene represents the SV40 promoter. The horizontal filled bars 5' ofthe SV40 promoter represent the DNA inserts to be tested for enhancer function.Horizontal arrows denote genomic (- ) or reverse genomic (-) orientation of the inserts. pAiOCAT2, the enhancerless reference plasmid;pSV2CAT, CAT plasmid containing the SV40 enhancer (represented by the unfilled box); pA HSII or pA HSII, the CAT plasmid containing the1.9-kb DNA fragment spanning HSII; pA HSHI (0.8) orpA HSII (0.8), the 0.8-kb DNA subfragment directly underlying HSII inserted in pAlOCAT2;pA HSII (1.1), the 1.1-kb subfragment, which does not span any hypersensitive sites, inserted in either genomic (-) or reverse genomic (-)orientation in pAlOCAT2. (C) Recombinant CAT plasmids containing the e-globin promoter. pep, The enhancerless reference CAT plasmidcontaining the E-globin promoter, which is represented by the dotted box; pep HSII or pEp HSII, the recombinant plasmid containing the 1.9-kbenhancer fragment spliced 5' of the E-globin promoter in pEp; pEp HSII (0.8), the 0.8-kb enhancer fragment spliced in pep.

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Table 1. Relative CAT activities of CAT plasmids driven by the SV40 or the E-globin promoterCAT activity

K562 cells HeLa cells THP-1 cells MEL cells

Copy no. HGH Copy no. HGHCAT normal- normal- normal- normal- 3T3 cells

mRNA* Average izedt Average izedt Average izedt Average izedt Average_pAlOCAT2 1 1 1 1 1 1 1 1 1 1pSV2CAT 8.4 ± 5.4 4.8 160 ± 40§ 110 2.3 ± 1.1§ 4.7 37 ± 9 32 87 ± 4pA HSII 67 ± 22 78 ±33 8.8 ± 5§ 0.4 ± 0.25§ 0.8 14 ± 7 12.8 1.4 ± 0.4pA HSII 26 ± 12 35 0.7 ± 0.3§ 16 ± 11pA HSII(0.8) 56 ± 30 88 ± 45 13 ± 5 15.4 18 ± 8pA HSII (0.8) 15 ± 4 32 ± 10 3 ± 1.5 1.37 13 ± 5pA HSII (1.1) 1.1 ± 0.2 0.7pA HSII (1.1) 1.7 ± 0.3 0.7 0.30 0.4 0.1 0.9 ± 0.05pep 1 1 1

pep HSII 270 ± 70 1 ± 0.1 21pep HSII 245 ± 75 0.9 ± 0.2 0.31pep HSII (0.8) 248 ± 80 0.25 + 0.02¶0 0.7

HGH, Human growth hormone. The relative CAT activity of a test plasmid is defined as the percentage of total input cpm in[14C]chloramphenicol that are converted to the acetylated forms (see spots a and b in Figs. 2 and 3) divided by the percentage of conversionby the enhancerless pAlOCAT2 or pep plasmid whose CAT activity is set equal to 1. Average values of relative CAT activities were determinedfrom at least two separate experiments. 0, Mock transfection.*The numbers are relative amounts of CAT mRNAs obtained from RNA protection assays.tThese CAT values have been further normalized with respect to copy numbers of the transfecting plasmids.MThese CAT values have been normalized with respect to the expression of a cotransfecting plasmid containing the HGH gene.§These values have been reported (20).These CAT values were obtained from plasmids introduced into THP-1 cells by electroporation.

fragment contains sequence features such as inverted re-peats, enhancer core sequences, and alternating purine andpyrimidine bases (7, 13), which are also found in othereukaryotic enhancer elements (22, 23). This 0.8-kb enhancerfragment is bordered on the 5' side by a 2.3-kb HindIllfragment and on the 3' side by the 1.1-kb subfragment of the1.9-kb HindIII fragment (see Fig. 1A), neither of whichshows enhancer activity (20). HSII has been mapped byGrosveld et al. (24) to an alternate position between -11.5and -12 kb 5' of the E-globin gene and thus to a positionwithin the 2.3-kb HindIII fragment; this map position wouldfail to reveal the correspondence between DNase I hyper-sensitivity and enhancer activity.

Erythroid Specificity of the Enhancer Element. To assess theerythroid specificity of the enhancer element, the aboverecombinant CAT plasmids were also transfected into HeLacells. In HeLa cells, the 0.8-kb fragment underlying HSII ingenomic orientation in pA HSII (0 8) and the 1.9-kb fragmentin genomic orientation in pA HSII (Table 1) do not showenhancer activity. The same DNA fragments in reversegenomic orientation, in pA HSII (0.8) and pA HSII (Table 1),however, stimulate CAT activity by approximately 10-foldover the enhancerless pAlOCAT2 plasmid. The cause of thisorientation-dependent activation of the globin enhancer ele-ment in HeLa cells is presently unknown. In a nonerythroid,monocytic leukemia cell line, THP-1, the 1.9-kb DNA frag-ment in reverse genomic orientation in pA HSII does not showenhancer function (Table 1).

Globin promoters have been reported to confer erythroid-specific expression on a linked gene (25). The activity of theenhancer element when it is coupled in reverse orientation tothe heterologous SV40 viral promoter in HeLa cells promptedus to construct another series of recombinant CAT plasmids,in which the enhancer element is coupled to the homologouse-globin promoter (see Fig. 1C)._When transEcted into K562cells, the CAT plasmids pep HSII and pep HSII, containingthe 1.9-kb fragment in either genomic or reverse genomicorientation coupled to the e-globin promoter, activate theCAT gene by up to 300-fold more than does the enhancerless

pep plasmid, which contains only the e-globin promoter 5' ofthe CAT gene (Fig. 2). When transfected into nonerythroidHeLa cells, these same plasmids do not activate the CATgene (Fig. 2 and Table 1). When transfected into the non-erythroid THP-1 cells of human monocytic lineage, theenhancerfragment in genomic orientation in pep HSII orin pep HSII (0.8) again does not show enhancer activity; theenhancer fragment in reverse genomic orientation in pepHSII exhibits very weak enhancer activity of -2-fold (Table

HeLa

K562

b Fir-~~~~~TLC nf ('AT enzUnmatic~activitv.

c

Y (2, ,QIH

r 1%U. Z . I JL%. %1 w-ry I ws,>1& H...a.11 "%L VIY

in K562 and HeLa cells, of recombinant* plasmids containing the e-globin promoter or

the SV40 promoter. Each lane represents theCAT activity of the respective plasmid asmarked. pA, pA1OCAT2; pSV, pSV2CAT.

29 If Acetylated chloramphenicol spots in theAt' chromatograph are marked by a and b, and

the unacetylated chloramphenicol spots area marked by c at the left.

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1), which is <1% of that of the same plasmid in erythroidK562 cells, as shown above. Thus, when the enhancerelement is combined with the homologous globin promoter,its activity and erythroid specificity appear to be increased.To compare the respective enhancer functions of the

various transfecting CAT plasmids, we have calculated therelative CAT enzymatic activities of these plasmids in refer-ence to the enhancerless pAlOCAT2 or pEp plasmids (Table1). To minimize variation in relative CAT values due to thevariability in transfection efficiencies between experiments,we have determined the CAT enzymatic activities fromextracts containing approximately equal numbers of thevarious transfecting plasmids by preparing the extracts forthe assays from aliquots of equal numbers of host cellstransfected by the various CAT plasmids under identicalconditions. We have further normalized the relative CATenzymatic activities with reference to the copy numbers ofthe various transfecting plasmids in the host cells, as deter-mined by the DNA dot-blot hybridization technique, or withreference to amounts of the expression product, humangrowth hormone, of a cotransfecting reference plasmid (26).In Table 1 the values of normalized CAT activities and thevalues of CAT activities determined by equalizing host cellnumbers used for preparing the CAT extracts agree with eachother within the experimental variations of the assay andproduce consistent trends in relative CAT activities.

In addition, the relative amounts of the CAT messages asdetermined by RNA protection assays (27) are also propor-tional to the CAT enzymatic activities of the respective CATplasmids as determined by the CAT assays (Table 1).

Developmental-Stage Independence ofthe Enhancer Element.To determine if the enhancer element functions in a develop-mental-stage independent manner-i.e., if it activates CATtranscription not only in erythroid cells expressing the embry-onic e- and fetal y-globin genes as in K562 cells but also inerythroid cells at the adult stage ofdevelopment expressing theadult f3-globin gene-we have transfected various recombi-nant CAT plasmids into mouse erythroleukemia (MEL) cells.MEL cells express the adult mouse p-globin gene and possessproperties characteristic of adult erythroid cells (28). In MELcells, the 1.9-kb and the 0.8-kb fragmentsijn either reversegengomic or genomic orientation, in pA HSII, pA HSII, pAHSII (0.8), or pA HSII (0.8), respectively, all activate CATgene transcription by 10- to 20-fold; the SV40 enhancerelement in pSV2CAT, by comparison, activates the CAT geneby =40-fold (Fig. 3 and Table 1). Compared to enhanceractivities in K562 cells, the enhancer activity in MEL cellsseems to be reduced. This apparent reduction in enhanceractivity may be due to the possibility that this very farupstream enhancer, at -53 to -54 kb 5' to the adult P-globingene, may require additional cis regulatory elements closer toand within the 3-globin gene (29, 30) for optimal activity in

MEL

a *#.* *ob** 0

* 0

c.00 it Otg~ ~

44+ oef ti b o

q P ""

4 k

FIG. 3. CAT enzymatic activities, in MEL and mouse 3T3 cells,of recombinant CAT plasmids containing the SV40 promoter. Theenzymatic activity was determined by TLC as described in Fig. 2.pA, pAlOCAT2; pSV, pSV2CAT; 0, mock transfection.

adult erythroid cells. Our preliminary results indicate thatwhen the enhancer element is coupled to the adult p-globinpromoter its activity in MEL cells appears to be increased (G.Huang and D.Y.H.T., unpublished results).

In mouse fibroblast cells, the 1.9-kb enhancer fragment inpA HSII does not activate the CAT gene, whereas the SV40enhancer element activates the CAT gene by =80-fold (Fig.3 and Table 1). Erythroid specificity of the human enhancerelement thus appears to be preserved in mouse cells.We have also used normal erythroid cells from the hamster

yolk sac (31) as transfection hosts for CAT plasmids con-taining the 1.9-kb or the 0.8-kb enhancer fragment. Ourpreliminary results (T. Boussios and D.Y.H.T., unpublisheddata) indicate that the human erythroid enhancer, whencoupled to the human embryonic e- or adult f3-globin pro-moter, is also functional in embryonic hamster erythroidcells, which express the embryonic as well as the adulthamster ,-like globin genes (31). A chicken globin enhancerelement has recently been shown also to display develop-mental-stage independence (32).

DISCUSSIONDNA sequences in or close to the globin genes including theglobin promoters have been reported to be sufficient fordetermination of erythroid-specific and developmental-stage-specific gene transcription (25). Yet, as shown in Figs.2 and 3, the transcriptional level of a test (CAT) gene, drivenby either the E-globin promoter or the SV40 promoter in theenhancerless plasmid pEp or pA1OCAT2, is low not only innonerythroid HeLa cells but also in erythroid K562 cells.However, when the E-globin or SV40 promoter is coupledwith the erythroid enhancer, the transcription of the linkedCAT gene is increased by 200- to 300-fold or 30- to 80-fold,respectively (Table 1). Furthermore, the heterologous com-bination of the erythroid enhancer and the SV40 promoter ofbroader cell type specificity also activates the CAT gene inthe nonerythroid HeLa cells, although at a greatly reducedlevel of 30- to 10-fold (Table 1). The above analysis suggestssynergy between the enhancer and the promoter sequence. Inthe absence of the enhancer, the SV40 or e-globin promoteralone is incapable of activating the transcription of a cis-linked gene at a significant level. In the absence of theappropriate (or homologous) promoter sequence (i.e., whenthe enhancer sequence is linked to a heterologous promoter),the transcriptional activation potential and tissue specificityof the enhancer are diminished. In erythroid cells, theapparent synergy between the globin enhancer, which mayrespond to transcriptional activation signals, and the globinpromoter sequences, which respond to developmental-stagespecific signals (33), may thus be capable of activating thetranscription and sequential switching of the respectiveembryonic, fetal, or adult al-like globin gene during variousstages of erythroid development.The apparent developmental-stage independence in eryth-

roid cells of the globin enhancer suggests that in the humangenome the enhancer element may be the primary sequenceinvolved in transcriptional activation not only of the proximalembryonic e-globin gene but also of the adult ,B-globin gene at53 to 54 kb downstream. Evidence in support ofthis suggestionis provided by the molecular defects ofthree reported cases ofy~p-thalassemias. In the Dutch (34, 35), the English (36), andthe Hispanic (37) y8,8-thalassemias, large DNA regions includ-ing the region spanning the enhancer element are deleted. Theapparently normal 3-globin genes in these y8p8-thalassemias(34-38) are flanked by normal DNA 3' ofthe ,8-globin gene and5' of the B3-globin gene for 2 kb (Dutch), 25 kb (English), and50 kb (Hispanic). Yet the in vivo expression of these ,3-globingenes is suppressed. In these cases of thalassemias, theenhancer sequence 3' of the Ay-globin gene and/or the

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enhancer 3' of the 83-globin gene (29, 30, 39) are present, yettheir presence does not seem to be sufficient to offset the effectof the deletion of the far upstream DNA in allowing fullexpression of the B-globin genes. The (commonly) deleted farupstream DNA is located in a region more than 7 kb 5' of theE-globin gene (34-37) and spans the enhancer element under-lying HSII as well as more upstream DNA sequences, which,however, do not exhibit significant enhancer activity (ref 20;D. Oh and D.Y.H.T., unpublished data). The deletion of theenhancer element underlying HSII may thus cause the sup-pression of the p-globin genes in these thalassemias, and itspresence in the normal genome appears to be of pivotalimportance to the transcriptional activation of the far down-stream P-globin gene. How the enhancer element exerts itsregulatory activity over 54 kb of intervening DNA and acrossfour other P-like globin genes is presently unknown andremains to be elucidated.

Further evidence suggesting a dominant role of the globinenhancer in transcriptional regulation of the adult P-globingene is provided by transgenic experiments. In the transgenicmice, a human 8-globin transgene in a transchromosomalminilocus, which is flanked on the 5' side by 22 kb of DNAspanning erythroid-specific major hypersensitive sites I-IV,has recently been shown to be transcribed in mouse erythroidcells at a level equivalent to that of the endogenous mouseP-globin gene (24). In mouse erythroid cells, a single copy ofthe P-globin transgene, stably integrated into the host chro-mosome without the presence of the 22 kb of 5' flankingDNA, is inefficiently transcribed at 0.4-4% of the transcrip-tional efficiency of the endogenous normal P-globin gene (1-5, 24). The 25- to 250-fold increase in transcriptional effi-ciency of the integrated ,8-globin transgene in the transchro-mosomal minilocus may be due to the presence of theerythroid enhancer sequence located at -11 kb 5' of thetransgene, since this enhancer element has been estimated byour transient transfection assays to be capable of transcrip-tionally activating a cis-linked gene by up to 300-fold. As inthe three cases of y8/3-thalassemias, the regulatory sequence3' to the ,3-globin gene (29, 30), which is also present in theminilocus, again does not appear to be the dominant regula-tory element involved in transcriptional activation of the,/-globin transgene (24).

It is possible that the erythroid enhancer element in 0.8 kbof DNA may constitute the sole sequence requirement forefficient transcription of a cis-linked and integrated P3-globintransgene in erythroid cells. The enhancer element may thushold promise for ultimate gene therapy in human geneticdisorders of erythroid cells, such as the thalassemia syn-dromes and sickle cell anemia. When integrated into the hostgenome, the enhancer sequence as well as the P3-globintransgene, however, is probably complexed to chromosomalproteins and may be subject to more structural and functionalregulation than a transiently transfected sequence. For suchan integrated enhancer to be transcriptionally active, thechromosomal structure of the enhancer may first need to bemodified or opened. In contrast, the enhancer sequence intransiently transfected plasmids, as we have studied in thisreport, may be viewed as already existing in an accessiblestructure, since it is presumably not stably integrated into thehost chromosome and is probably free of the chromosomalconstraint of an integrated sequence. One possibility is thatthe enhancer element in 0.8 kb of DNA, when integrated intothe host genome, can assume an open and accessible chro-mosome structure in erythroid cells and can therefore tran-scriptionally activate a cis-linked transgene at a level that wehave observed in the transient assays. The alternative pos-sibility is that one or more sequences, such as those under-lying the other far upstream major hypersensitive sites HSI,HSIII, and HSIV, are required for full transcriptional en-hancement of a stably integrated transgene.

We thank Dr. B. Clark for assistance with electroporation intoTHP-1 cells; Drs. N. Hopkins, L. Gehrke, H. Goodman, and P.Leder for critical review of the manuscript; and L. Cobb and A.Miller for typing the manuscript. D.P.L. was sponsored by theMassachusetts Institute of Technology Undergraduate ResearchOpportunities Program. This work was supported in part by Mas-sachusetts Institute of Technology intramural funds, by grants toI.M.L. from the National Institutes of Health (DK 16272) andJohnson & Johnson, and by a grant to D.Y.H.T. (HL 39948) from theNational Institutes of Health.

1. Chada, K., Magram, J. & Costantini, F. (1986) Nature (London)319, 685-689.

2. Dzierzak, E. A., Papayannopoulou, T. & Mulligan, R. C. (1988)Nature (London) 331, 35-41.

3. Kollias, G., Wrighton, N., Hurst, J. & Grosveld, F. (1986) Cell 46,89-94.

4. Magram, J., Chada, K. & Costantini, F. (1985) Nature (London)315, 338-340.

5. Townes, T. M., Lingrel, J. B., Chen, Y. H., Brinster, R. L. &Palmiter, R. D. (1985) EMBO J. 4, 1715-1723.

6. Tuan, D. & London, I. M. (1984) Proc. Natl. Acad. Sci. USA 81,2718-2722.

7. Tuan, D., Solomon, W. B., Li, Q. L. & London, I. M. (1985) Proc.Natl. Acad. Sci. USA 82, 6384-6388.

8. Forrester, W., Takegawa, S., Papayannopoulou, T., Stamatoyan-nopoulos, G. & Groudine, M. (1987) Nucleic Acids Res. 15, 10159.

9. Banerji, J., Rusoni, S. & Schaffner, W. (1981) Cell 27, 299-308.10. Wang, X. F. & Calame, K. (1985) Cell 43, 659-665.11. Pinkert, C., Ornitz, D., Brinster, R. & Palmiter, R. (1987) Genes

Dev. 1, 268-276.12. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell.

Biol. 2, 1044-1051.13. Li, Q. L., Powers, P. A. & Smithies, 0. (1985) J. Biol. Chem. 260,

14901-14910.14. Baralle, F., Shoulders, C. & Proudfoot, N. (1980) Cell 21, 621-630.15. Green, H. & Kehinde, 0. (1974) Cell 1, 113-119.16. Krakauer, T. & Oppenheim, J. J. (1983) Cell. Immunol. 80, 223-228.17. Wigler, M., Silverstein, S., Lee, L. S., Pellicer, A., Cheng, Y. &

Axel, R. (1977) Cell 16, 777-785.18. Sussman, D. J. & Milman, G. (1984) Mol. Cell. Biol. 4, 1641-1643.19. Potter, H., Weir, L. & Leder, P. (1984) Proc. Natl. Acad. Sci. USA

81, 7161-7165.20. Tuan, D., Abeliovich, A., Lee-Oldham, M. & Lee, D. (1987) in

Developmental Control of Globin Gene Expression, eds. Stamato-yannopoulos, G. & Nienhuis, A. W. (Liss, New York), pp. 211-220.

21. Benz, E. J., Murnane, M. J., Tonkonow, B. L., Berman, B. W.,Mazur, E. M., Cavallesco, C., Jenko, T., Snyder, E. L., Forget,B. G. & Hoffman, R. (1980) Proc. Natl. Acad. Sci. USA 77, 3509-3513.

22. Gluzman, Y. & Shenk, T., eds. (1983) Enhancer and EukaryoticGene Expression (Cold Spring Harbor Lab., Cold Spring Harbor,NY).

23. Gillies, S. D., Folson, V. & Tonegawa, S. (1984) Nature (London)310, 594-597.

24. Grosveld, F., van Assendelft, G. B., Greaves, D. R. & Kollias, G.(1987) Cell 51, 975-985.

25. Rutherford, T. & Nienhuis, A. W. (1987) Mol. Cell. Biol. 7, 398-402.26. Selden, R. F., Howie, K. B., Rowe, M. E., Goodman, H. M. &

Moore, D. D. (1986) Mol. Cell. Biol. 6, 3173-3182.27. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T. &

Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7056.28. Marks, P. & Rifkind, R. A. (1978) Annu. Rev. Biochem. 47,419-437.29. Trudel, M. & Costantini, F. (1988) Genes Dev. 1, 954-961.30. Kollias, G., Hurst, J., deBoer, E. & Grosveld, F. (1987) Nucleic

Acids Res. 15, 5739-5748.31. Boussios, T., Condon, M. & Bertles, J. (1985) Proc. Natl. Acad.

Sci. USA 82, 2794-2797.32. Nickol, J. M. & Felsenfeld, G. (1988) Proc. Natl. Acad. Sci. USA

85, 2548-2552.33. Emerson, B. M., Lewis, C. D. & Felsenfeld, G. (1985) Cell 41, 21-

30.34. Van der Ploeg, L. H. T., Lonings, A., Oort, M., Roos, D., Bernini,

L. & Flavell, R. A. (1980) Nature (London) 283, 637-641.35. Vanin, E. F., Henthorn, P. S., Kioussis, D., Grosveld, F. &

Smithies, 0. (1983) Cell 35, 701-711.36. Curtin, P. & Kan, Y. W. (1988) Blood 71, 766-770.37. Driscoll, M. C., Dobkin, C. & Alter, B. P. (1987) Blood 70, 74

(abstr.).38. Kioussis, D., Vanin, E., deLange, T., Flavell, R. A. & Grosveld,

F. G. (1983) Nature (London) 306, 662-666.39. Bodine, D. M. & Ley, T. (1987) EMBO J. 6, 2997-3004.

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