Vol. 10, No. 3MOLECULAR AND CELLULAR BIOLOGY, Mar. 1990, p. 1199-12080270-7306/90/031199-10$02.00/0Copyright © 1990, American Society for Microbiology

Correlation between Patterns of DNase I-Hypersensitive Sites andUpstream Promoter Activity of the Human e-Globin Gene at

Different Stages of Erythroid DevelopmentPIERRE BUSHEL, KIM REGO, LAUREL MENDELSOHN, AND MAGGI ALLAN*

Departments of Genetics and Medicine, College of Physicians and Surgeons of Columbia University,630 West 168th Street, New York, New York 10032

Received 17 August 1989/Accepted 28 November 1989

DNA 5' to the human r-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in threehuman erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE)stages of erythroid development. We have mapped 10 r-globin DHS in K562 cells, in which the r-globin geneis maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 basepairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and-1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the r-globin gene is expressed atextremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the-200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major capsite DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site isdetectable at all three stages of erythroid development, including KMOE cells in which r-globin synthesis isundetectable. We suggest that this site may be implicated in maintaining the entire j8-globin cluster in an activechromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters.Thus two distinct types ofDHS exist-promoter positive and promoter negative. In HEL cells, all the upstreampromoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. Thissuggests that the maintenance of DHS and regulation of their associated promoters occur by independentmechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains activerepresents a unique pattern of expression and suggests that HEL cells possess regulatory factors whichspecifically down regulate the e-globin upstream promoters.

The human p-like globin genes extend over approximately60 kilobase pairs (kbp) of chromosome 11. The genes arearranged 5-,E-G'Y_A-y-5.i_33, in the same order as they aredevelopmentally expressed. The E-globin gene is expressedup to 10 weeks gestation and is then silenced for theremainder of the lifetime of the individual. This gene pos-sesses 12 known DNase I-hypersensitive sites (DHS) be-tween the major cap site and -15 kbp upstream of the gene(16, 36, 41). Five of these DHS contain upstream promoterslocated -4.5, -2.2, and -1.48 kbp and -900 and -200 basepairs (bp) from the major cap site (1, 2; Fig. 1). The -2.2-kbppromoter, located within an Alu repetitive element, is tran-scribed by PolIll (12) in the opposite direction from theE-globin gene, giving rise to 350-bp transcripts which arenonpolyadenylated, nucleus confined, and detectable in vivoonly when the e-globin gene is fully active (5). The remainderof the upstream promoters give rise to transcripts whichextend through the gene and are polyadenylated. In thehuman erythroleukemia cell line K562 or in normal embry-onic red blood cells, 10 to 15% of E-globin transcriptsoriginate from the upstream sites (2). With the exception ofthe Alu PolIII promoter, none of the upstream initiation sitesare associated with any known regulatory motifs and, in-deed, it has been shown that the upstream promoters can beregulated independently of the major promoter in responseto a number of cis- and trans-acting factors (4). Minortranscription initiation sites occur within DHS elements in anumber of genes. Examples are thymidine kinase (27, 31),polyomavirus early gene (10), immunoglobulin R.u transcripts

* Corresponding author.

(28, 30), human -y-globin (18, 22), and a-globin (20). All theabove-mentioned DHS have been shown to regulate theirassociated genes, and we have recently shown by usingtransient expression assays that at least three of the E-globinupstream DHS-promoters are involved in regulating theE-globin gene at different stages of erythroid differentiation(40; M. Allan, G. J. Grindley, P. Bushel, J. Wu, and L.Mendelsohn, submitted for publication; P. Bushel, L. Men-delsohn, and M. Allan, manuscript in preparation). In eachcase, regulation is directly related to transcription from theappropriate upstream promoter. It has previously been sug-gested that low-level transcription initiation from DHS re-sults from the relatively nonspecific recognition by polymer-ase of accessible or open chromatin (3, 39). However, atleast three of the E-globin DHS have no associated initiationsites, and we conclude that at least two different types ofDHS exist, with and without associated promoters. Thesetwo types of DHS may have different mechanisms by whichthey exert their regulatory effect and may indeed be active atdifferent stages of development and differentiation. Forexample, at least one of the promoterless DHS (-6.5 kbpupstream of the E-globin gene) has no effect in a transientexpression assay but may be implicated in maintaining thewhole 3-globin cluster in an active chromatin conformation(19, 37).

In this paper, we have compared E-globin DHS in cell linesrepresentative of embryonic (K562), fetal (HEL), and adult(KMOE) erythroid development. The accessibility of eachDHS is directly compared, both with activity of its associ-ated promoter and with transcription from the e-globin major

1199

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

1200 BUSHEL ET AL.

K562

2 J

-

FIG. 1. Correlation of E-globin upstream initiation sites with DHS in K562 cells. The line drawing shows data summarized from Allan etal. (2), Allan and Paul (5), and Zhu et al. (41). Transcription initiation sites are represented by small closed circles. The distance from the majorcap site is indicated, and the direction of transcription is represented by horizontal arrows. DHS are indicated by vertical arrows, the widthof which represent intensity of subbands. A', Polyadenylated transcripts; A-, nonpolyadenylated transcripts; K, KpnI; Bg, BglII; E, EcoRI;T, TaqI; P, PvuII; C, ClaI. Nuclei (25 x 106) from K562 cells were suspended in 1 ml of RSB buffer (see Materials and Methods) and incubatedfor 5 min at 4°C with increasing units of DNase I (0 to 30 U). After the DNA was extracted and purified, 15 p.g was digested with a threefoldexcess of KpnI and electrophoresed through a 0.7% agarose gel. Fractionated DNA was transferred to nitrocellulose and hybridized overnightat 65°C to an a-32P-labeled 1.17-kbp BgIII-EcoRI probe from the 3' end of the r-globin gene (see line drawing). Filters were washed to a finalstringency of 0.3 x SSC at 65°C and autoradiographed for 3 days at -70°C with intensifying screens. Observed bands are the parent (P) andsubbands representing DHS no. 1 through 9. Distance from the major cap site is indicated in brackets to the right of the autoradiograph. Actualsize of the subbands is shown to the left of the autoradiograph. DNase I concentrations are shown above the appropriate lane of theautoradiograph.

cap site. Our findings for specific DHS-promoters are dis-cussed with regard to their regulatory functions.

MATERIALS AND METHODS

Cell culture. Cell lines were grown in SLM mediumsupplemented with 10% fetal bovine Serum (GIBCO). K562,HEL, and KMOE cell lines were a gift from Tim Rutherford.The K562 clone used in this study expresses approximately200 copies of s-globin mRNA per cell and can be induced byhemin to express 1,500 to 2,000 copies per cell. This cell linehas undetectable levels of ,-globin RNA. The HEL cellclone expresses approximately 20 copies of e-globin RNA,2,000 copies of y-globin, and 100 copies of P-globin per cell.The KMOE clone has undetectable levels of r-globin, 200copies of y-globin, and 100 copies of ,-globin per cell andcan be induced by 1-p-arabinofuranosylcytosine to expressapproximately 400 copies of ,-globin mRNA per cell. Thecells used in this study were analyzed in the absence ofinduction with chemical agents.

S1 mapping. RNA was prepared by a variation of themethod of Chirgwin et al. (8). S1 mapping was carried out bythe method of Berk and Sharp (6) as modified by Weaver andWeissman (38).

The DNA probes were dephosphorylated, labeled at the 5'end by T4 polynucleotide kinase and [-y-32P]ATP (>5,000Ci/mmol), and strand separated as described by Maxam andGilbert (26).

Total RNA was annealed to end-labeled probe DNA for 16h in 10 jil of formamide hybridization buffer (80% formam-ide, 0.4 M sodium chloride, 0.04 M PIPES [piperazine-N,N'-bis(ethanesulfonic acid)] [pH 6.4], 0.001 M EDTA). Allhybridizations were made up to a total of 20 ,ug of nucleicacid by the addition of yeast tRNA.

Hybridization was terminated with 250 RI of ice-coldnuclease assay buffer (0.25 M NaCl, 0.03 M sodium acetate[pH 4.6], 0.001 M ZnSO4, 200 ,ug of calf thymus DNA perml), and incubated with 500, 1,000, 2,500, or 5,000 U of S1nuclease at 37°C. After 1.5 h of incubation, samples wereextracted with phenol, ethanol precipitated, and analyzed on6% polyacrylamide gels.

Primer extension. cDNA was synthesized by reverse tran-scriptase, as described in detail by Ghosh et al. (17). TheDNA primer was labeled at the 5' ends, as described above,the strands were separated, and the anti-mRNA strand washybridized for 16 h at 57°C to RNA in 10 ,ul of 80%formamide hybridization buffer. Reactions were quenched

MOL. CELL. BIOL.

1.9

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

CORRELATION OF r-GLOBIN DHS-PROMOTERS 1201

with a 0.5 volume of 0.3 M sodium acetate, extracted withphenol, and precipitated with ethanol. Samples were sus-pended in 200 ,Rl of 60 mM NaCl-50 mM Tris hydrochloride(pH 8.3)-10 mM dithiothreitol-6 mM sodium acetate-i mMeach of dATP, dCTP, dGTP, and dTTP-25 U of reversetranscriptase and incubated at 41°C for 3 h. After a further1-h incubation in 0.2 N NaOH, the reaction was neutralizedwith 1 N HCl, extracted with phenol, precipitated withethanol, and analyzed on a 6% polyacrylamide gel.DNase I analysis. Harvested cells (2 x 108 to 4 x 108) were

aliquoted for each DNase I concentration point and thenpelleted by centrifugation at 1,000 rpm for 10 min. The cellpellets were washed with phosphate-buffered saline, equili-brated with RSB buffer (10 mM Tris [pH 7.4], 10 mM NaCl,3 mM MgCl2), and then the cellular membranes were lysedwith RSB NP-40 (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mMMgCl2, 0.5% Nonidet P-40) at 4°C for 5 min at a concentra-tion of 12.5 x 106 cells per ml for each step. Nuclei werepelleted by centrifugation at 2,000 rpm for 10 min and thenwashed with RSB. The nuclei pellets were then resuspendedin RSB to a final concentration of 25 x 106 nuclei per ml andhomogenized by passing repeatedly through 18- and 23-gauge needles. Pancreatic DNase I (Boehringer MannheimBiochemicals) was added at increasing unit concentrations,and the mixture was incubated at 4°C for 5 min. Reactionswere terminated, and nuclei were lysed with the addition of2 volumes of proteinase K buffer (50 mM Tris [pH 7.5], 100mM NaCl, 1 mM EDTA [pH 7.0], 0.5% sodium dodecylsulfate). Samples were deproteinated with the addition of100 ,ug of proteinase K (Boehringer) per ml and wereincubated at 55°C for 18 h. DNA was extracted and purifiedwith equal volumes of phenol-chloroform isoamyl alcohol(1:1) and dialyzed against 0.1x SSE (15 mM NaCl, 1.5 mMEDTA, pH 7.0) overnight. DNA was precipitated with 0.1 MNaCl and 2 volumes of absolute ethanol at -20°C overnight.DNA was pelleted, washed with 70% ethanol, and redis-solved in 0.1x SSE. Fragmented DNA was transferred tonitrocellulose (35) and hybridized at 65°C overnight to nick-translated probes, as described by Maniatis et al. (24). Blotswere washed to a final stringency of 0.3 x SSC (lx SSC is0.15 M NaCl plus 0.015 M sodium citrate) at 65°C. Probeswere nick translated (33) exactly as directed by the Boehr-inger nick translation kit, using all four [a-32P]deoxynucleo-side triphosphates (>800 Ci/mmol).

RESULTS

Correlation of e-globin DHS and promoters in K562 cells.To map e-globin DHS in K562 cells, nuclei were isolatedfrom K562 cells which were expressing e-globin at highlevels, as detected by S1 mapping (see Fig. 5). The nucleiwere digested with a range of DNase I concentrations asdescribed in Fig. 1; DNA was purified and digested with athreefold excess of KpnI. Digestion products were separatedon 0.7% agarose gels and transferred to nitrocellulose filters(35). Fragmented DNA was hybridized to a 1.17-kbp BglII-EcoRI fragment (labeled by nick translation) containingintron 2 and exon 3 of the e-globin gene (see line drawing,Fig. 1). The filters were washed to a final stringency of 0.3 xSSC and autoradiographed for 3 days. The autoradiograph inFig. 1 shows the 13.6-kbp parent band present at all DNaseI concentrations and shows initial digestion at 10.0 U ofDNase I per 25 x 106 nuclei. Nine subbands are detected,which represent preferential digestion of chromatin byDNase I, and, therefore, represent DHS. Eight of these DHSare located -11.7, -10.5, -6.5, -4.5, -2.2, and -1.48 kbp

DHS K562 HEL KMOEa b c a b c a b c

(1) -11.7 kb 0.1 4 _ _(2) -10.5 kb 0.1 4 - _ _(3) -6.5 kb 0.1 4 20.0 4 - 10.0 -(4a) -5.5 kb 7.5 --_(4b) -4.5 kb 0.1 # + _ _(5) -2.2 kb 0.1 4 + -_ _(6) -1.48 kb 10.0 + 30.0 . _(7) -900 bp 7.5 4 + 30.0 4 _(8) -200 bp 0.1 4 + 30.0 4 - _(9) cap 7.5 4 + 30.0 4 +

FIG. 2. Relative sensitivity of £-globin DHS and associatedpromoter activity in cell lines representing the various stages oferythroid development. E-globin DHS are numbered 1 through 9 (asin Fig. 1), and distances from the major cap site are given in column1. a, DNase I concentration (in units) at which DHS first appear(derived from Fig. 1 through 5); b, width of arrows representsmaximum intensity of DHS; c, promoter activity associated witheach DHS (derived from Fig. 6).

and -900 and -200 bp upstream of the e-globin gene, andthe ninth is located directly over the major cap site. Ther-globin DHS are not detected in nonerythroid cells, such asHeLa (41) or HL60 cells (36), but are specifically correlatedwith expression of the r-globin gene. The positions of thee-globin DHS in K562 cells are represented by verticalarrows in the line drawing in Fig. 1. Also shown are thepositions of r-globin upstream promoters (2, 5; see Fig. 5).Six of the s-globin DHS (including the major cap site)contain transcription initiation sites, five of which transcribethrough the gene and are polyadenylated, and one whichoriginates within a PollI promoter associated with the Alurepetitive element located -2.2 kbp upstream of the gene.The 350-bp Alu transcript is nonpolyadenylated, nucleusconfined, and transcribed in the opposite direction from thegene. The results described in Fig. 1 confirm all of the DHSpreviously reported by us (41) and by others (16, 36). Inaddition, we now resolve the band stretching between -200bp to the major cap site into two separate DHS, one of which(the -200-bp DHS) appears at the lowest concentration ofDNase I (0.1 U), and is therefore approximately 100-foldmore sensitive to DNase I than the major cap site DHS.Furthermore, two additional DHS at -11.7 and -10.5 kbpupstream of the r-globin gene are reported in this study.These sites have previously been reported by Tuan et al. (36)in both K562 and HEL cells and by Grosveld et al. (19) inHEL cells.As summarized in Fig. 2, and shown in Fig. 1, the major

DHS located at -11.7, -10.5, -6.5, and -2.2 kbp and -200bp upstream of the cap site also appear at the lowest DNaseI concentration (0.1 U). The major cap site is also locatedwithin a prominent DHS but does not appear until digestionwith 10 U of DNase I. The minor DHS located -4.5 kbpupstream of the gene appears after digestion with 0.1 U ofDNase I, while the minor DHS at -1.48 kbp and -900 bp areapproximately 75- to 100-fold less sensitive to DNase I (Fig.1 and 2). DNase I hypersensitivity does not correlate withpromoter activity. For example, the major cap site produces80% of e-globin transcripts, while the -200-bp promoter,which is 100-fold more sensitive to DNase I, produces onlyS to 10% of transcripts. Similarly, the major DHS (located-11.7, -10.5, and -6.5 kbp upstream of the gene) do not

VOL. 10, 1990

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

1202 BUSHEL ET AL.

have associated promoters, while the minor DHS (located-4.5 and -1.48 kbp and -900 bp upstream of the gene) do.Loss of e-globin specific DHS during erythroid develop-

ment. A major purpose of this study was to detect changes inthe pattern of the c-globin DHS during the developmentalinactivation of the gene. To do this, we have chosen theK562 cell line as a model for the embryonic stage ofdevelopment (34), the HEL cell as a model for the fetal stage(25), and the KMOE line as a model for the adult stage (21).

Nuclei isolated from K562, HEL, and KMOE cells weredigested with increasing concentrations of DNase I. DNAwas purified and digested with a threefold excess of Asp718,a KpnI isoschizimer. Fragmented DNA was separated on0.6% agarose gels, transferred to nitrocellulose, and hybrid-ized to the nick-translated BglII-EcoRI E-globin probe pre-viously described for Fig. 1. As was found with KpnI, a13.6-kbp parent band is revealed and is preferentially di-gested at higher DNase I concentrations (Fig. 3A). Themajor DHS located -11.7, -10.5, and -2.2 kbp upstream ofthe cap site are no longer detectable in HEL cells or KMOEcells. This has been confirmed in multiple experimentswhere the gels were run out to reveal subbands close to theparent band (not shown). The -6.5-kbp DHS is still visiblein HEL cells, although it is approximately 200-fold lesssensitive to DNase I. The -200-bp DHS is also visible inHEL cells but is 300-fold less sensitive to DNase I, while themajor cap site is only 4-fold less sensitive. The minor site-4.5 kbp upstream of the cap site is undetectable in HELcells, while the minor DHS at -1.48 kbp and -900 bp arestill detectable in HEL cells, although they are reduced insensitivity by three- to fourfold. Only the -6.5-kbp DHS isstill detectable in KMOE cells but is significantly reduced inintensity compared with HEL cells. The results summarizedin Fig. 2 are derived from at least 4 to 5 independentexperiments.As an independent confirmation that the observed loss of

e-globin DHS in HEL and KMOE cells did not result fromdifferences in general accessibility to DNase I, the blotsshown in Fig. 3A were rehybridized to an 837-bp XmaIII-Sau3A probe derived from intron 1 of the c-Ha-ras oncogene(23). The c-Ha-ras gene is expressed equally in all three celltypes and is therefore expected to have a similar DHS profilein each cell line. A 5-kbp parent band and a 1.6-kbp subbandare revealed after hybridization to the c-Ha-ras probe (Fig.3B). The subband defines a DHS located at the 5' boundaryof the c-Ha-ras promoter. We have recently shown that thisregion is responsible for up regulating the multiple c-Ha-rasinitiation sites located upstream of the donor splice site,indicating the functional relevance of this site (23). Thec-Ha-ras parent and subband are present in all three lines,supporting the view that the loss of specific r-globin DHS in

HEL and KMOE cells is a real reflection of developmentalchanges in the chromatin upstream of this gene.

Detailed analysis of the DHS within 1,500 bp upstream ofthe e-globin major cap site during the embryonic to fetalswitch. To independently confirm the changing pattern ofDHS during the embryonic to fetal switch shown in Fig. 3and to obtain better resolution of the DHS close to thee-globin gene, we digested DNA from K562 and HEL cellswith a threefold excess of EcoRI after DNase I digestion, asdescribed in the legend to Fig. 4. The digested products wereelectrophoresed on a 0.6% agarose gel and transferred tonitrocellulose. The fragmented DNA was hybridized to thenick-translated BglII-EcoRI E-globin probe previously de-scribed (see line drawing, Fig. 4).

In K562 cells, the -200-bp DHS appears at 0.1 U ofDNase I and reaches maximum intensity at 7.5 U. The majorcap site subband appears at 7.5 U and (at this concentration)is of equal intensity to the -200-bp band. In HEL cells, boththe -200-bp and major cap site DHS appear at 30 U ofDNase I and are less intense than in K562 cells. Thus, the-200-bp DHS becomes 300-fold less sensitive to DNase I asthe embryonic to fetal switch occurs, while the major capsite DHS is approximately 4-fold less sensitive.The minor subbands representing DHS located -900 bp

and -1.48 kbp upstream of the cap site are still visible asminor bands in HEL cells, although these sites show afourfold reduction in DNase I sensitivity.

Detailed analysis of the DHS far upstream of the E-globingene. As seen in Fig. 3, the -6.5-kbp DHS decreases200-fold in sensitivity to DNase I during the embryonic tofetal switch. To further investigate this region and to obtainbetter resolution of the far upstream DHS, we digested DNAisolated from K562 and HEL nuclei with a threefold excessof EcoRI (following digestion with DNase I as described inMaterials and Methods). Digested products were separatedon 0.6% agarose gels and hybridized to a nick-translated577-bp TaqI-PvuII 5' r-globin probe. The probe was sub-cloned to avoid contamination with the neighboring Alurepetitive sequences (see line drawing, Fig. 5). Figure 5confirms that the sensitivity of the -6.5-kbp DHS decreases200-fold during the inactivation of the E-globin gene in HELcells. In addition, the -6.5-kbp subband is around 100-foldless intense in HEL cells. Better resolution has revealed anadditional DHS (no. 4b) located -5.5 kbp from the major capsite. This site is not detectable in HEL cells. The subbandsrepresenting the -4.5-kbp and the -2.2-kbp Alu DHS arealso undetectable in HEL cells.Thus, by using independent enzymes and probes, we have

confirmed that only the -6.5 and -1.48-kbp and the -900-and -200-bp and major cap site DHS remain sensitive toDNase I in HEL cells. These HEL cells, which can be

FIG. 3. (A) Patterns of DHS upstream of the E-globin gene in cell lines representing various stages of erythroid development. DNA wasisolated from nuclei derived from K562, HEL, and KMOE cells after digestion with increasing concentrations of DNase I (described in thelegend to Fig. 1). Fifteen micrograms of this DNA was digested with a threefold excess of Asp718, electrophoresed through 0.6% agarose gels,and transferred to nitrocellulose. Fragmented DNA was hybridized overnight at 65°C to an a-32P-labeled 1.17-kbp BgIII-EcoRI probe fromthe 3' end of the E-globin gene, and filters were washed to a final stringency of 0.3 x SSC at 65°C. Blots were autoradiographed for 3 days at-70°C with intensifying screens. Parent band (P) and DHS no. 1 through 9 are indicated, as in Fig. 1. DNase I concentrations are shown aboveeach lane of the autoradiograph. The DHS observed in each cell line are denoted by numbered arrows above each line drawing. The thicknessof each arrow indicates the intensity of the DHS. A, Asp718; Bg, BgIII; E, EcoRI; C, ClaI. (B) DNase I sensitivity of the c-Ha-ras promoterin K562, HEL, and KMOE cells. The blots shown in Fig. 3A were washed to remove the E-globin probe and rehybridized overnight to anax-32P-labeled 800-bp XmaIII-Sau3A probe derived from intron 1 of the c-Ha-ras oncogene. Filters were washed to a final stringency of 0.3 xSSC at 65°C and autoradiographed for 5 days at -70°C with intensifying screens. The 5-kbp parent band (P) and 1.6-kbp subband areindicated, as in Fig. 3A. The position of the DHS is denoted by a bold arrow on the line drawing. A, Asp718; B, BamHI; D, donor splice site;X, XbaI; Ac, acceptor splice site; S, Sau3A; exl, exon 1 of the c-Ha-ras gene; boxed P, c-Ha-ras promoter. The line drawing is numberedwith relation to the BamHI site 1,700 bp upstream of the first ras coding exon.

MOL . CELL . BIOL .

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

HIEL

CORRELATION OF E-GLOBIN DHS-PROMOTERS 1203

KMOE

s'. G 2^i5^0 4C053_~~~~~~~~~~~3 _r_o_ -P

-3

7- .B_s 4'343 Ut

5 6 7 8 9

,._+E B EA

_J -

HEL

KMOE _

B 62

o 0.1 10.0

s.o

_,,_ -

67wl8 ¶+ E A

-A A- .-f-c

3

ffEL

p

t. ~,-_- -~ _~I,)

KMOE

0.0 o O. 1 10 10.020o0 300.

[-P -P

KM A B D X

A' S C

HE A B D

*: p

KMQEP. .. * t p ;LI

1... .-

Ac S A

-Y1! 0 c Ha-ras;6S!* t 1?3' 1%

x

V-n:

Ac S A

c-Ha-aso-

Il e1 i, -f

x Ac S A

c-Ha-ras6 ^ }-k-

VOL. 10, 1990

A K562

- P

L-K- 3

- 4

- S

A2K562 A

1

+

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

1204 BUSHEL ET AL.

K562 HEL

pp

- 6

9

6 B9

- t +-.~~ ~r.--1 _

FIG. 4. Changing DNase I sensitivity of the r-globin major promoter and the -200-bp promoter regions during the embryonic to fetalswitch. DNA (15 p.g) was isolated from nuclei derived from K562 and HEL cells following digestion with different concentrations of DNaseI. DNA was digested with a threefold excess of EcoRI, electrophoresed through a 0.6% agarose gel, and transferred to nitrocellulose.Fragmented DNA was hybridized to an a-32P-labeled 1.17-kbp BgIIl-EcoRI probe from the 3' end of the r-globin gene and washed to a finalstringency of 0.3x SSC at 65°C. Blots were autoradiographed for 3 days at -70°C with intensifying screens. Parent band (P) and subbandsrepresenting DHS are numbered to the right of the autoradiograph. The actual sizes of subbands are indicated to the left of the autoradiograph.Concentrations of DNase I are shown above each lane of the autoradiograph. The DHS observed in each cell line are denoted by numberedarrows above each line drawing. The thickness of each arrow indicates the intensity of the DHS. A, Asp718; Bg, BgII; E, EcoRI; C, ClaI.

considered functionally equivalent to fetal erythroid cells,lack the DHS located -11.7, -10.5, -5.5, -4.5, and -2.2kbp upstream of the gene (results are summarized in Fig. 2).Our results differ significantly from those of Tuan et al.

(36), who found that the DHS at -11.7, -10.5, and -6.5 kbpare present at all stages of erythroid development, suggest-ing that all of these sites may be important in activating theentire ,-globin cluster. However, this report did not relatethe pattern of DHS to the transcriptional status of theE-globin gene. Since it has been reported that particularly inHEL cells, E-globin expression is extremely variable (14), itseemed important to assay for transcriptional activity of thisgene in relation to the pattern of DHS.

Correlation of r-globin multiple promoter activity andDNase I hypersensitivity during the embryonic to fetal switch.The DHS located -4.5, -2.2, and -1.48 kbp and -900 and-200 bp upstream of the e-globin cap site contain upstreaminitiation sites in K562 cells. To determine whether thecorrelation between DHS and promoter sites was maintainedduring the embryonic to fetal switch, we assayed for tran-scriptional activity from the upstream promoters in K562and HEL cells. In this series of experiments, we alsoaccurately quantitated levels of r-globin RNA originatingfrom the major cap site in K562 and HEL cells. A series ofrestriction fragments spanning the 5 kbp upstream of ther-globin gene (Fig. 6) was gel purified. These fragments were5'-end labeled, strand separated, and used as hybridizationprobes, either for S1 analysis or primer extension analysis,of RNA derived from K562 or HEL cells. Results repre-sentative of a number of experiments are given in Fig. 6.When the 371-bp MboII-MboII fragment spanning the £-globin cap site is used for Si analysis, a number of Siproducts are seen in K562 cells (Fig. 6A). The major bands,129 to 131 bp long, map to the canonical cap site (-55 to -53from ATG) (1, 2), while the longer protected fragment

represents an RNA species initiating 200 bp upstream of thecap site. When an identical amount of RNA derived fromHEL cells is analyzed by using this probe (lane H2), themajor cap site bands are 100-fold less intense than in K562RNA. When 4 jig of poly(A)+ RNA from HEL cells isanalyzed, although the major cap site bands are now equiv-alent to the K562 cell bands (lanes K and Hi), the -200 capsite remains undetectable. Even with 20 ,ug of poly(A)+RNA from HEL cells, the -200 cap site remains undetect-able (not shown). Thus, the -200 cap site RNA species hasdecreased by at least 500-fold in HEL cells. Using the 190-bpMboII-XbaI fragment for primer extension analysis, wedetect an RNA species mapping -900 bp upstream of thee-globin gene in K562 cells, whereas in HEL cells, thisspecies is undetectable (Fig. 6B). The sensitivity obtained byusing this probe allows us to estimate at least a 500-foldreduction in activity of this promoter. Similarly, by using the200-bp Hinfl-Hinfl fragment for primer extension analysis,we detected an RNA species initiating -1.48 kbp upstreamof the gene (within the first Alu repeat) in K562 cells but notin HEL cells (Fig. 6C). This promoter shows at least a200-fold reduction in activity. Finally, the 310-bp TaqI-TaqIprimer extension probe detects RNA species mapping -4.5,-4.3, and -4.2 kbp upstream of the s-globin gene. Thesespecies are reduced in HEL cells by at least 100- to 200-fold(Fig. 6D). Thus, none of the n-globin promoters upstream ofthe major cap site are detectable in HEL cells, and weestimate a reduction in activity of at least 100- to 500-foldcompared with K562 cells. The major cap site is 100-fold lessactive in HEL cells but is detectable at low levels. Each ofthe promoters detected in K562 cells has been previouslyconfirmed by using both S1 analysis and primer extensionanalysis (2). The -2.2-kbp promoter has previously beenshown by Allan and Paul (5) to be undetectable in any celltype (including HEL cells) in which the r-globin gene is not

..I n I ;. - - r, " .1 -

i. , -, I.; "I '-' %...

MOL. CELL. BIOL.

z- 1.

9

67- 8 9

;p e.gi .., A .--c:obln

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

CORRELATION OF £-GLOBIN DHS-PROMOTERS 1205

K562

6.5---.

6.3-

4.3-

HEL

PP

__ 5

_ 3

4b

- - 4a2.3

3

,.I P.

T P E- A: .0

F 4i34b 48 T jIIAu.

ifcFIG. 5. Patterns of DHS in the far upstream region of the E-globin gene during the developmental inactivation of the gene. DNA (15 ,ug)

was isolated from nuclei derived from K562 and HEL cells following digestion with increasing concentrations of DNase I. DNA was digestedwith a threefold excess of EcoRI, electrophoresed through a 0.6% agarose gel, and blotted onto nitrocellulose. Fragmented DNA washybridized overnight at 65°C to an a-32P-labeled 577-bp Taql-PvuII probe located upstream of the more distal Alu repeat, as shown in the linedrawing. Filters were washed to a final stringency of 0.3 x SSC at 65°C and autoradiographed for 3 days at -70°C with intensifying screens.The undigested parent band (P) and subbands representing DHS are numbered to the right of the autoradiograph. Actual sizes of bands, inkilobase pairs, are indicated to the left of the autoradiograph. Concentration of DNase I (in units) is shown above each lane. The line drawingshows the 5'-flanking region of the E-globin gene. The DHS observed in K562 and HEL cell lines are denoted by vertical arrows, as in Fig.1. E, EcoRI, Taq TaqI; and Pvu, PvuII.

maximally active. This series of experiments indicates thatthere is no clear correlation between DHS and associatedpromoter activity. Irrespective of the alteration in DNase Isensitivity of particular sites, the associated upstream pro-moters are, without exception, inactivated in HEL cells. Themajor cap site shows a 4-fold reduction in DNase I sensitiv-ity but a 100-fold reduction in promoter activity; on the otherhand, the -200 cap site shows a 300-fold reduction in DNaseI sensitivity and at least a 500-fold reduction in promoteractivity. It is tempting to speculate that the reduction in the-200 cap site DNase I sensitivity is related to the reductionin transcription from the major cap site. Both the -900-bpand -1.48-kbp DHS are reduced 3- to 4-fold in sensitivity toDNase I and at least 200- to 500-fold in promoter activity. Itis clear from a comparison of the -200- and -900-bp and-1.48-kbp sites that inactivation of the upstream promoterscan precede the disappearance of the associated DHS.Finally, the -4.5- and -2.2-kbp DHS are undetectable inHEL cells and promoter activity is similarly undetectable. Inthe KMOE cells used in these experiments, e-globin RNA isundetectable either from the major cap site or from any ofthe upstream promoters (not shown).

DISCUSSION

We have detected 10 DHS upstream of the e-globin gene inK562 cells in which the gene is maximally transcribed. Themajor sites are located -11.7, -10.5, -6.5, and -2.2 kbpand -200 bp upstream of the e-globin gene and directly overthe major cap site (summarized in Fig. 2). Minor sites aresituated -5.5, -4.5, -2.2, and -1.48 kbp and -900 bpupstream of the cap site. The positioning of the r-globinDHS concurs with previous reports (16, 36, 41). However,

there is some disagreement regarding the relative sensitivityof sites. Tuan et al. (36) claim that the most sensitive sitesare located far upstream of the gene (- 11.7, - 10.5, and -6.5kbp), while the sites closest to the gene are described asminor. Furthermore, one diffuse site is reported between-200 bp and the major cap site. Our results clearly show twoindependent sites, at -200 bp and directly over the majorcap site, which differ substantially in their sensitivity toDNase I (Fig. 1, 2, 3, and 4). The -200-bp site appears at thelowest concentration of DNase I (as do the far upstreamsites) and matches the -6.5-kbp site in intensity. The capsite DHS is also a major site. It seems likely that the -200-bpand cap site DHS have been previously underestimated.The pattern of e-globin DHS in HEL cells (in which

e-globin transcription is extremely low, as confirmed by Sianalysis) differs substantially from that in K562 cells (Fig. 3,4, and 5). The major sites at -11.7, -10.5, and -2.2 kbp andthe minor site at -4.5 kbp are undetectable in HEL cells.The major sites at -6.5 kbp and -200 bp and the major capsite are still visible in HEL cells but are 200- to 300-fold (or,in the case of the major cap site, 4-fold) less sensitive toDNase I. Thus, the developmental inactivation of the r-globin gene is accompanied by very large reductions insensitivity of the DHS located -11.7, -10.5, -6.5, -4.5,and -2.2 kbp and -200 bp upstream of the r-globin gene andsmaller reductions in the -1.48-kbp, -900-bp, and majorcap site DHS. Our results, again, differ from previousreports (36), which suggest that all the minor sites closest tothe gene disappear in HEL cells, while the sites located-11.7, -10.5, and -6.5 kbp upstream of the gene remainstrong in HEL cells. It may be significant that these authorsfailed to confirm e-globin levels in the HEL cell clones used

VOL. 10, 1990

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

1206 BUSHEL ET AL.

B

.p

cap v

FIG. 6. Si and primer extension analysis of e-globin RNA obtained from K562 and HEL cells. RNA was hybridized at 57°C to a seriesof gel-purified single-stranded DNA probes 5'-end labeled with polynucleotide kinase and [y-32P]ATP. The position of labeling is denoted byan asterisk on probes shown above the line drawing of the 5'-flanking region of the e-globin gene. Following hybridization of the 371-bpMboII-MboII DNA probe, hybrids were digested at 37°C for 1 h with 1,000 U of Si nuclease (Boehringer). Hybridization of probes B, C, andD was followed by extension with reverse transcriptase as described in Materials and Methods. Products of Si digestion or primer extensionwere separated on 6% denaturing polyacrylamide gels and autoradiographed for 48 h at -70°C with an intensifying screen. Autoradiographsare shown above the appropriate probe. Probe A: lane K, 4 ,ug of K562 total RNA; lane Hi, 4 ,ug of HEL poly(A)+ RNA; lane H2, 4 jig ofHEL total RNA. Probe B: lane K, 5 ,ug of K562 poly(A)+ RNA; lane H, 5 jig of HEL poly(A)+ RNA. Probe C: lane K, 5 ,ug of K562 poly(A)+RNA; lane H, 5 ,ug of HEL poly(A)+ RNA. Probe D: lane K, 5 ,ug of K562 poly(A)+ RNA; lane H, 5 ,ug of HEL poly(A)+ RNA. Markers(M) are HaeIII fragments of 4X174. P, Primer. Positions of RNA initiation sites are denoted on the line drawing by vertical arrows. Thedistance from the major cap site is indicated on the line drawing and in bold numbers to the left of each autoradiograph.

to analyze DHS. HEL cells vary in e-globin levels fromalmost undetectable to equivalent to that of induced K562cells (14), and it seems possible that the HEL cell clonesused for DHS analysis continued to express significant levelsof e-globin RNA. This is supported by the findings ofForrester et al. (16), who report the continued presence ofonly the -6.5-kbp DHS in normal fetal erythroid cells, inwhich transcription from the e-globin gene was undetect-able. A subsequent report from the same laboratory (15)suggests the continued presence of the -10.5-kbp DHS infetal and adult red blood cells, although the -11.5-kbp site isfound only in K562 cells. It is unclear to what extentconflicting reports as to the presence or absence of the farupstream sites reflect the phenotypic differences betweencultured cell lines and their normal counterparts or theconsiderable potential for contamination of normal red bloodcells with other members of the blood cell series (15). Insummary, while there is some conflict as to the behavior ofthe -10.5-kbp site, the continued presence of the -6.5-kbpDHS at all stages of erythroid development has been con-firmed both in cell lines and normal red blood cells.

All known regulatory elements reside within regions ofDNase I hypersensitivity (13), and one purpose of this studywas to distinguish between those DHS potentially involvedin maintaining the s-globin gene transcriptionally active,those DHS potentially involved in inactivating the gene at 10weeks gestation, and those DHS potentially involved in

maintaining the activity of the entire ,-globin gene cluster.We have recently shown that the -4.5-kbp DHS-promoterdown regulates the r-globin gene 20- to 30-fold, probably bytranscriptional interference. Down regulation is overcome inmature erythroid cells by the transcriptional activation of the-2.2-kbp Alu DHS-PolIII promoter (40; Allan et al., submit-ted). Since the -4.5-kbp and -2.2-kbp DHS and promotersare undetectable in HEL cells (Fig. 5 and 6), this interactingsystem apparently is not involved in inactivating the geneduring the embryonic to fetal switch but is more likely tooperate in erythroid stem cells. Fragments containing the-1.48-kbp and -900-bp DHS-promoters up regulate thee-globin gene 20-fold in nonerythroid cells, again directly asa result of promoter activity (Bushel et al., in preparation).These relatively nonspecific DHS are maintained in HELcell lines in which the e-globin gene is still active at lowlevels, although the associated promoters are undetectablein HEL cells (Fig. 6). The major DHS at -200 bp isdecreased 300-fold during the embryonic to fetal switch,while the major cap site is reduced in sensitivity only 4-fold.A silencer of e-globin transcription has recently been local-ized close to the -200 DHS-promoter (7). In view of thefinding that the -4.5-kbp down regulator operates by tran-scriptional interference, it is intriguing that the -200 pro-moter decreases in activity as the major cap site increasesduring induction of K562 cells with hemin (Allan et al.,submitted). Although there is some dispute in the literature

D C

r;.

A

--.:

e- -

_

-..w

2_a m

probe -1-2r00C!

i

4 A4 .4A* 4

MOL. CELL. BIOL.

*1

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

CORRELATION OF E-GLOBIN DHS-PROMOTERS 1207

as to the fate of the far upstream DHS, in our hands, the-6.5-kbp DHS is the only site maintained in cells repre-sentative of all stages of erythroid development. Previousreports suggest that this site may be implicated in maintain-ing the globin cluster in an active chromatin conformation(16). The decrease in intensity of this DHS throughoutdevelopment could suggest a greater influence at the earlystages, possibly in activation of the entire cluster. In studieswhere it has been confirmed that fetal erythroid cells usedfor DNase I analysis have either extremely low or undetect-able levels of E-globin RNA (16; present study), the DHSlocated -11.7 and -10.5 kbp upstream of the gene are

absent in these cells. It has been suggested that these sites,together with the -6.5-kbp DHS, maintain activity of thewhole cluster (19, 36). We would, however, suggest thatwhile it is possible that these sites may be implicated in theearly activation of the cluster, they become quiescent duringthe embryonic to fetal switch. Fragments containing the-6.5-kbp site have no effect in regulating the e-globin gene intransient expression assays (Allan et al., submitted). How-ever, large fragments containing this site have been shown tostrongly up regulate any covalently linked gene in erythroidcells (19). It should be pointed out that the globin mini-locusused in these experiments contains not only the far upstreamDHS but also the so-called minor sites up to and includingthe -900-bp DHS. As discussed above, the -2.2 and -1.48-kbp and -900-bp DHS could together have the capacity toup regulate an associated gene at least 50-fold. The -2.2-kbpAlu element would be of particular significance in transgenicmice or stable transformants where the transfected gene may

be integrated close to another gene and, thus, may besusceptible to transcriptional interference (9, 11, 32). Insummary, we suggest that the elements from -6.5 kbpthrough -900 bp have a major effect in erythroid-specificregulation of the globin genes.

Five of the ten E-globin DHS upstream of the major cap

site also contain minor promoters. Therefore, at least twodistinct classes of DHS exist, and we have recently shownthat the four DHS-promoters so far examined exert theirregulatory effect (both positive and negative) as a directresult of promoter activity (Allan et al., submitted; Bushel etal., in preparation). DHS with associated promoters havebeen reported in several genes (18-20; 28-30), and this typeof control mechanism may be seriously underreported. Thee-globin upstream promoters lack any known regulatorymotifs, and we originally proposed that polymerase com-

plexes bound nonspecifically to open chromatin. This clearlyis not the case, since the major DHS upstream of the-4.5-kbp DHS lack initiation sites. There is no correlationbetween sensitivity to DNase I and upstream promoteractivity, since the minor sites at -4.5 and -1.48 kbp and-900 bp contain promoters, while the major sites at -6.5,-10.5, and -11.7 kbp do not. It is possible (though at

present untestable) that these far upstream sites have asso-

ciated promoters at very early stages of erythroid develop-ment. In HEL cells, all the upstream promoters disappear,although the -200- and -900-bp and the -1.48-kbp DHSand the major cap site promoter are still detectable. Thus,inactivation of the upstream promoters can precede thedisappearance of the associated DHS, supporting the viewthat the alterations in chromatin structure which result inhypersensitivity to DNase I and regulation of upstream

promoter activity occur by independent mechanisms. Thepattern of DHS and promoter activity in HEL cells is, in our

experience, unique. For example, when the e-globin gene istransfected into nonerythroid cell lines, all DHS are present

and transcription from the upstream promoters is signifi-cantly greater than from the major cap site (4, 41). Similarly,a number of nonerythroid cell lines have been shown toinappropriately express the E-globin gene. Without excep-tion, the appropriate DHS are present and transcriptionoriginates exclusively from the upstream promoters (2). Thecurrent finding that the -1.48-kbp and -900- and -200-bppromoters are completely inactivated in HEL cells, whilethe associated DHS are still detectable, suggests the pres-ence, in HEL cells, of regulatory factors which specificallydown regulate the upstream promoters. The existence of atleast one such factor which down regulates the -200-bppromoter in K562 cells has been demonstrated by using an invitro competition assay (40). Since the -1.48-kbp and -900-bp elements apparently up regulate the e-globin gene as adirect result of the activity of their promoters, we arecurrently testing whether these elements become nonfunc-tional in HEL cells despite the continued presence of theassociated DHS.We suggest the following sequence of events leading to the

activation of the 3-globin cluster: (i) the DHS located at-6.5, -10.5, and -11.7 kbp activate the 3-globin cluster; (ii)the upstream promoters become transcriptionally active; (iii)in embryonic erythroid stem cells, transcription from the-4.5-kbp promoter inactivates the gene by transcriptionalinterference; (iv) this is overcome in mature embryonicerythroid cells by activation of the -2.2-kbp Alu PollIlpromoter; (v) in mature erythroid cells, the -900-bp and-1.48-kbp DHS-promoters up regulate the r-globin gene;(vi) during the embryonic to fetal switch, the -2.2-, -4.5-,-10.5-, and -11.7-kbp DHS disappear first; (vii) all of theupstream promoters become inactive; (viii) the -200- and-900-bp and -1.48-kbp DHS disappear as the gene becomestotally inactive. The -6.5-kbp DHS is present at all stages oferythroid development, suggesting a possible role in regulat-ing not only the r-globin gene but the entire 3-globin cluster.

ACKNOWLEDGMENTS

Support for this research was provided by Public Health Servicegrant ROI HL37023 from the National Institutes of Health.Thanks are also due to Nick Shelness for assistance in preparing

the manuscript.

LITERATURE CITED1. Allan, M., G. J. Grindlay, L. Stefani, and J. Paul. 1982. Epsilon

globin gene transcripts originate upstream of the mRNA cap sitein K562 cells and normal human embryos. Nucleic Acids Res.10:5133-5147.

2. Allan, M., W. G. Lanyon, and J. Paul. 1983. Multiple origins oftranscription in the 4.5 kb upstream of the E-globin gene. Cell35:187-197.

3. Allan, M., P. Montague, G. J. Grindlay, G. Sibbet, M. Donovan-Peluso, A. Bank, and J. Paul. 1985. Tissue specific transcriptionof the human r-globin gene following transfection into theembryonic cell line K562. Nucleic Acids Res. 13:6125-6136.

4. Allan, M., P. Montague, J. D. Zhu, and J. Paul. 1984. Differen-tial response of multiple r-globin cap sites to cis- and trans-acting controls. Cell 38:399-407.

5. Allan, M., and J. Paul. 1984. Transcription in vivo of an Alufamily member upstream from the human r-globin gene. NucleicAcids Res. 12:1193-1200.

6. Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of earlyadenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721-732.

7. Cao, S. X., P. D. Gutman, P. G. Harish, 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.

VOL. 10, 1990

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.

1208 BUSHEL ET AL.

8. Chirgwin, J. M., A. E. Pryzbyla, R. J. MacDonald, and W.Rutter. 1979. Isolation of biologically active ribonucleic acidfrom sources enriched in ribonuclease. Biochemistry 18:5294-5299.

9. Corbin, V., and T. Maniatis. 1989. Role of transcriptionalinterference in the Drosophila melanogaster Adh promoterswitch. Nature (London) 337:279-282.

10. Cowie, A., C. Tyndal, and R. Kamen. 1981. Sequences at thecapped 5' ends of polyoma virus late region: an example ofextreme terminal heterogeneity. Nucleic Acids Res. 9:6305-6322.

11. Cullen, B. R., P. T. Lomedico, and G. Ju. 1984. Transcriptionalinterference in avian retroviruses-implications for the pro-moter insertion model of leukaemogenesis. Nature (London)307:241-245.

12. di Segni, A., G. Carrara, G. R. Tocchini-Valentini, C. C.Shoulders, and F. E. Baralle. 1981. Selective in vitro transcrip-tion of one of the two Alu family repeats present in the 5'flanking region of the human E-globin gene. Nucleic Acids Res.9:6709-6722.

13. Elgin, S. C. R. 1984. Anatomy of hypersensitive sites. Nature(London) 309:213-214.

14. Enver, T., J.-W. Zhang, N. P. Anagnou, G. Stamatoyannopou-los, and T. Papayannopoulou. 1988. Developmental programs ofhuman erythroleukemic cells: globin gene expression and meth-ylation. Mol. Cell. Biol. 8:4917-4936.

15. Forrester, W. C., S. Takegawa, T. Papayannopoulou, G. Stam-atoyannopoulos, and M. Groudine. 1987. Evidence for a locusactivating region: the formation of developmentally stable hy-persensitive sites in globin expressing hybrids. Nucleic AcidsRes. 15:10159-10175.

16. Forrester, W. C., C. Thompson, J. T. Elder, and M. Groudine.1986. A developmentally stable chromatin structure in thehuman ,-globin gene cluster. Proc. Natl. Acad. Sci. USA83:1359-1363.

17. Ghosh, P. K., J. B. Reddy, M. Piatak, P. Lebowitz, and S. M.Weissman. 1980. Determination of RNA sequences by primerdirected synthesis and sequencing of their cDNA transcripts.Methods Enzymol. 65:580-595.

18. Grindlay, G. J., W. G. Lanyon, M. Allan, and J. Paul. 1984.Alternative sites of transcription initiation upstream of thecanonical cap sites of the human -y-globin and 3-globin genes.Nucleic Acids Res. 12:1811-1820.

19. Grosveld, F., G. B. van Assendelft, D. R. Greaves, and G.Koilias. 1987. Position-independent, high level expression of thehuman 3-globin gene in transgenic mice. Cell 51:975-985.

20. Hess, J., C. Perez-Stable, A. Deisseroth, and C. K. J. Shen. 1985.Characterization of an unique RNA initiated immediately up-stream from human al-globin gene in vivo and in vitro: poly-merasell-dependence, tissue specificity, and sub-cellular loca-tion. Nucleic Acids Res. 13:6059-6074.

21. Kaku, M., K. Yagawa, K. Nakamura, and H. Okano. 1984.Synthesis of adult-type haemoglobin in human erythremia cellline. Blood 64:314-317.

22. Lachman, H. M., and J. G. Mears. 1983. DNase I hypersensi-tivity in the -y-globin gene locus in K562 cells. Nucleic AcidsRes. 17:6065-6077.

23. Lowndes, N. F., J. Paul, J. Wu, and M. Allan. 1989. c-Ha-rasgene bidirectional promoter expressed in vitro: location andregulation. Mol. Cell. Biol. 9:3758-3770.

24. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

25. Martin, P., and T. Papayannopoulou. 1982. HEL cells: a newhuman erythroleukemia cell line with spontaneous and inducedglobin expression. Science 216:1233-1235.

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

27. McKnight, S. L., M. Chao, R. W. Sweet, S. Silverstein, and R.Axel. 1983. Transcriptional control signals of a eukaryoticprotein-coding gene, p. 73-85. In M. Miwa and S. Nishimura(ed.), Primary and tertiary structure of nucleic acids and cancerresearch. Japan Scientific Society Press, Tokyo.

28. Nelson, K. J., J. Haimovitch, and R. D. Perry. 1983. Character-ization of productive and sterile transcripts from immunoglob-ulin heavy-chain locus: processing of L,u and ,u mRNA. Mol.Cell. Biol. 3:1317-1332.

29. Osborne, T. F., and A. J. Berk. 1983. Far upstream initiationsites for adenovirus early region 1A transcription are utilizedafter onset of viral DNA replication. J. Virol. 45:594-599.

30. Parslow, T. G., and D. K. Granner. 1983. Structure of anuclease-sensitive region inside the immunoglobulin kappagene: evidence for a role in gene regulation. Nucleic Acids Res.11:4775-4792.

31. Pelham, H. R. B. 1982. A regulatory upstream promoter elementin the Drosophila Hsp 70 heat shock gene. Cell 30:517-528.

32. Proudfoot, N. J. 1986. Transcriptional interference and termina-tion between duplicated a-globin gene constructs suggests anovel mechanism for gene regulation. Nature (London) 322:562-565.

33. Rigby, P. W. J., M. Dieckmann, C. Rhoquades, and P. Berg.1977. Labelling deoxyribonucleic acid to high specific activity invitro with DNA polymerase I. J. Mol. Biol. 113:521-560.

34. Rutherford, T. R., J. B. Glegg, and D. J. Weatherall. 1979. K562human leukaemic cells synthesize embryonic haemoglobin inresponse to haemin. Nature (London) 280:164-165.

35. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

36. Tuan, D., W. Solomon, Q. Li, and I. M. London. 1985. The",3-like-globin" gene domain in human erythroid cells. Proc.Natl. Acad. Sci. USA 82:6384-6388.

37. van Assendelft, B. G., 0. Hanscombe, F. Grosveld, and D. R.Greaves. 1989. The 3-globin dominant control region activateshomologous and heterologous promoters in a tissue-specificmanner. Cell 56:969-977.

38. Weaver, R. F., and C. Weissman. 1979. Mapping of RNA by amodification of the Berk-Sharp procedure: the 5' termini of 15S3-globin mRNA precursor and mature 10S ,-globin mRNA haveidentical map co-ordinates. Nucleic Acids Res. 6:1175-1193.

39. Weintraub, H. 1983. A dominant role for DNA structure informing hypersensitive structures in chromatin. Cell 32:1191-1203.

40. Wu, J., G. J. Grindlay, P. Bushel, L. Mendelsohn, and M. Allan.1990. Negative regulation of the human r-globin gene by tran-scriptional interference: role of an Alu repetitive element. Mol.Cell. Biol. 10:1209-1216.

41. Zhu, J., M. Allan, and J. Paul. 1984. The chromatin structure ofthe human e-globin gene: nuclease hypersensitive sites correlatewith multiple initiation sites of transcription. Nucleic Acids Res.12:9191-9204.

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 17

Nov

embe

r 20

21 b

y 11

2.78

.70.

240.