DNase I hypersensitivity in the γ globin gene locus of K562 cells

Volume 11 Number 17 1983 Nucleic Acids Research

DNase I hvpersensitfvit) In the 7 globin gene locus of K562 cefls

Herbert M.Lachman and J.Gregory Mears* +

Division of Experimental Hematology, Department of Medicine, Albert Einstein College ofMedicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Received 20 April 1983; Revised and Accepted 1 August 1983

ABSTRACTWe have probed the chromatin conformation of the y- y-t-6

globin gene locus of K562 cells, a human hematopoietic cell line,with the enzyme pancreatic DNAse I. This enzyme preferentiallydigests genes in an active configuration. We have found that inK562 cells, which produce embryonic and fetal but not adulthemoglobins, both the active y and inactive 8 genes are DNAse Isensitive. However, only the active y genes have DNAse Ihypersensitive regions. The hypersensitive regions have beenmapped to an area approximately 100 base pairs 5' to the y and ygenes.

INTRODUCTION

The control of human globin gene expression is a poorly

understood process. Despite an impressive accumulation of

knowledge of the fine DNA structure of normal and mutant human

globin genes in recent years the mechanism responsible for the

precise, efficient regulation of these genes in the developing

erythron has not been elucidated.

It has been shown in other eucaryotic gene systems that the

chromatin conformation of expressed or developmentally committed

genes is altered, as defined by sensitivity to DNAse I digestion

(see 1 for review). When nuclei are partially digested with DNAse

I, actively expressed genes and genes that will be developmentally

activated, are digested faster than non-active genes (2-5). The

domain of increased sensitivity includes the structural genes and

large portions of the 5' and 3' regions (3,6,7). DNAse I

sensitive regions are also undermethylated (7-9) and bind the

non-histone proteins HMG 14 & 17 (10-12).

With minimal DNAse I digestion specific double stranded

(hypersensitive) DNA cleavages are often seen (6,7). These appear

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Nucleic Acids Research

as unique bands on Southern blots. Hypersensitive cleavages have

been observed near genes that are actively transcribing mRNA (6,7)

or near genes like the drosphila heat shock genes that can be

activated within minutes (13). For example, in chick embryo red

blood cells (RBCs) the active embryonic (j globin gene has a

hypersensitive cleavage near the 5' end of the gene. In adult

cells new hypersensitive cleavages are seen 2 and 6 kilobases (kb)

5' to the adult B globin gene (6). Since DNAse I hypersensitive

cleavages are associated with transcribed genes, there may be

particular conformational changes in these regions important for

gene expression.

We report a study of the chromatin conformation of the human

globin genes in K562 cells, a cell line capable of embryonic

(t2l2' a2 e2' ^2^2^ a n d f e t a l hemoglobin ( O 2 Y 2 ) , but not adult

hemoglobin (ao*2' a2^2^ synthesis (14). Hemoglobin levels are low

under normal growth conditions but increase markedly with the

addition of hemin or other inducing agents (15).

The study demonstrates preferential DNAse I digestion of theGT» A T , (GY - glycine at codon 136, A T - alanine at codon 136), «,

and B gene loci compared to a control nonhematopoietic gene locus

as well as DNAse I hypersensitive regions near the t gene loci.

The K562 cell line, derived from a patient with chronic

myelogenous leukemia (16) is being studied as a model for the

fetal-adult hemoglobin switch. This system may help us understand

developmental gene switching in general and may have important

clinical implications for the treatment of serious B globin gene

defects such as sickle cell anemia and 6 thalassemia.

MATERIALS AMD METHODS

K562 cells (clone R from Dr. A. Skoultchi) were grown in RPMI

1640 and 10% fetal calf serum (GIBCO, Grand Island, NY) containing

210 mg/1 NaHCO , 5.94 g/1 HEPES (Sigma), and 1 mg/l garamycin•3 Q

(Schering). 1-3 x 10 cells were harvested for each experiment.

The cells were either grown without inducing agents or induced in

log phase with 25-50 pM hemin (Sigma). Before induction 0.1% of

cells were benzidine positive. After induction 60-90% were

positive and the cell pellet was obviously hemoglobinized.

To isolate nuclei, K562 cells were washed free of media with

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phosphate buffered saline, then lysed after a 15 minute incubation

in reticulocyte standard buffer (RSB) S. 0.5% nonidet. Nuclei were

pelleted and washed 3 times in RSB then resuspended in RSB to

yield a DNA concentration of 0.5-1.0 mg/ml. Bovine pancreatic

DNAse I (Boehringer Mannheim, West Germany, lot # 1201427) was

added either at a fixed concentration (0.1-2.0 pg DNAse i/mg DNA)

with aliquots removed at 0, 1/2, 1, 3, 5, 10, 15, 20, and 30

minutes or at increasing concentration (0, 0.5, 1, 2, 5 yg DNAse

i/mg DNA) for 5 minutes. Experiments were performed at 37° C.

50x aliquots were removed and added to 50i of 0.1 M EDTA and

centrifuged. DNA in the supernatant was determined by optical

density at 260 nM and percent solubility obtained in order to

monitor the activity of DNAse I. The reactions were stopped by

adding 3 volumes of PK buffer (0.05 M Tris, pH 7.5, 0.1 M NaCl,

0.001 M EDTA, 0.5% SDS) and 50 yg/ral proteinase K (Boehringer).

Solutions were incubated overnight at 55° C and the DNA purified

as previously described (17).

15 yg of DNA were restriction digested with either Eco RI,

Hpa I, Bam HI, or Bgl II (New England Biolabs), using sufficient

enzyme to ensure complete digestion. DNA was electrophoresed on a

0.9% agarose gel for 18 hours at 15-20 volts, then blotted to

nitrocellulose paper (Schleicher & Schuell) as previously

described (18). Filters were hybridized to 32P-labelled probes

(Amersham) prepared by nick translation (19). The probes used

were human 8 globin (4.4 kb Pst I fragment), T globin (Bam HI-Eco

RI IVS-2 fragment), and human insulin (pH INS 214). The 8 and y

probes were kindly provided by Dr. A. Bank and the insulin probe

by Dr. J. Cohen. The specific activity after nick translation wasg

l-5x 10 cpm/yg DNA.

Human peripheral blood leukocytes (WBCs), pelleted following

NH4C1-

NH^COj lysis of RBCs were used as control cells.

RESULTS

Figure 1 shows a DNAse I digestion pattern for uninduced

(Fig. 1A) and induced (Fig. IB) K562 cells. In Fig. 1A the nuclei

were partially digested with 0, 0.5, 1.0, and 5.0 yg DNAse i/mg

DNA whereas in Fig. IB digestion with 0, 0.5, and 1.0 ug DNAse

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B Induced KDb<i - y inironA Uninduced K 5 6 2 - r Intron

7.2kb- b2.7 kb— 1

*l.5kb- * *'-5kb"

1 2 3 4 1 2 3

Figure 1. DNAse I sensitivity of y globin gene loci inuninduced and induced K562 cells. Uninduced K562 nuclei weredigested with 0, 0.5, 1.0, and 5.0 wg DNAse i/mg DNA (A, channels1-4, respectively) whereas induced K562 nuclei were digested with0, 0.5, and 1.0 yg DNAse i/mg DNA (B, channels 1-3, respectively).DNA samples were subsequently analyzed by Eco RI digestion,Southern blotting and hybridization to P-y IVS-2 probe.Substantial loss of the normal 7.2 and 2.7 kb fragments isobserved by the last channel of each blot. A new 1.5 kb Eco RIhypersensitive fragment (*) is prominent by channel 2 of eachblot.

I/mg DNA is illustrated. Following DNAse I digestion, DNA was

extracted, restriction digested with Eco RI, Southern blotted and

hybridized to 32P-y IVS-2. The normal 7.2 kb (5' Gy gene) and 2.7

kb (5' y gene) fragments are seen (20). In addition, a new faint

fragment is apparent in both experiments which is 1.5 kb in size.

Figure 2 shows the DNAse I digestion pattern for nuclei from

normal WBCs following progressive digestion with 0-15 yg DNAse

I/rag DNA and analyzed in an identical fashion to Fig. 1. Again

the normal 7.2 kb and 2.7 kb Eco RI fragments are visualized.

However, by contrast to Fig. 1, the relative intensities of the

two fragments persist with appreciably more DNAse I digestion.

Other experiments with genes known to be expressed in WBCs showed

substantial DNAse I sensitivity with 5 Mg DNAse i/mg DNA (J.G.

Meara, unpublished observations). In addition, no 1.5 kb Eco RI

fragment is observed.

Two further control experiments were performed to be sure

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WBCs-Y Intron

2.7 kb — • - #

1 2 3 4 5 6 7

Figure 2. DNAse I sensitivity of y globin gene loci innormal WBCs. Nuclei of WBCs were digested with 2, 5, 7.5, 10,12.5, and 15 vg DNAse i/mg DNA (channels 2-6, respectively).Channel 1 is DNA prepared directly from fresh K562 cells. DNAsamples were analyzed as in Fig. 1. Identical 7.2 and 2.7 kb EcoRI fragments are seen in K562 and WBC DNA. No appreciable loss ofthe y globin gene related Eco RI fragments is seen withsubstantial DNAse I digestion and no 1.5 kb hypersensitivefragment is observed.

K562-lnsulin

9.5 kb—1

1 2 3 4 5 6 7

Figure 3. DNAse I sensitivity of the insulin gene in K562cells. Uninduced K562 nuclei were digested with 0, 1, 2, 5, 7.5,10, and 12.5 yg DNAse i/mg DNA (channels 1-7, respectively). DNAsamples were digested with Bgl II, Southern blotted and hybridizedto P- INS 214. The normal 9.5 kb fragment is seen in allchannels without a significant decrease in intensity by channel 7.Parallel 3 and B globin gene experiments with the same DNA samplesyielded results similar to those seen in Figures 1 and 7.

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Figure 4. Comparison of controlK562 DNA to DNAse I digested K562 DNA.Channel 1 is DNA directly prepared

^^^ from fresh K562 cells whereas channel7.2 kb—^ «^i ^ ^ 2 is DNA prepared from K562 nuclei

following digestion with 2 pg DNAseI/mg DNA. Both DNAs were analyzed asin Fig. 1. No 1.5 kb Eco RIhypersensitive fragment is seen in

2.7 kb—•" «*• fl^B control DNA whereas it is prominentin partially DNAse I digested DNA.

— 1.5 kb*

that the differences between K562 cells and human WBCs were not

artefactual. Figure 3 shows the DNAse I digestion pattern for

K562 nuclei following progressive digestion with 0-12.5 ug DNAse

i/mg DNA. DNA was purified, digested with Bgl II, Southern

blotted, and hybridized to P- INS 214. The insulin gene is not

expressed in hematopoietic cells (21,22) and is associated with a

9.5 kb Bgl II fragment (23) as seen here. Note that this fragment's

intensity does not appreciably diminish with increasing DNAse I

digestion as compared to the preferential loss of the T globin

gene related fragments illustrated in Fig. 1.

Since a very faint 1.5 kb Eco RI fragment is occasionally

observed in the 0 point of the y globin gene experiments (Fig. IA,

channel 1), K562 DNA was prepared directly from fresh K562 cells

avoiding the steps involved in nuclei preparation. Figure 4

compares K562 DNA prepared directly (channel 1) or following

nuclei preparation and 2 |ig DNAse i/mg DNA (channel 2). Ho 1.5 kb

Eco RI T globin gene related fragment is observed in fresh K562

DNA as contrasted to the appearance of the 1.5 kb Eco RI fragment

with partial DNAse I digestion. Since this fragment is not seen

in DNA prepared from human WBCs or fresh K562 cells and becomes

prominent after partial DNAse I digestion, it represents a

fragment bounded by a hypersensitive region.

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Ikb

G r

probe yIVS2 I 1 I 1

Hi- -I 5---Eco RI I » 1 I x 1

72 27

•-I 3-Hpol —/f X 1 1

24 5 I

4 5Bom HI I x—I TV—

5 1 15 5

5 0 i 6 36 7----

113

B q l l I x x 113

Figure 5 • Restriction map of y- y gene locus andhypersensitive sites. T- Y gene locus is at top. Black boxesrepresent exons whereas clear boxes represent interveningsequences. The Bam HI- Eco Rl r - IVS- 2 probe encompasses thearea shown by the bar under the genes. The restriction enzymesused are listed on the left with the expected fragments indicatedby the solid lines with their respective sizes below the lines(29). Hypersensitive fragments are shown by broken lines with theapproximate sizes in kilobases above the broken lines.

One can see from the map in Fig. 5 that the new 1.5 kb Eco RI32

fragment which hybridizes to the P~r IVS- 2 probe can be

generated by the presence of a hypersensitive region about 100G A

base pairs 5' to the y and/or Y structural loci. The 5' end of

the 1.5 kb fragment would be generated by DNAse I activity at the

hypersensitive region and the 3' end by the internal Eco RI site,

which is present in both Y globin gene loci, since the y- IVS- 2

probe is bounded at its 3' end by this Eco RI site. The loction

of the hypersensitive region(s) was confirmed by Hpa I digestion

which normally generates 24 and 5.1 kb fragments extending 5 from

sites within the IVS- 2 of the G T and A Y globin gene loci,

respectively (Fig. 5). The IVS- 2 Hpa I sites are approximately

200 base pairs 5' to the internal Eco RI sites. Following partial

DNAse I digestion of K562 nuclei a new 1.3 fragment is generated

which is consistent with hypersensitive region(s) 5' to the Y

globin gene loci (Fig. 6). We have tentatively assigned this 1.3

kb fragment only to the T globin qene since 3.8 and 1.3 kb

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Bam HI Bgl 11

I-4.5* • - . 6 . 7

— 11.3*

-6.7*-6.3*

Figure 6. Mapping of hypersensitive sites. Channels 1, 3,and 5 are Hpa I, Bam HI, and Bgl II digests of control K562 DNA.Channels 2, 4, and 6 are digests of K562 DNA extracted from nucleithat had been digested with 0.5 jig DNAse i/mg DNA. The left sideof each group indicates the size of the known restrictionfragments in kb for the respective enzyme and y- IVS- 2 probe(29). On the right are the new hypersensitive fragments and theirapproximate sizes. Each channel is from a separate blot althoughexperimental conditions were identical, i.e., 0.9% agarose run at15 volts for 18 hours. Channel 6 however was run on 0.7% agaroseat 20 volts for 18 hours to separate the two largest fragments.The 24 kb Hpa I fragment is not visualized because of theinefficient transfer of large fragments.

fragments would be generated if the 5.1 kb y globin gene Hpa I

fragment were cleaved by DNAse I at the predicted site.

In order to determine precisely whether the hypersensitive

region occurs 5' to one or both y globin gene loci, the following

experiments were performed. Bam HI generates a 5.1 kb fragment

which extends 3' to the \ globin gene (Fig. 5). In some, but not

all experiments a new 4.5 kb fragment was generated which is

consistent with a hypersensitive region 5' to the y globin gene

(Fig. 6). Since this fragment was inconsistently seen, the

hypersensitive region associated with the y globin gene may be

less accessible.

Bgl II normally generates a 13 kb fragment which encompasses

both y globin gene loci (Fig. 5). Following partial DNAse I

digestion of K562 nuclei, Bgl II produced in addition to the

expected 13 kb fragment, 11.3, 6.7, 6.3, and 5.0 kb fragments

(Fig. 6). All new fragments are consistent with the presence of

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A uninduced KDc^- p oene g Induced K562- (3 Gene

_, 5.2 kb-3 6 k b^ 3.6 kb-

2.3kb— 2.3kb-

1 2 3 4 1 2 3

Figure 7. DNAae I sensitivity of the 6-B globin gene loci inuninduced and induced K562 cells. Uninduced K562 nuclei weredigested with 0, 0.5, 1.0, and 5.0 ug DNAse i/mg DNA (A, channels1-4, respectively) whereas induced K562 nuclei were digested with0, 0.5, and 1.0 ug DNAse l/mg DNA (B, channels 1-3, respectively).DNA samples were analysed by Eco RI digestion. Southern blottingand hybridization to P- B globin gene probe. Preferential lossof the normal (5.2 kb fragment - 5'6 gene, 3.6 kb fragment = 3'Bgene) and t (2.25 kb fragment » 5's gene) gene fragments isobserved as compared to the larger 9.5 kb insulin fragment seen inFig. 3. No new hypersensitive fragments are observed.

hypersensitive regions approximately 100 base pairs 5' to both the

Y and T globin genes (Fig. 4).

A similar series of experiments was performed to analyze the

6-8 globin gene pair. Figure 7 shows a DNAse I digestion pattern

for uninduced (Fig. 7A) and induced (Fig. 7B) K562 cells.

Following DNA purification, Eco RI digestion and Southern

blotting, the filters were hybridized to the P-B globin gene

probe. the normal 5.2 kb (5'B globin gene), 3.6 (3'B globin

gene), and 2.25 kb (5'6 globin gene) Eco RI fragments are seen

(24). « globin gene specific fragments are difficult to visualize

because of differences in gene structure to the 3 globin gene

probe (24). As was seen with the y globin genes, there is

preferential digestion of the B- like globin gene fragments when

compared to the unexpressed insulin gene fragment (Fig. 3). The

5.2 kb Eco RI fragment is more intense because of greater

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representation in the 6 globin gene probe. No 6- like

hypersensitive fragment is observed although a 1.5 kb Eco RI 6

globin gene fragment would be expected if a hypersensitive^site

similar to those observed for the y globin genes existed 5 to the

B globin gene. Because of the previously mentioned differences in

homology with S globin gene related fragments we cannot rule out

the existence of a i globin gene related hypersensitive region.

DISCUSSION

Our findings that both the y- y and J-B gene pairs are DNAse

I sensitive in uninduced and induced K562 cells suggests that the

gene pairs share a single chromosomal domain of altered chromatin

conformation despite the lack of 6 globin gene expression. Since

we find hypersensitive regions associated only with the actively

expressed y globin gene loci, one implication is that human globin

gene expression requires the presence of one or more

hypersensitive regions within the chromosomal domain. Thus,

developmental changes in human globin gene expression such as the

fetal to adult hemoglobin switch may be accompanied by changes in

the location of hypersensitive regions within a larger domain of

altered chromatin which encompasses all genes which have the

potential for expression within a genetic program. A recent study

suggests the presence of nascent i globin RNA transcripts in K562

cells which may not be full length and demonstrates that no B

globin RNA is detectable (25). One prediction from this would be

the presence of a & globin gene related hypersensitive region.

Use of a B globin gene related probe does not allow us to

determine whether such a region exists.

Our results in the K562 cell line are similar to those of H.

Weintraub in the chick embryo system (2) and D.M. Miller, et al.

in mouse erythroleukemia cells (MELC) (26) that an entire

developmental locus is DNAse I sensitive whether or not RNA is

being transcribed. We are presently attempting to determine the

extent of DNAse I sensitivity. It is possible, as others have

shown, that the degree of DNAse I sensitivity may vary somewhat

within the chromosomal domain in addition to extending for many

kilobases beyond the genes of interest (3,6,7,).

In normal humans, both y globin genes are expressed although

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the ratio of their protein products varies according to the stage

of development. In fetuses, in which the y globin genes are the

major active non-o globin genes, the ratio is typically T/ T =

7/3 whereas in adults, in which the T globin are expressed at a

low level, the Gy/AT " 2/3 (27). It is interesting to note that

in K562 cells, which display many characteristics of fetal

development including a Gy/AY = 7/3 (28), both T globin genes are

flanked at their 5" ends by hypersensitive regions. Thus, if

hypersensitive regions are necessary for expression, one

conclusion from our study may be that the y globin genes possess

the potential for independent regulation of expression rather than

a single regulatory area responsible for fetal or adult type y

globin gene expression. We presently do not understand why the y

globin gene related hypersensitive region is less easily

demonstrable by our methodology. Both the Hpa I and the Bam HI

studies are consistent with the presence of a less accessible

hypersensitive region. It is possible that this region is not as

extensive as that related to the y globin gene. Further studies

are required to determine if possible differences in the two Y

globin gene hypersensitivve regions are related to the

developmentally changing ratios of their protein products.

Both T globin gene hypersensitive regions map to 5' flanking

regions which are highly conserved in their DNA structure and

which have been shown to contain two putative promotor sequences,

the ATA sequence and the CCAAT sequence 30 base pairs and 80 base

pairs 5' to the cap site, respectively (29). Since we have not

yet identified any other hypersensitive regions near the y globin

genes, it is possible that the presently described hypersensitive

regions which are closely allied to putative promotor sequences

are sufficient for active y globin gene expression.

The K562 cell line is viewed by many investigators as a

useful model for the study of human globin gene expression,

particularly since globin gene expression is enhanced by the

addition of hemin and other inducing agents in a manner similar to

that described for MELC (30). However, induction in MELC has

recently been shown to be accompanied by a 6 to 10 fold increase

in the generation of hypersensitive regions 5' to the a and S

major globin genes (9), whereas we have not been able to identify

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a similar trend between uninduced and induced K562 cells. ThuB,

induction as defined by the activation of a genetic program of

differentiaition, may not occur in K562 cells. Rather, inducing

agents produce an augmentation of the low level expression of the

globln genes detectable in uninduced cells. In addition, it is

unlikely that inducing agents select for a subpopulation of cells

capable of hemoglobin production since the hypersensitive regions,

if considered a marker for active y globin gene expression, are

equally discernible in uninduced and induced K562 cells. This is

consistent with the finding that uninduced K562 cells have uniform

amounts of globin mRNA as detected by in situ hybridization (31).

The K562 cell line is an interesting substrate for further

studies of differential human globin gene expression at the level

of chromatin. Experiments designed to study differences in the

5'regions of the y globin gene versus S globin gene loci which

relate to the presence or absence of hypersensitive regions may be

useful in understanding the mechanism(s) of the fetal to adult

hemoglobin switch in humans. Insight into the fetal to adult

hemoglobin switch may point to future therapy which can favorably

modify the clinical course of the common genetic disorders of

hemoglobin synthesis.

ACKNOWLEDGEMENTS

We would like to thank Kim E. Schaefer for excellent

technical assistance. This work was supported by NIH grants GM

28841 and AM 7294 and the Sinsheimer Foundation. Dr. Mears is a

Hartford Fellow.

A preliminary report describing parts of this paper has

appeared in an abstract (32).

*To whom correspondence should be addressed

+ Present address: Columbia University College of Physicians and Surgeons, Department ofMedicine, Hammer Health Sciences Center, Room 1602, 701 West 168th Street, New York,NY 10032, USA

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DNase I hypersensitivity in the γ globin gene locus of K562 cells

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