Regulation of murine α-, βmajor-, and βminor-globin gene expression

Vol. January

150,

No.

1, 1988

BIOCHEMICAL

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RESEARCH

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15, 1988

Pages

REGULATION OF MURINE a-, Nadine

Weich,

RMAJOR-,

Paul

and BMINOR-GLOBIN

A. Marks,

and Richard

GENE EXPRESSION

A. Rifkind

Dewitt Wallace Research Laboratories Memorial Sloan-Kettering and the Sloan-Kettering Graduate School of Medical Sciences, Cornell New York, New York 10021 Received

November

Division University

of the

6, 1987

SUMMARY : Transcriptional properties of the mouse globin genes and their promoters were examined in COS cells using transient expression assays. Transfected intact mouse al-globin genes generate transcripts, whereas pmaj and pmin globin transcripts are detected only if the genes are associated with a strong exogenous (SV40) enhancer sequence. Under these conditions the ratio of accumulated tmaj and jimin mRNA sequences approximates the 4:l ratio observed in reticulocytes and murine erythroleukemia cells (MELC) induced to differentiate by hexamethylene bisacetamide (HNBA). As determined using hybrid genes consisting of globin gene 5' regions fused to the chloramphenicol acetyltransferase (CAT) structural gene, this 4:l ratio appears dependent upon the relative activities of the two 5' promoter regions. 0 1988 Academic Press, Inc.

Mechanisms and the

two

examined

linked

in controlling g-globin

adult

are

differences To gain

not

fully

genes

further

their

assays

transcriptional

control

among the

present

transient

transcriptional

and pmin-globin differ

genes,

in sequence

accomplish

the

sequences

were

of each measured suggest

that

have

and (2)

second fused

to the

different

mechanisms

0006-291x/88 $1.50 Copyrighr 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

intact

fimaj

CAT structural

as CAT enzymatic

genes

their

are

204

(4-6).

mouse

al-,

promoters, (2,3,7).

(1)

$majwhich To

and @min 5' regulatory gene

activity

in In the

to investigate

activity al,

expression

differences

human globin

to examine

objective,

gene

(2,3).

properties.

suggested

and transcriptional

for

and 13min genes

was used

been

the

evidence

globin

of cloned

have

Although

transcriptional

expression

properties

of the mouse al-

and pmin)

is

)3maj,

al,

of regulated

examined

expression

studies

there

of mouse

understanding

mouse we have

($maj

MELC (1).

understood,

in regulation

Transient

the

expression

in differentiation-inducible

mechanisms

in the

involved

(a).

responsible

and the influence The results for

204-211

BIOCHEMICAL

Vol. 150, No. 1, 1988

transcriptional

control

Following transfection,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the mouseal- and p-globin genes. al-globin

l,rnaj and pmin genes require

mRNAis detected,

an exogenous enhancer.

levels of 5maj and 9min mRNAreflect

closely

their

whereas the The relative relative

expression in HMBA-induced MELCand in normal erythroid

cells.

Each level of expression appears determined, at least in part, sequences S' to the structural

by

genes.

Materials

and Methods

Globin and hybrid gene plasmids. Plasmids were prepared by alkaline lysis and two cesium chloride-ethidium bromide equilibrium centrifugations (9,lO). Recombinant PSVO-cat, lacking the SV40 early transcription unit, was obtained from PSV2-cat as described (8). PSV2-cat containing the SV40 early transcription unit was the source of SV40 DNA sequences. A 198 bp fragment containing the origin of replication and a 216 bp fragment containing the 12 bp tandem repeats were ligated into the BamHI sites of plasmids pSR327 (11) and PSVil-cat. Recombinants pBR327SHand ?SVSH-cat consist of pBR327 or PSVO-cat sequences and the SV40 origin of replication. Recombinants pBR327NPand PSVNP -cat contain the SV40 transcription enhancer. A 3 kb Sac1 fragment containing the al-globin gene (121, a 7 kb EcoRI fragment containing the pmaj-globin gene (131, and a 6.2 kb EcoRI-Hind111 emin fragment (13) were inserted into each pBR327 plasmid (Fig. 1A). The globin regulatory sequences(GRS) (5' promoter and cap sites) studied were: a 700 bp NcoI al fragment; a 550 bp HincII emaj fragment; and a 600 bp BamHI-HincII emin fragment. These were inserted into the Hind111 site of each PSV-cat plasmid (Fig. 1BI. DNA transfection and CAT assay. One day before transfection, COS cells, in Dulbecco's modified Eagle's medium and 10% fetal calf serum, were plated (4x104 cells/cm2). For transfection, 1Opg of plasmid DNAadded to CaC12 and Hepes buffer Lontaining phosphate (141, was then added to the cells. After four hours exogenous DNA was removed and Hepes-buffered 15% glycerol was layered wer the cells for 1 min. Cells were harvested for RNApreparation and CAT assay (8) 48 hours after transfection. RNAPreparation and RNAProbes. DBA/2 mice were injected intraperitoneally with 0.1 ml 1% phenylhydrazine on days 0, 4 and 7, and bled on day 9. Red cells were washed in phosphate buffered saline and total RNA prepared (15,161. MBLCDS-19 (subclone 9) cells were maintained in mediumlacking nucleosides (Gibco Laboratories) with 10% fetal calf serum. Cells were induced with 5mMHMBA(96 hrs), and total RNA prepared.- To construct templates for complementary strand RNA probes a pGEM4vector (Promega Biotec) containing the Sp6 (17) and T7 promoters flanking a pUCl8 multiple cloning region was used. Probes al-pGEM4 and alXl-pGEM4 were linearized with HindIII prior to transcription from the Sp6 promoter. fimaj-@EM4 (T7 promoter) and fimin-pGEM4 (Sp6) were linearized with EcoRI and HindIII, respectively. The probes were synthesized as suggested by Promega Biotec except that a32P-UTP (3000 Ci/mmole) was substituted for a32P-CTP. Transcription was at 37'C and the hybridization and RNase protection assays performed as described (11). 205

Vol. 150, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/\ sv40 ORI

sv40 EN”

ORI

40 EN”

Figure 1. Globin gene and globin-CAT hybrid gene plasmids. (A) or bmin pBR327 constructs: the arrow represents ~1, Pmaj, direction of transcription, the thin line, sequences from pBR322; the thick line, mouse sequences flanking the globin genes; ORI, origin of replication; ENH, transcription enhancer. (B) “1, praaj, or IjminPSV-cat constructs: GRS represents globin 5' regulatory sequences. The slash through restriction endonuclease cleavage sites indicate loss of that site during plasmid construction (B-BamHI; E-EcoRI; H-HindIII).

Results Globin

in mouse __--- cells of globin mRNA was determined

uniformly

labeled

and dmin homology detect

A

Discussion

gene expression Accumulation

with

and

non-homology

is

RNA probes about

and 3).

Although

RNase protection

assay

(Fig.

98%, the

of 4 consecutive

by RNase protection

2

bases.

For

example,

pmaj can when the

CY1-Globin Gene

B 135nt 126nt

a Psi I 3’ /CNVCMN 128nt

BamHt 5’ Probe ProtectedFfagmeIItS

p-minor

p-major Gtobin Gene

Globin Gene

ExonI HaeIll 3’ 1 2 3

4 5 6

ExonI 5’

135nt

Probes

Hae Ill Ha; 3’ vrvymr

ProtectedFragments

-

135nt

RNase protection analysis of total RNA from DBA/2 mouse Figure 2. Protected RNA fragments were analyzed on reticulocytee and MELC. The RNA probes used are shown an 8% polyacrylamide8M urea gel. (A) Reticulocyte RNA (lpg): (Lane 1) cr; (lane 21 in the diagram. (B) HNBA-induced MELC RNA (5pg): (Lane 4) Irmaj; and (lane 3) pmin. 5) pmaj; (lane 6) pmin. a, ; (lane

206

5’

Vol. 150, No. 1, 1988

M

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SH NP al -Globin Gene Exon II

Exon I

Pst I

Pst I 3’

m

5’ Probe Protected Fragments

.115 nt

128 nt 123

RNA from cells transfected with al-globin gene. Figure 3. Cells were transfzted with lflvg DNA. alpEW (lane 11, alpBR327SH (lane 2), or a~pBR327NP (lane 3). 2Opg RNA (lanes 1 6 31 or 6pg RNA (lane 2) were analyzed by RNase protection, on an 8% polyacrylamide -8M urea gel. The CXJ probe used in this experiment is shown in the diagram. 144nt fragments are produced from incorrectly initiated and/or spliced globin mRNA. Correctly processed mRNA yields 128nt and 115nt fragments. Lane M: size markers from 32P-labeled pBR322 DNA cut with MspI.

smaj

probe

full

length

occurs

is

hybridized

135nt

with

fragment

at several

sites

Rmin mRNA and treated

is

not

detected;

of non-homology

with

RNase the

instead,

digestion

and smaller

fragments

are

detected. RNA

or from

from

DSA/2 mouse reticulocytes

MELC exposed

was assayed

for

~1,

(lmin mRNA was 4:l mRNA

in the

and 1:l Globin

to HMBA for

not

pmaj

yet

fully

2, lanes

1, 2 and 3),

(Fig.

2, lanes

4, 5 and 61,

The ratio

of Rmaj to

sequences.

both

mature

(Fig.

sources.

The ratio

HIMRA-induced

of a to p

MELC was 2:l

(18,19)

reticulocytes.

gene expression To study

in COS cellseffect of regulatory

the

intact

mouse al-,

pmaj-,

series

of plasmids

that

each

and pmin

in RrJA from

in mature

96 hrs

series,

one construct sequence Since

the

for

gene the

amplification

constructs

assayed (Fig. to

sequence genes

with

genes, for

1A) or

SV40 origin

(201,

SV40 ennancer

of some heterologous contains

each

were

contains

regions

and pmin-globin

insure markedly

in COS cells that

enhancer. 207

on transcription

two

expression.

In

transient each 5' of

sequence

replication

sensitivity stimulates (5,21,22),

of

we constructed (Fig.

fori) for

lB),

for

expression. transcription

each

series

also

Vol. 150, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

p-major

p-minor

SH NP

SH NP

123

4 56

Figure 4. E analysis of transfected pmajand pmin-globin genes. Cells were transfected with 1084 DNA. BmajpBR327 (lane l), idmajpBR327SH (lane 2), ijmajpBH327NP (lane 31, pminpBR327 (lane 4), BminpBR327SH (lane 5), or pminpBR327NP (lane 6). Each lane was loaded with 2Opg RIJA. RNase digestion products were analyzed by electrophoresis on an 8% polyacrylamide -8fl urea gel.

We first with

investigated

neither

ori

nor

al mRNA was detected 3, lane 4).

or

neither

the

2) was at least ori.

upstream

al-globin

transcripts

fragment

is

fragment

reflects

the

of the

3' cryptic

Both

1

length

and

the

3, lane SV40

initiated 9majpBR327SH

only

Tine 211nt

within

and

aberrant,

probe,

sites

detected

without

2 and 5).

splice

were 4, lanes plasmid,

transcripts,

transcripts, full

(Fig.

a1 ~RNA (Fig.

plasmid

cap site.

4, lane

(Fig.

the

of

(jmaj

while the

the

kmaj

9Ont first

(6,7,23,24). The third

globin

genes

alpBR327NP generated

set

generated by the

1 and 3).

proper

transcripts 4:l.

of plasmids

to an exogenous

lanes roughly

from

no normal

homolog

competent

aberrant

gene

generated

upstream

exon

that

(Fig.

initiated

genes level

alpBR327

transcripts

replication

we detected

oE the

$minpBR327Sd

the

low

with

cells

of correctly

100 fold

In addition

pmin

transfected

with

amount

of ~RNA by ylobin A distinct

transfected

Rmaj nor

pminpBR327

In transfectants

alpBR327SH,

generation sequences.

in COS cells

11, whereas

in 13majpBR327

the

enhancer

roughly

30-fold

(Fig.

4, lanes was not

the

plasmid

and BminpBR327NP 3 and 61,

response

of

with (Fig. also

at a pmaj:@min

due to differences

208

the

CZJ mRNA transcripts

enhancerless

9majpBR327NP

ratio

examined Transfection

enhancer.

nonreplicating, Both

This

(NP series)

3,

generated ratio

in transfection

Of

BIOCHEMICAL

Vol. 150, No. 1, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS C

B

A

45 30

I

01

51-nn-l5

I-

P maI

Pm

01

Pw

PSVdcat

Pm,

PSVsn.cat

PSV,+cal

Figure 5. Expression of CAT at 48 hrs in COS cells transfected Thin layer chromatography with 1Ovg globin(GRS)-CAT hybrids. separates chloramphenicol (CM) from its acetylated forms. spots were excised from the gels, quantitated by scintillation counting, Each oar represents the and converted to percent CM acetylated. average from three experiments. efficiency

since

or RNA recovery,

plasmid

(data

not

shown)

a cotransfected

generated

al reference

equivalent

a: mRNA in both

transfectants. Glonin

promoter-directed To

the

determine

whether

mouse globin

transcription

genes

expression

from

plasmlds,

which

express

insignificant

(PSVSti-cat

activity

neither levels all

active

promoter

that for

it

--in vivo. To insure

directed

adult

that

in

an inverted

enhancer

a-globin half

by the

is the

less

(a, active

potential

(Fig. with

(25).

the SV40

the

most

clpSVNP-cat

ancl

Since

and q), than

it the

o promoter

CAT transcription

containing

209

SV40 ori

5C) and differential

regulatory

orientation

SA).

plasmids

while

genes

globin

construct

(Fig.

sequences,

respectively.

13.0%,

enhancer-dependent

a control

promoter

produced

conversion),

a3 promoter

represents

exclusively

transfected sequence

the

(Fig.

QmajPSVNP-cat 20% and

differential

CAT expression

globin-CAT

of

QminPSVO-cat

or

nor

increases

was substantial

mouse

pmaj,

promoters

CAT gene

by globin al,

three

the

the

of CAT activity

from

converted

for

SV40 ori

modestly

was unaffected.

surprising found

with

(56% chloramphenicol

two

within

we assayed

directed

only

(NP series)

QminPSVNP-cat are

contain

series)

expression

genes

in COS cells

located

genes,

transfected

CAT activity

enhancer

ylobin

hybrid

Cells

sequences

may be responsible

of intact

sequences.

5s).

CAT gene expression

sequences, the

pmin-globin

This

construct

there is

not

Smaj activity is we 5' generates

Vol. 150, No. 1, 1988

BIOCHEMICAL

no increased

CAT activity

course,

aberrant

that

may also

affect

globin

gene

relative

expression located

level

the 5'

in the

is

in part,

excluded,

from

genes

these

on intact suggest

that

in COS cells

determined

compared

gene one

enhancer-like

to the

and,

by sequences

of al-globin

region,

of

in RNA stability

upon two activities,

provides

regulatory

of expression

not

regions

globin The level

which 5'

is

observations

flanking

three

regions.

cap site,

It

generated

cells,

dependent,

and another

relative

of the

coding

appears 3' to the

(26),

together,

in mouse erythroid

5' to the

shown).

and differences

and on the

expression

by inference,

not

CAT activity

taken

expression

differential located

(data initiations

the

Nevertheless,

genes.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity

which

influences

8-globin

genes.

by grants

from

the

Acknowledgements These National

studies Cancer

Bristol-Myers

were Institute

Cancer

supported,

in part,

and CA-087481

(CA-31768

Research

the

and the

Award.

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