Different Combinations of Regulatory Elements May Explain Why ...

BIOLOGY

OF

Different

JOHN

(1991)

Combinations of Regulatory Elements May Explain Why Placenta-Specific Expression of the Glycoprotein Hormone a-Subunit Gene Occurs Only in Primates and Horses1

NILSON,2

H.

231-237

44,

REPRODUCTION

A. BOKAR,

JOSEPH

Department

M. CLAY,

COLIN

DEBORA

TODD

L. HAMERNIK,

of Pharmacology,

Case

A, FARMERIE,

and

Western

Cleveland,

RUTH

Reserve

Ohio

ROBERT

A. FENSTERMAKER,3

A. KERI

University,

School

of Medicine

44106

ABSTRACT Expression

and

horses.

are

required

CRE

binds

In

glycoprotein

humans,

two

for

also

that

indicates

a-subunit

gene.

change that renders In contrast, although CRE

but

appears

with

the

horse

the

of expression

to retaln

has

of the

sequence

of the

of

URE.

been

retained has

Chorionic

gonadotropin

protein hormones unit, Expression tary

of all mammals

and

been

gene

This

suggests

FSH, and a-subunit and

to the TSH) gene

that

either

a different a completely

glyCo-

ular

expression trast, while

progress events

in placenta

has been underlying

of primates

and

their

tissue-specific

search

(D.L.H.),

accessory

Grant

C. Clay Ohio

P30

is the

CA 43730,

recipient

School

John

of Medicine, OH

44106.

3Current 2074

and

the

of a grant

Institutes RA.F.), Ohio

from

the

Abington

Case FAX:

H. Nilson, Western

Ph.D., Reserve

Division

Road,

Cleveland,

of Neurosurgerv, OH

and

express

to cAMP. a functional

lacks

cognate

to

nuckotide

responsiveness

region

the

a number Indirect

fail

to a single

protein

interacts

elements

less

is known

of Health GM-07382

Edison

Grants (JAB.,

involved

primates

and

AM- 28559

is required

Biotechnology

American

Heart

Center Association,

Professor, University,

Department 2119

(J.H.N.). Northeast

Road,

University

Hospitals

equine

to be cloned. understanding

the

synthesis

This

has

of CG,

involved

of

CG molec-

we

have

why it is exin placenta of systematic

anal-

-146 and

Cleve-

and

origin.

These

elements

is referred

to as the

to bind

of Cleveland,

tinct 231

protein

[16].

located

upstream placenta regions,

of different reflecting

are

human in the

-180 and The element

a protein

result probably

elements

of the are

binding [10, 14, 18-

Expression

sequence

expression

cells and, presumably, be subdivided into two

44106.

is the

evolutionary

between nucleotides site of transcription, appears

primates elements,

DNA

DNA-protein animals

major findings that have of the a-subunit gene

regulatory

for Placenta-Speqflc a-Subunit Gene two

least

studies, transgenic

summarize the that expression

of horses

gion start

Re-

of Pharmacology,

Abington

encoding

gene and addressed of all mammals but

transfection by PCR, and

in placenta

[10-151.

(J.H.N.),

Cancer

genes

in the

horses.

combinations

Requirements of the Human

about

RA.K.),

the

genes have yet approach toward

an independent

216-368-3395.

address:

from

that

of regulatory

of

21, 351. Below, we led us to conclude

Affiliate. 2Correspondence:

land,

(TA.F.,

set

(CRE),

whereas

expression.

mammals be traced

element

different

including gene assays, cloning

made in understandplacenta-specific

Even

by National

DK-0731

can

on the a-subunit in the pituitary

At

This work was supported

gene

the protein that confers the promoter.regulatory

placenta-specific HD07138-03

to placenta

primates

ysis of promoter-regulatory regions of the genes encoding the a-subunits of humans, cattle, and horses. Our approach has entailed the use of a variety of experimental techniques

and characterized [5, 8], little mechanisms responsible for

expression,

cattle

mechanisms

only

mammals known the a and CGI3

of the human a-subunit gene [10-181. In conthe genes encoding the 13-subunits of human

LH and CG have been cloned is known about the molecular

element

a-subunit

from

expression

focused pressed

[1,6]. Recently, much the molecular

and

(eCG) as these As an initial

protein

pituitary

genes are located on different chromosomes [9]; presumably this also applies to equidae, Although there are reports of gonadotropin activity in placenta of other mammals, CG has been defined chemically in only primates and horses

ing

of only

response

for placenta-specific

in placenta

of rodents

in placenta

unique

of the

potential

even

or that

and a unique 13-suboccurs in the pitui-

The latter are also the only CG13 genes [1, 5, 81, In humans,

possess

a protein

region the

suggests

conserved,

in placenta

expression

is a heterodimeric

common

[1-61

binds

regulated

[6,7],

equidae to

(LH, of the

URE

but cAMP

and

in horses.

(CG)

of an a-subunit

The

(URE)

promoter-regulatory

OVERVIEW composed

of all mammals

element

these genes incapable of binding gene occurs in horse placenta,

placenta-specific

expression

gene.

of the

protein

pituitary

in the

regulatory

a-subunit

a-subunit

a-subunit

a functional

to provide

for placenta-specific

URE

occurs

upstream

analysis

URE-binding

the CRE-like expression

URE

of the

Comparative

a functional

gene

termed

expression

that

Lack

a-subunit

elements,

protein.

indicates

evidence

hormone

different

placenta-specific

a ubiquitous

of mammals the

of the

5’-flanking

for

gene re-

-100 relative to the between -180 and

regulatory

unique

required

a-subunit

element

(URE)

to choriocarcinoma

[10, 11, 15]. The URE may each of which binds a dis-

232

NILSON

the

Alone,

URE

has

no

URE activity is completely quence element located

effect

on

[10, 11].

transcription

dependent between -146

ET AL.

on the adjacent and -110. This

quently,

acting

se36-

the

a tripartite elements

human

domain

composed

defines

a-subunit

the

of two

placenta-specific

different

cLc-

enhancer

of

gene.

bp sequence is composed of two 18-bp direct repeats Containing a conserved palindrome TGACGTCA. Each repeat is designated

as a cAMP

a single

18-bp

response

element

can

element,

confer

or

GRE,

either the minimal a-subunit promoter (-100 a heterologous promoter [10-12]. Furthermore, binds

a ubiquitous

binding protein base within the

43-kDa

because

cAMP-responsiveness

nuclear

to

The Proximal Promoter from Cattle and Rodents

to +

44) or

each

phosphoprotein

CRE (CRE

unit

or CREB [22-24]). Changing virtually any core palindrome disrupts binding of CREB

moter mouse

The

CREs differ

from

URE

the

because

they

function

dependently to confer cAMP responsiveness to ogous promoter [10, 11]. Thus, cAMP responsiveness sue-independent expression. and one promoter

and

superimposed

on

in-

or to a heterologous CREs they

promoter

are essential unmask the

dif-

-

of the a-subunit genes of bovine, only a single homolog of the human

homologs

from

these

three

species,

rat, and a CRE.

however, of the transition

have

To test tors

this

shown

possibility,

we

constructed

in Figure

1 and

tested

the bovine and, preof the human a CRE absence

the their

of a func-

expression

activity

after

vectrans-

-cAMP (-3l3)

pBo(CAT

-

-

,_..

(+48)

-E-

+

cAM P

I---

CAT -

(-hO)

(-100/-114)

(+48)

(-170)

(-100/-114)

(+48)

pHuBOCCAT

p(AI8)HuBcCAT

(-166)

pBo(*CAT

---j

i-..

-:.LJ””

(+48)

0

0.5

1.0

CAT

1.5

a

human a disrupts

1---

CAT

-

human a-subproximal-pro-

tional GRE in the proximal-promoter region of the a-subunit gene of bovine, rat, and mouse may explain why the a-subunit gene is silent in the placentas of these mammals.

for placenta-specific exprestissue-specific properties of

URE. In addition, the URE and CREs together have all properties of a classical enhancer [10, 11, 17]. Conse-

pSVOCAT

feature of the [18] that the

the perfect palindrome and renders sumably, the mouse and rat homologs incapable of binding CREB [18]. Because the URE alone is inactive,

tissue-specific

[11, 17]. Stated

a unique we reported

of the a-Subunit Gene a Functional CRE

C to T transition at the fourth position TGACGTCA sequence [18, 25, 26]. This

a heterolis tis-

The latter property emerges only when the URE or two CREs are linked to the minimal a-subunit

ferently, the sion because

CREs are Recently,

regions contain

The

[14],

the the

Tandem gene.

Region Lacks

5.0

10.0

Activity

FIG. 1. A single nucleotide transition creates a functional CRE and restores activity to the bovine n-subunit promoter after transfection in human choriocarcinoma cells. pSVOCAT and pBaCAT were constructed by fusing the indicated promoter-regulatory regions from either the Rous sarcoma virus or the bovine n-subunit gene by ligation into the Hindlhl site in pSVOCAT 110, 18). pHuBaCAT contains the human a sequence (-170 to -100) linked directly to the downstream sequence (-114 to +48) of the bovine promoter, forming a hybrid promoter-regulatory region containing the human URE-CRE linked to the bovine promoter. p(s18)HuBaCAT is identical to pHuBoCAT except that one copy of the tandem CREs has been deleted. pBa*CAT contains the bovine a 5’-flanking sequence (-166 to +48), with a point mutation that creates a functional CRE (T to C at position -136), linked to CAT. The resulting constructs were transfected into BeWo choriocarcinoma cells 110, 18). Each transfected plate was subcultured; half of the cells were treated with medium containing 1 mM 8-Br-cAMP, and the other half were given control medium. Cell lysates were prepared and subsequently tested for CAT activity (271. The values reported for CAT activity are means and SEM from an average of six transfections (minimum of three). CAT activity is expressed as percent conversion per hour per 100 micrograms of protein. Reproduced with permission from Bokar et al. 1181,

a-SUBUNIT

fection into observations

human choriocarcinoma that warrant consideration.

specific

enhancer

100; minimal

URE plus bovine

of the

human

GENE

cells.

IN PRIMATE

There are three First, the placenta-

a-subunit

two tandem GREs) a-subunit promoter

EXPRESSION

gene

conferred (region

HORSE

AND

to determine of functional

(-170

to

activity -114 to

to a + 48;

if there is a correlation between the presence GRE and placental expression of the a-subunit

gene. Oligodeoxyribonucleotides flanking the human a-subunit served

between

the

construct pHuBaCAT). This suggested that inactivity of the parent bovine construct, which contains 313 bp of 5’-flanking sequence (pBaCAT) was due to a defective placentaspecific enhancer. Second, a truncated human a-subunit

used as primers primate genes

enhancer containing ferred activity to the

zee)

the URE and only one GRE still bovine promoter (p(M8)HuBaCAT),

but to a lesser extent. This suggested tional GRE was required to unmask tivity of the human URE. bovine a-subunit promoter

GRE. This change shown). Activity

(pBa*CAT)

was

restored of this

identical

of the cells

a single nucleotide change sequence of the bo-

CREB binding activity (data modified bovine promoter

to activity

of the

minimal

Thus,

unit

for placenta-specific

gene

confers

potential

Mouse Placenta Factors Required a-Subunit Gene Although plain

why

dents critical

and cattle, trans-acting

that

the

is also

gene

placentas factors,

required

for

from such

from

for CAT activity, tive, consistent above human the

URE-binding

centas. binding expression

used

monkeys [33], these data in conjunction with a URE,

gene

of tandem

of

lower

GREs

was

primates

a recent

indicates

that

evolutionary

P1

P3

p

TATA

-149

103

ttacacca

in placentas

of ro-

Gorilla

ate

aaattgacgtcatggtaa

aaattgacgtcatggtaa

ttacacca

these species may as the URE-binding

also lack protein

Baboon

ate

aaattgacgteatggtaa

ttacacea

Rhesus

ate

aaattgacgteatggtaa

ttaeaeca

Horse

ate

aaattgaIgteat-taa

ttaea3a

Cattle

at

aaattgalgteatggtaa

ttaIQa

Sheep

aQ

aaattgalgteatggtaa

ttaIQa

Pig

ate

aaattgalgtcatggtaa

ttaeaea

Rabbit

ate

aaattgalgtcatggtaa

ttacaeca

Dog

ate

aaattgaIgtcatgtaa

ttacaa

Mouse

ate

Rat

ate

further important

expression.

test

To

examined

in mammalian suggests for

genes

PCR [28, 29] to survey regions from several

r

P2

aaattgacgtcatggtaa

is conserved

ac-

event.

aaattgacgtcatggtaa

was acreported

as well.

Gene Event

aattgalgtcatggtaa aaatgaIgtcatggtaa

ttalaeea ttalaeea

pla-

that the UREplacenta-specific

The Tandem CREs of the Human a-Subunit to be the Product of a Recent Ez’olutionary We have moter-regulatory

and rhesus GRE, acting

ate

is silent

of

direct placenta-specific expression of the Furthermore, the presence of a single GRE

a-subunit

a

b

cells. Thus, expression of the in mouse placenta suggests that

of other

genes

P.Chimp

is sufficient

transgene studies

be

Since

[31,32] a single

a-subunit

and rhesus monkey) contain GG is synthesized in placentas

to ex-

GRE

only the human a CAT with the gene transfection

of a number

In contrast,

ttacacca

were

Such conservation protein may

GREs. (baboon bona fide

genes of chimpan-

aaattgacgtcatggtaa

embryos

protein

18] were

aaattgacgtcatggtaa

mouse

for choriocarcinoma a CAT transgene

[2-4,

atc

placenta-specffic

transgenic

the

genes

expression.

this possibility, we established lines of transgenic mice containing chimeric constructs composed of the chloramphenicol acetvltransferase (CAT) gene [27] linked to the promoter-regulatory region of either the human (-1 500 to +45) or bovine (-313 to +48) a-subunit gene [18]. When placentas

GRE.

is sufficient to a-subunit gene. in

bovine

Human

of a functional

a-subunit

tandem monkeys

of baboons suggest that

quisition

and

GRE (Fig. 2b [30,35]). The a-subunit higher primates (gorilla and pygmy

contain World

a single

human

to regions relatively con-

(P3 and P2; Fig. 2a). Sequence analysis of revealed at least one copy of a perfectly con-

retention of a functional region of the bovine a-sub-

Contains All the Necessary trans-acting to Support Expression of the Human

absence

18-bp and

corresponding GRE that were

bovine

promoter linked to the truncated human placenta-specific enhancer (pM 8). This suggests that the bovine URE was as active as the human URE even though their sequences differed by two nucleotides. URE in the promoter-regulatory

served humans Old

funcac-

Finally, complete rescue in human choriocarcinoma

could be achieved by introducing (1 to C) in the core palindromic-like vine not

that only a single the placenta-specific

con-

233

PlACENTA

Appear

the proximal prodifferent mammals

FIG. 2. PCR analysis of the proximal promoter regions of mammalian a-subunit genes. (a) The proximal promoter-regulatory region of the human glycoprotein hormone a-subunit gene contains two major regulatory elements-an upstream regulatory element (URE) and a tandemly repeated cAMP response element (CRE). Arrows indicate regions over which the primers (P1, P2, and P3) extend and the direction of synthesis during primer amplification 129, 30, 351. (b) Sequences of the primate CREs and nonprimate CRE homologs. Numbers indicate nucleotide position with respect to the transcription start site and capital letters denote nucleotide differences relative to the human gene (P. Chimp = pygmy chimpanzee). Reproduced with permission from Fenstermaker et al. 1351.

234

NILSON

The Horse a-Subunit Despite the Absence In addition to homolog contains tive GREs of cattle, horse GRE homolog

ET AL.

Gene Is Expressed in Placenta of a Functional CRE a few other changes, the same C/T transition sheep, an pigs fails to displace

genes

the CREB

GRE. This suggests do not compensate

restore GREB binding activity the horse GRE homolog fails

whether

the

horse

(Fig.

Each

of these

into human from further

that the horse a protein, even when

tracts

URE.

that

binds

specifically

to the

function

ilar

human

to the

extracts whether

a GRE, we conclude that if the horse a GRE homolog binds a protein unique to horse placenta, this protein must be quite different from the CREB family of proteins found in rat brain [23] and liver [14], human placenta [14,22], and

placenta-specific

To test

this

vectors

was

a series of of the GRE a-subunit

transfected

evidence nuclear

to to

3).

The horse a-subunit gene (Fig. 4a), suggesting that this

a protein

could

choriocarcinoma cells. These results indicated that and rhesus GRE are active whereas the GRE homologs horse, sheep, and pig are inactive. This provides

bind any nuclear protein when incubated with choriocarcinoma cell extracts or with extracts prepared from fetal equine kidney cells (FEK) (data not shown). Since FEK excontain

GRE homolog

if linked to a functional human URE, we made vectors containing a human URE linked to each homologs isolated from the various mammalian

horse a GRE as do the inac-

(Fig. 2b). Similarly, binding of human

protein [22] from the human a-subunit that these other nucleotide differences for the C/T transition and the horse GRE. In addition,

the

determine

GRE is incapable of binding linked to a functional URE. also contains a URE homolog sequence binds a protein simprotein

possibility

a

that

indirectly,

binds

we

from human choriocarcinoma the horse URE can bind the

the

prepared

cells protein

human nuclear

to determine that binds

to

a radioactive human URE sequence. As shown in Figure 4b, gel-mobility shift analysis indicates that the radioactive human URE binds to at least three different proteins or pro-

FEK. Cooperative binding occurs between proteins that bind to the URE and GRE of the human a-subunit gene [15]. To

tein complexes as distinguished molecular weights (bands 1,2,

by and

3).

differences Bands

in 1 and

native 3 are

pSVI

I

I

I

____

I-i

41--AT

ATAAT

0

J-cAMP

AT

+cAMP P I

0

-

10

.I

I

20

30

40 CAT

FIG.

3.

ActIvity

of the

CPE4Ike

sequences

found

In the

proximal

promoter.regulatory

region

‘

I

of several

50

60

I

“150

300

Activity mammalian

a-subunit

genes. Construction

of

the pSV1 vector (pXSV1CAT) has been described previously I0l. Polymarase chain reactions (29, 351 wIth primers specific for the URE and TATA box of the human a-subunit gene (P1 and P2, FIg. 2a( were used to amplify regions containing the CRE or CRE-like homologs (C) from genomic DNA of humans (H, hatched bars), rhesus monkeys (R, vertical bars), horses (E, horizontal bar.), sheep (0, backhatched bars), and pigs (P, shaded). After IsolatIon and purification, the amplified DNA fragments were cloned Into the Xba I site of the parent pSV1 vector (351. To ensure that the SV 40 early promoter directed transcription of the CAT gene, the PCR DNA fragments were cloned In the opposite orientation relative to the TATA box of the human a-subunit gene which Is present In each of the amplified DNA5. Call culture conditions, transfection, and CAMP treatments were performed as described previously 1101. All CAT activity v&uea ware within the ((near range of the assay (27); values are expressed as percent conversion of substrate, per microgram protein, per hour, relative to the parent pSV1 vector. Each value represents the mean plu, SEM of 3 Independently transfected plates. Reproduced with permission from Fensterrnaker at al. (351.

a-SUBUNIT

GENE

EXPRESSION

IN PRIMATE

HORSE

AND

235

PLACENTA

a -147

-201

Human

cacctgaaaatggctccaaac-aaaaatgacctaagggttgaaacaagataagatc

Equine

cGcctgaaaatggctcaaaacaaaaaatgatctaagAgttgaaacaagataagatc

Bovine

caTctgaaaatggctcaaaacaaaaaatAatctaagggctgaaacaagataagatA

b. hU

eU II

Competitor

50*

-

100*

250*

500x

MSV

‘l8

500*

500*

I

00*

50*

250*

500*

2 3 -

URE.

FIG. 4. The horse URE binds the same nuclear protein(s) in human (a) Comparison of the horse, cattle and human URE sequences.

choriocarcinoma Numbers indicate

spect to the transcription start site of the human a-subunit gene and capital letters relative to the human gene. (b) Gel mobility shift assay of nuclear extracts prepared cells using hU as the radioactive probe. Lane 1, no competitor; Lanes 2-5, increasing competitor; Lanes 6-9, increasing molar excess of eU competitor; Lanes 10-11, 500x competitor (MSV = CCAAT-box sequence; a18 = a-subunit CRE 110, 341). Reproduced maker et al. 1351.

sequence-specific

because

increasing

radioactive

human

URE

radioactive

human

URE.

displaces plexes the

equine

URE

when

binds

protein a 500-fold

irig elements

were

Nonradioactive

sequence

same

excess equine

human Although

URE

to the

by the

lack

molar

of two

different

added

to the

binding

reaction

a-subunit

also

expressed

human

of competi(the

If the

of the

URE to comless efficient,

specifically

excess

of non-

binding

as evidenced

box from MSV [34], and gether, these data indicate recognize the choriocarcinoma

molar displaces

binding of the radioactive 1 and 3, but less efficiently.

URE-binding tion

efficiently

After

ment fer

CCAAT

moter,

sequence-specific protein(s) in human cells, but with different affinities.

denote nucleotide differences from human choriocarcinoma molar excess of unlabeled hU molar excess of heterologous with permission from Fenster-

promoter-regulatory

lacks a functional

gene

in placenta? There radiation of oddand

binding protein may the URE now functions

cz-act-

human a GRE [10]). Taken tothat both horse and human UREs

proximal

cells that binds to the human nucleotide position with re-

like

the

GRE.

URE-binding

If true,

unmasks

with

another

in horse

activity

may

a variety cis-acting

is the

horse gene

are at least three possibilities. even-toed ungulates, the URE-

the

horse

expression

protein

interacting Thus,

only

of the

why

have evolved independently such without the aid of an accessory

placenta-specific

but

region GRE, then

URE alone to

placental

a

cells.

should

heterologous

that eleconpro-

Alternatively,

the

be promiscuous and capable of of different DNA-binding proteins. element

of the horse

may

have

emerged

URE. Finally, a URE

may

that exist

NILSON

236 in the horse

gene,

but this element

in placenta-specific expression.

may

not be involved

If so, another

element

ET AL. 8. Fiddes

JC, Talinadge

human

must

9. Naylor

provide that function. We possibilities.

are currently testing each of these

BJ. Bokar

lation

of the

URE

A functional

centa-specific Expression sory

to unmask

protein. gene

centa

clearly

of the bovine ates a functional

that

promoter confers

and

this

for

study

cells.

play

ies [18].

Murine

comes

role

placenta

our

contains

crea-

a protein

mice

stud-

binds

spe-

that

suggests

portant

for

that

structural in

tion

of the

unit

gene

quired

the

of other

placenta-specific or

in conjunction

element/trans-acting

factor

JP,

and

comments

(ed),

on

Chorionic

2. Goodwin

level

of transcriptional

glvcoprotein four

Moncman

sequence

3. Fiddes

a

21.

JA,

1981:

256:5121-6127.

Imura 6. Gharib

H (ed),

in normal and

The

Pituitan ME.

gonadotropins.

7. Wurzel

JM, Curabla

activity

of rat

placenta

1983:

113:l854-l8V.

Res

1983;

i Mol

1980:

AppI

studies Segal

of the

Gland. Shupnik

Endocrine LM. Gurr is not

New MA, Rev

York: Chin

1981;

25. Si

in the placenta

1990:

bovine

nu-

26.

pituitary

due

to

:t

chorionic

AM,

Hanson

Acad

RW, NilsonJH.

the

genes

for

Char-

the

a-subunit

(GTP). J Biol

carboxykinase JF. The

that

Chem

interact

1988;

a

basal

263:9879-9886. and

Enhancer

hormone

gonadotropin to modulate

monophosphate

promoter

mediated

a-gene.

JF.

Distinct

Mol

ele-

and

cell-

Endocrinol

adenosine

signal

transduction

by the

interactions

3,5’-

pathways

1989;

monophos-

converge

at the

of DNA-binding

RA, Andersen

TA, Fenstermaker

il-I.Expression

proteins.

B, Hamernik

of the glycoprotein

a functional

requires

GC, Andersen

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