Short sequence-paper Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebra¢sh,...

www.bba-direct.com

Biochimica et Biophysica Acta 1676 (2004) 193–202

Short sequence paper

Cloning and characterization of the promoter region of the human

prostaglandin F2a receptor gene

Dean B. Zaragozaa,*, Robyn Wilsona, Kathleen Eysterb, David M. Olsona,c,d

aPerinatal Research Centre, CIHR Group in Perinatal Health and Disease, Department of Obstetrics and Gynecology, University of Alberta,

Edmonton, AB T6G 2S2, CanadabSystems Physiology and Structural Biology Research Group, Division of Basic Biomedical Sciences, University of South Dakota School of Medicine,

Vermillion, SD, USAcDepartment of Pediatrics, University of Alberta, Edmonton, AB, CanadadDepartment of Physiology, University of Alberta, Edmonton, AB, Canada

Received 6 August 2003; received in revised form 22 October 2003; accepted 7 November 2003

Abstract

The promoter region of the human prostaglandin F2a receptor (FP) gene was isolated, sequenced, and characterized. The 5V-flanking regionhas minimal homology to the bovine, mouse, and rat FP promoters with the exception of a 100-bp region. The human promoter similarly lacks

a canonical TATA-box and a CAAT-box. Potential binding sites for SP-1, GATA-1, STAT-1, and AP-1 are present in the 5V-flanking region.

One major transcription start site was identified using 5V RLM-RACE analysis and mapped to an adenine residue 262 nucleotides upstream

from the initiator codon in exon 2. Transfection of HeLa cells with FP promoter-GFP deletion constructs indicates that the � 2437/� 1946

region contains repressor activity. DNase I footprinting analysis of this region identifies a footprint over the GATA-like site at � 2400. This

suggests repression of basal FP transcription may be mediated by a GATA binding site.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Human FP promoter; Prostaglandin F2a; Transcriptional regulation

In human pregnancy, prostaglandin (PG)F2a is recognized

as one of the ‘‘triggers’’ of labor [1] because the myometrium

contracts in response to exogenous PGF2a, in vivo and in

vitro [2,3]. PGF2a acts through binding to its own plasma

membrane receptor termed the PGF2a receptor (FP). FP is a

member of a group of proteins collectively known as uterine

activation proteins (UAPs) that are involved in preparing the

uterus for the contractions of labor. As such, FP is believed to

play an important role in the maintenance and termination of

pregnancy and in women. Current data suggest that FP

expression in the human uterus is suppressed during preg-

nancy resulting in relaxation of the uterus and pregnancy

maintenance [4]. Increases in FP expression at the time of

labor are believed to contribute to uterine contraction and the

termination of pregnancy [5]. Understanding how FP is

regulated at the transcriptional level may be critical to our

understanding of how pregnancy is maintained and subse-

0167-4781/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbaexp.2003.11.004

* Corresponding author. Tel.: +1-780-492-0029; fax: +1-780-492-

1308.

E-mail address: [email protected] (D.B. Zaragoza).

quently terminated resulting in expulsion of the baby. Clues

to this regulation can be obtained from studying the FP

promoter.

FP is encoded by a single gene and is a member of the

seven-transmembrane domain receptor superfamily of G-

protein coupled receptors. The human, bovine, mouse and rat

FP gene structure has been well characterized and consists of

three exons and two introns [6–9]. Transcription initiation

sites for the FP gene have been identified for the rat, mouse,

and bovine gene. In the mouse, several sites were identified

between 222 and 274 bp upstream of the translational start

codon with the major start site 232 bp upstream of the ATG

start codon [7]. In the rat, tissue-specific transcription start

sites were identified in intron 1 as well as the typical region

f 263 bp upstream of the start ATG [9]. The bovine gene

also contains multiple initiation sites in exon 1 ranging from

98 to 265 bp upstream of ATG. Similar to the rat gene, the

bovine gene has transcription initiation sites located in intron

1 [8]. Transcription start sites have not yet been identified in

the human FP gene. In contrast to the gene, the FP promoter

is not as well characterized. Four groups have examined

sequence from the 5V flanking region of the rat [9], mouse

D.B. Zaragoza et al. / Biochimica et Biophysica Acta 1676 (2004) 193–202194

[7], bovine [8] and human [6] FP gene. A DNA fragment

containing 2590 bp of 5V flanking sequence of the rat FP

gene has been tested for regulating both basal and IL-6

mediated FP transcription. Deletion analysis identified both

positive and negative regulatory regions within this fragment

[9]. A fragment containing 1500 bp of 5V flanking region of

the mouse FP gene has been tested for promoter activity and

found to regulate transcription in a tissue dependent manner

[7], however, no specific regions or cis-acting elements were

shown to be important for regulating FP gene transcription.

Two regions of the bovine FP gene were examined for

promoter activity, 1818 bp upstream of exon 1 and 1086

bp upstream of exon 2 [8]. Again, while the region upstream

of exon 1 was found to be responsive to phorbol ester and

contain several putative transcription factor binding sites, no

specific regions or cis-acting elements were directly linked to

transcriptional regulation. While 370 bp of 5V-flankingregion of the human FP gene has been reported [6], this

region was not tested for promoter activity and no transcrip-

tion initiation start site or transcription factor binding sites

were identified. Furthermore, the authors do not suggest that

this sequence contains the entire FP promoter. It is likely that

regulatory elements could be located further upstream of the

gene and within intron 1. Crucial to the understanding of the

regulation of this molecule across human pregnancy is the

understanding of the transcriptional regulation of the gene.

Currently, very little is known about the human FP promoter.

The purpose of this study was to identify regions in the 5V-flanking sequence of the human FP gene important for

transcriptional regulation. To this end, we isolated and

characterized a larger region of 5V flanking sequence of

the human FP gene than previously reported, and examined

its ability to regulate transcription of a GFP reporter con-

struct. Upon identifying functional regions of the promoter,

we examined these regions for protein binding activity by

DNase I footprinting analysis.

The transcription initiation site of the human FP gene was

determined by Ambion’s First Choice 5V RLM-RACE

method. Fig. 1 shows the results of the analysis. A 258-bp

fragment was obtained from nested PCR reactions using the

5V RACE inner primer/hFP nested reverse primer pair (Table

1) and cDNA derived from tobacco acid pyrophosphatase

(TAP) treated myometrial RNA (Fig. 1A, lane 1). No product

was obtained when RNA was not treated with TAP (lane 2)

indicating that the 258-bp product was derived from full-

length mRNA. Lanes 3 and 4 contain nested PCR reactions

using mouse CXCR-4 primers (Table 1, 5V RACE Control

Primers) to serve as a control to validate the assay and the

expected 301-bp product was obtained in the + TAP reac-

tions (lane 3). A no-template control PCR reaction using the

hFP-RACE primers was also included in lane 5. After

cloning the 258-bp FP PCR product into pGEM-T, 20 clones

were sequenced using the hFP nested reverse primer. Of the

20 clones only five (clones 1, 2, 6, 12, and 17) contained the

adapter sequence and all of these clones mapped the 5V end

of the mRNA to an adenine 262 nucleotides upstream of the

ATG initiator codon (Fig. 1B). Eleven clones contained hFP

sequence but no adapter sequence, and for that reason we did

not believe these to contain true 5V ends. These were most

likely the result of incomplete reverse transcription. The

other four clones contained nonsense sequence containing

repeats of the hFP nested reverse primer. These clones

contained no actual FP sequence. We believe these clones

to have resulted from primer dimer artifacts of the PCR

reactions. Because our 5V RLM-RACE method gave rise to 5

clones out of 20, it is possible that we may have missed a

small population of minor transcription start sites upstream

of adenine � 265. Indeed, minor transcription start sites

have been identified upstream of the major site in the mouse,

rat, and bovine FP gene. To test for this possibility in the

human FP gene, we performed conventional RT-PCR using

two sense primers (one upstream of the adenine at � 265 and

one downstream) and one common antisense primer (hFP

nested reverse primer). Fig. 1C shows the results. Both RT-

PCR reactions using either sense primer gave rise to the

expected size PCR products. This indicates that a small

population of transcription start sites exists upstream of

adenine � 262 that was missed by our RLM 5V RACEanalysis.

Our RLM 5V RACE results are in agreement with the

major transcription initiation sites mapped for the rat (270 bp

upstream of ATG), mouse (232 bp upsteam of ATG), and

bovine (265 bp upstream of ATG) FP gene [7–9]. In addition

to minor upstream start sites, these studies also report

multiple downstream transcription initiation sites which

may have resulted from incomplete reverse transcription or

degraded FP mRNA. By only examining clones that

contained the adapter sequence, we ensure that our clones

resulted from the RT reactions which fully extended to the

true 5V end of the human FP mRNA. The RLM 5V RACE

technique also controls for false-positives resulting from

degraded mRNA.

In addition to the major transcription start sites loca-

ted f 260 bp upstream of the ATG initiator codon, the rat

and bovine FP genes have additional transcription start sites

that map to intron 1 [7,9]. The authors attribute their findings

to the existence of alternate promoter regions which may be

tissue-specific. While our analysis suggests that only one

promoter is operating in human myometrial tissue, alternate

tissue-specific promoters may still exist. An analysis of

human FP transcription initiation sites in other tissues may

reveal initiation sites in intron 1 and the existence of tissue-

specific promoters for the human FP gene.

The 5V-flanking region of the human FP gene was

isolated and cloned using a Genome Walking protocol

(Clonetech, Palo Alto, CA, USA). Using this protocol, we

obtained a 4106-bp fragment containing the first 33 bp of

exon 2, a 1450-bp intron 1, a 187-bp exon 1, and 2436 bp

upstream of the transcription start site (Fig. 2). Analysis of

putative binding sites for transcription factors was carried

out using MatInspector V2.2 [10]. As shown in Fig. 2, the

sequence lacks a TATA-box and a CAAT-box, as is the case

Fig. 1. Identification of the transcription initiation start site of the human FP gene by 5V RLM-RACE. (A) Nested PCR products from 5V RLM RACE analysis

separated by agarose electrophoresis. Calf intestinal phosphatase (CIP)-treated RNA isolated from human myometrial tissue (lanes 1 and 2) or control mouse

thymus tissue (lane 3 and 4) was left untreated (lane 2 and 4) or treated with tobacco acid phosphatase (TAP) (lanes 1 and 3). Following reverse transcription

reactions and primary PCR reactions, nested PCR reactions were performed using the 5V RACE Inner primer/hFP Nested Reverse primer pair (lanes 1 and 2) or

5V RACE Inner primer/5V RACE Inner control primer. No-template reactions (NT) were performed using the 5V RACE Inner primer/hFP Nested Reverse

primer pair (lane 5). Samples were run against a 123-bp ladder (lane 6). (B) hFP 5V RLM-RACE products were cloned into pGEM-T and sequenced. Sequence

results of the five clones that were positive for the 5V RACE adapter (5V RACE Inner Primer, boxed region) are shown. All positive clones map the 5V end of

human FP mRNA to an adenine residue 262 base pairs upstream of start ATG (designated by an arrowhead). (C) Conventional RT-PCR on RNA isolated from

human myometrial tissue. RT-PCR was performed using either a sense primer located upsteam of adenine-262 (Table 1, upstream primer) or a sense primer

located downstream of adenine-262 (hFP-GFP-f1) with a common antisense primer (hFP 1rst Reverse). Water controls for each primer pair are also included.

D.B. Zaragoza et al. / Biochimica et Biophysica Acta 1676 (2004) 193–202 195

for the murine and rat FP promoters [7,9]. Of the two

potential bovine FP promoters previously identified, the

upstream promoter is also TATA-less but the promoter

localized to intron 1 did contain a TATA-like sequence

and a CAAT-like box [8]. Although the human FP promoter

contains no conventional TATA sequence or CAAT se-

quence immediately upstream of the transcription start site,

there are two SP-1/GC elements located within 100 bp of

the transcriptional start site at positions � 114 and � 86

(Fig. 2). These GC box elements may explain the lack of a

Table 1

PCR primers

Primer Name Size (nt) Sequence (5V to 3V) Designed for: GenBank accession

number or source

Method used for:

5V RACE Outer

Primer

24 GCTGATGGCGATGAATGAACACTG RLM-RACE Adapter RLM-RACE kit RLM-5V RACE

5V RACE Inner Primer 35 CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG RLM-RACE Adapter RLM-RACE kit RLM-5V RACE

5V RACE Outer Control

Primer

24 (305) GATCACCAATCCATTGCCGACTAT (328) CXCR-4 D87747 RLM-5V RACE

5V RACE Inner Control

Primer

24 (263) GAAGTAGATGGTGGGCAGGAAGAT (286) CXCR-4 D87747 RLM-5V RACE

Upstream Primer 18 (� 93) AGGAAGAGCCGGAGGTCA (� 76) phFPprom-GFP – RT-PCR

hFP first Reverse 26 (231) GGAGATAAAAGCCAACCACTCAAGTC (206) Human FP L24470 Genome Walking and

RLM-5V RACE

hFP Nested Reverse 26 (198) TCTCAAAACTGTGCAGGATTGCAGTC (173) Human FP L24470 Genome Walking and

RLM-5V RACE

Adaptor Primer 1 (AP1) 22 GTAATACGACTCACTATAGGGC Genome Walking Adapter Universal Genome

Walking Kit

Genome Walking

Adaptor Primer 2 (AP2) 19 ACTATAGGGCACGCGTGGT Genome Walking Adapter Universal Genome

Walking Kit

Genome Walking

hFP-GFP-f1 19 (114) GGCTCCGTCTTCTGCTCCT (132) Human FP-GFP fusion L24470 Real Time PCR

hFP-GFP-r1 23 (1123) TCTTGTAGTTCCCGTCATCTTTG (1096) Human FP-GFP fusion PGlow-Topo Real Time PCR

pSV-Bgal-f1 23 CCGTAGATAAACAGGCTGGGACA pSV-h-galactosidase Promega Real Time PCR

pSV-Bgal-r1 19 CGTTGAAACGCTGGGCAAT pSV-h-galactosidase Promega Real Time PCR

hGAPDHs 19 (1457) GAAGGTGAAGGTCGGAGTC (1476) Human GAPDH J04038 Real Time PCR

hGAPDHa 20 (3412) GAAGATGGTGATGGGATTTC (3392) Human GAPDH J04038 Real Time PCR

D.B.Zaragoza

etal./Biochimica

etBiophysica

Acta

1676(2004)193–202

196

GC/SP-1 GC/SP-1

Adapter Primer 2

NF-IL-6

NFκB

NF-IL-6

NF B

AP-1

AP-1

AP-1

AP-1

AP-1 (-)

AP-1 (-)

CRE

GATA (-)

GATA

GATA (-)

GATA (-)

GATA

GATA

STAT

YGATG (-)

ACTATAGGGC ACGCGTGGTC GACGGCCCGG GCTGGTCCTC AGAAATAACA TCACACATCT ACAACCATCT GATCTTCAAC AAAACTGACA AAAACAAGCA

ATGGAGAAAG GATGTCCTAT TTAATAAATG ATGTTGGGAA AACTGGCTAG CCATATGCAA AAAACTGAAA CTGGACCCCA TTCCTTATAC CTTATACAAA

AATTAACTCA AGATGGATTA AAGACTTAAA TGTAAAACCT AAAATAATAA AAACTCTAGA AAAAAACATA GGTAATACCA TTCAGGACAT AGGCATGGGC

ATAGACTTTA TGACAAAAAC ACAAAAAACA ATGACGACAA AAACACACAA AAAAATGACA ACAAAGGCCA AAATTGACAC ATGGGGTCTA ATTAAACTAA

AGAACTTCTG TACAGCAAAA GAAACTATCA TCGGAGTGAA CAGGCAACCT AGAGAATGGG AGAAATACCT AATGTAGATG ATGGGTTGAC CACCATGGCA

CATGTATACC TATGCACATG TATCTCAGAA CTTAAAGTAT AATAATTTAA AAAAAGTTAA AATAAAATAT TAATGACTTT CAAAGAAAAA AAAAGTCAAG

AAACAATAGA TGCTGGCGAG GCTATGGAGA AATAGGAATG CTTTTACACT GTTGGTAGGA ATGTAAATTA GTTCAACCAA TATGGAAGAC GGCATGGTGA

TTCCTCAAGG ATCTAGAACC AGAAATATCA TTTGATCCAG CAATCCCATT ACTGGGTATA TACCCAAAGG AATACAAATC ATTCTATTAT GAAGACACAT

GCACACATGT GTTTACTGAA GCACTATTTA CAATAGCAAA GTCATAGAAC CAACCCAAAT GCCCATCAAT AATAGACTGG ATAAAGAAAA TGTGGTACAT

ATACACCACG GAATACTACA CAGCCATAAA AAGGAATGGG ATCATGTCCT TTGCAGGGAC ATGGATGAAC CTGGATGCTA TCATCCTCAG CAAACTAGCA

CAGGAACAGA AAACCAAACA CTGCATGTTT TCACTTATAA ATGGGAGTTC AGCAATGAGA ACACATGGAC ACAGGGAGAG GAACAACATG CACCAGGGCC

TGTTAGGGGG TGGGGGGCAG GGGGAGGGAA CTTAGAGGAC AGGTCAATAG GTGCAAAAAT TTCATGTTTT TTAAAAAAGA AAAAAATTAT ATATGCACTC

ACATATTTTA AAAAATAAAG GAAAGAATAG TACTCTGTGT CATTCATATT TTCAGTATAG CTTATCACAT AATTGCCTTT TGGATACTGG CCATTCATGT

ATAATATTTC TCTAGTAACC AGGTTATCTA ACCAAAATGT TTAATTCCCT CACCATAATT AAGTAACAGT TTAGTTTACC AGTTATGATC ATATCATTTA

AAAAAATCTC AATATCTACC ACAAGGGAAA ACTGCAATCA TTCCTTTCTA CAGTTAATGG CTAATTTATT TTATCCCGCT TTTTCATTTT TTGATCCCTT

CCAATTTCCA GGAATGTTAG AGGTCCCTTC TGGTTTTAGA ACGGAGAAAC AGGGTTCGTT TCCATGTGGT ACTGATTTTT CACATAGGTA GTGGCAACAT

TTTACTACAA TAAAAAGAGC AAAAGTCAAT AATAGATTCA CATAAGTGTT TAGCTACAAA AATGAGACAC AGCTAATGGG TGTGTGTCGA AGCAATATTA

GATAAAAGCA ATTACAGAAT AAAATGGCTC AAATCGTAAT TCCTAATGAA AAATAGATTA CTTCATCATA AATTTATCTC CACGAAATTT ATTAGACGCA

TATCTCTTGG GGTATACATA TCACTAGAAA TTCTCTCTCC AGTGGACTTC TAAAATCACT GAAATATTAC TAATCCCTTA TCTGCAGGTG TACTTCAAGA

AGTCATCTCA TCCCTTTTCC TTTTCATATT CCTTAGAAAG CTGGAAACAG TCCCAGTCCT CTGTGTGGCA GTGAACCCTG AGGCTACGGG TGTTTTCATT

TACAATACTG GGCTTGAATA CCTGTTTTTA ATTCCCTGGA GATATTTGCC ACAAAATGGC TCCCTCAAGC ACGCAACCTC TTTCTACCTG GTTTCTTTAT

GCGTTTCGAG CTGGACTTGG AATGAATTGT CTGCAAGCTA CCATCCGACA GCCTAACACA CCATCTTAGG CTTTCCTCGG CTCTCCAGAC TGCTGTCTTG

CATCCCCAGA GATGGCTTGG GGGATAAAAA TGAGTTTAAA ACCAAACTTT CTTTAGCTGG TCCAGCGGCT AAGTGCAGCT CTGATGCATC CGAGCACGGT

GCCCTCTTCT CTCCCAGTAC TGCTCTGTGC CCATGGAAAA GGGGGCGGAG GCGAGAGAGA AGAGGAAGAG GCGGAGGTCA CTCGCGCGCC CCATCCCTCC

CAGGCTGCCA GCGCAGCTAG GTGCAGAGGG ATCCCAGGAG CCGCGCGCGC CCCGCAGTTT CCGCGCTAAG GGAACGAGTG CGCGGAGGGG ACGAGCGGCT

GGACCACAGC CGGCGCCCGA TCAGGATCTC CGCGCTGGGA TCGGTGGAAC TTGAGGCAGC GGCGGCGCGG GGCGCCATGG CACACCGAGC GGCTCCGTCT

TCTGCTCCTC AGAGAGCCCG GCTGGCGGCC TGGGATGACA AGGTACCATC CCTCCAGAGG CTGATCCCAA TGCATCGGCT TCGCTTATCT AGACCCAGAT

TTAATCAACT CACCCCCTGA GACACCCAGC CACAGCTTCT GCTGGCTGAG CCTGAGCACT GAGTTTGGAC CAGATTCCCC AAGCTAGGGA GACGCATAGA

GAAAGAAGGG TACTCGAGCT AACCCATCCC CATTCTGAGC TGCTCTGCGG GGGATTGGGT AGAAAGGGTC TCAGAAGAGG GAAATCGGCA CAGATTCCGC

TTCTCGCCGT TGCGGGGTTC CCTGCGGGCG GCACTGCGAG TTCCTCACCC GGGCACGTCC TCAACTCCCC GTGCCCGGGA ATGAGGGAGA AAACAGCCCT

TTGGCGTTTG AGCAATTGCC AGAAAGTTTC AGCGAGTTCG TTAGTTTGGA GGGAATGTTG CGAGGCCAAG CCGAAGGGCC GGGGGGGATA GTAACGGGCG

AAAGTCGAAC TTGTGAGTAA GACCTTGGAG AAGGCGACCG TCCTGGGGTT AGCCTAGGCA GTTCCACTCG GGCTTGGGCG CAGGAAAGGC TGGCTTCCGG

AATTCCCAGC CTCCGGGAAA GCTAGCTGCT TGGGAAACGC CCAATCGAAT TAGCTCAGAG AAGAGTGGCC GTGGCCTTGT GTATCCAGTG TCTGTGCCTC

AGGCTTCAAC CCCCTAGGGA AAGAGTGACA AGCAAATACT TCGCCTTGAA TGTTGCTACT CTGAAGCTGT AACTGGAACA GAACTTGGGC CGAGTGGAAA

ATGAACGTGG GCCCGCCCCT TCCCCCACGC GAAACAAGAA TTCTACAACA GGGGCGCTAA GACCAAAAAC CTTTCCCAGA GACTCAACAC TCTGCTTTTC

ATGGGTTCAA CGTGTGTGTG GTTTGCTGTT ATTTGATTTA TGCTTAGAAA CCATATTCAG AAAATCCGCA TTTAACATCC AATGAACTTG CTGGTCCCCT

TTTCCAAAGT TTGGAGATAG TTATGTCTGC CTGACAAGCA GGTCGTTTAA CTGAGGAATA ATCTGCAGTT TTGGGTCAAA GCCCACCAGA GGAAAGAGAA

GACACTTTAA AAGGTCTCTC TCTCTTTTTT AATAGACTTA GGATTTAAAA AATCTTATTG GGTTTAAATC TAATCTTGGT AGCGATTTTG TAAAATTTGA

AAATTTTAAG GTGTCAGTGA CGTGTCAGGG GGACATTCAG GAGGAGGAGG AGGTGCTCAT TTGCAGAGGG GCCACAGAAG TTAACCAAGG GGTCTCAATT

TCAACAGCTT CTTCACCTTG AAAATGAAAT GTGGAACCGC AGGCAGATAT GAGCTGCTTT CAGTATGATG CAACCTTGCT TATGATAGGC CAGATAAAAC

CCATCCCACC ACTGGGTGCT GGGGCACGTC AGTCATAAAC GTCAGATGAG CAGTAATGCG GTGTTCATAA TTCCTCTCCC TTTATCCTAC AGATGTCTGG

ACTGCAATCC TGCACAGTTT TGAGA

-2455

-2355

-2255

-2155

-2055

-1955

-1855

-1755

-1655

-1555

-1455

-1355

-1255

-1155

-1055

-955

-855

-755

-655

-555

-455

-355

-255

-155

-55

46

146

246

346

446

546

646

746

846

946

1046

1146

1246

1346

1446

1546

1646

GC/SP-1 GC/SP-1

Adapter Primer 2

NF-IL-6

NF B

NF-IL-6

NFκB

AP-1

AP-1

AP-1

AP-1

AP-1 (-)

AP-1 (-)

CRE

GATA (-)

GATA

GATA (-)

GATA (-)

GATA

GATA

STAT

YGATG (-)











































-2455

-2355

-2255

-2155

-2055

-1955

-1855

-1755

-1655

-1555

-1455

-1355

-1255

-1155

-1055

-955

-855

-755

-655

-555

-455

-355

-255

-155

-55

46

146

246

346

446

546

646

746

846

946

1046

1146

1246

1346

1446

1546

1646











































-2455

-2355

-2255

-2155

-2055

-1955

-1855

-1755

-1655

-1555

-1455

-1355

-1255

-1155

-1055

-955

-855

-755

-655

-555

-455

-355

-255

-155

-55

46

146

246

346

446

546

646

746

846

946

1046

1146

1246

1346

1446

1546

1646

Fig. 2. Sequence of the 4106-bp hFP promoter DNA fragment that contains � 2436 bp of 5V-flanking sequence. Shown in grey and underlined is the Adapter

Primer used for the genome walking protocol. Bases are numbered relative to the transcription start site determined from 5V RLM-RACE analysis (arrowhead).

The fragment also contains all of exon 1 and the first 33 bp of exon 2 (shaded regions) as well as intron 1. Potential transcription factor binding sites are

underlined. Shown in bold italics is the homologous region found in the mouse, rat, and bovine FP promoter.



TATA box as they are found in many TATA-less promoters.

It is believed that SP-1 can function as a tethering moiety to

recruit the general transcription machinery to TATA-less

promoters [11–14]. This is also supported by the existence

of SP-1/GC box elements immediately upstream of the

transcription initiation sites for the TATA-less mouse, rat,

and bovine FP genes [7–9].

The human FP promoter bears no homology to the mouse,

rat, or bovine FP promoters with the exception of a region

Fig. 3. Ability of the hFP promoter fragment (4106 bp) to promote gene transcrip

phFPprom-GFP. (B) PCR primers (hFP-GFP f1/hFP-GFP r1) were designed acro

These primers will yield a 435-bp product if intron 1 is spliced out. (C) Results of R

with phFPprom-GFP. A minus reverse transcriptase reaction (�RT) was performe

PCR product was sequenced to verify that it was a FP-GFP fusion and that intro

between � 1013 and � 920 (Fig. 2, shown in bold italics).

This ~100-bp region is found at position � 989 of the mouse

promoter [7], � 1154 of the rat promoter [9], and � 1484 of

the bovine promoter [8], and therefore may play some

common regulatory role in transcription (see below). How-

ever, the overall lack of homology among promoters sug-

gests that regulation of transcription of the FP gene differs

among species. Nevertheless, similar transcription binding

sites are found in all of these promoters. Several GATA and

tion. (A) The hFP fragment was cloned into a GFP reporter vector to make

ss intron 1 in order to detect FP-GFP fusion mRNA in RT-PCR reactions.

T-PCR reactions from RNA isolated from HeLa cells transiently transfected

d in parallel to ensure that the 435-bp product was derived from RNA. The

n 1 was spliced out.


AP-1 sites are present throughout the human FP promoter

(Fig. 2). GATA sites were also found in the rat promoter [9]

and in the bovine FP promoter although they were not

reported [8]. AP-1 sites were also found in rat FP promoter

[9]. In addition, we identified a STAT site at position � 950

and a cAMP response element (CRE) was found at position

� 2124 (Fig. 2). A CRE site was also present in the rat

promoter [9]. Interestingly, this STAT site at � 950 and the

GATA site at � 984 are located in the region homologous to

the mouse, rat, and bovine promoter. Whether these sites

have any functional significance remains to be determined.

Intron 1 of the human FP gene was also examined for

potential transcription factor binding sites and found to

contain two NFnB sites at + 746 and + 777, and two NF-

IL-6 sites at + 593 and + 1102 (Fig. 2). The NFnB sites may

be important for the IL-1h induction of human FP mRNA in

human granulosa-luteal cells [15]. We have observed a

similar effect of IL-1h in human myometrial cells (unpub-

lished data). The NF-IL-6 sites may also be important given

recent evidence showing IL-6 regulation of FP transcription

in the rat [9]. Mutational analysis of these sites under

conditions of IL-1h or IL-6 treatment will provide more

information on their functional relevance.

The basal transcriptional activity of the 4106-bp hFP

DNA fragment was assessed by transfecting GFP reporter

constructs into HeLa cells and measuring mRNA levels of

hFP-GFP fusion mRNA. HeLa cells were used because of

the high transfection efficiency achieved, and RT-PCR

analysis of RNA isolated from HeLa cells indicated that

these cells do express FP (data not shown). The full-length

construct contains 2436 bp of sequence upstream of the

Fig. 4. Transcriptional activity of the 5V-flanking region of the human FP gene. He

5V deletions of the hFP promoter. Cells (1�105) were plated in each well of si

antimycotic (Invitrogen) for 24 h (f 60% confluent). Cells were co-transfected wi

(Promega, Canada) (to normalize for transfection efficiency) using Effectene Trans

with Qiagen’s EC buffer (Qiagen proprietary compound mixture) to 100 Al. Twelveto each sample and incubated 5 min at room temperature. Fifteen microliters of

incubation at room temperature. After addition of 600 Al of growth media (D-MEM

to the HeLa cells and incubated overnight. Following a 19-h incubation, cells were

determined by real-time RT-PCR. Both FP-GFP mRNA levels and h-galactosidasewere then corrected for transfection efficiency by dividing this value by GAPDH

independent experiments. Values greater than two standard deviations from the m

Student’s t tests were performed on consecutive paired samples.

transcription initiation site as well as 1450 bp of intron 1

(Fig. 3A). RT-PCR reaction primers (Table 1, hFP-GFP f1/

hFP-GFP r1) spanned intron 1 and were designed to detect a

FP-GFP fusion mRNA of 435 bp if intron 1 was properly

spliced out of the fusion mRNA (Fig. 3B). We were able to

detect a single RT-PCR product, f 435 bp, from RNA

isolated from HeLa cells transfected with the full-length

phFPprom-GFP construct (Fig. 3C). Sequencing confirmed

that this product was a FP-GFP fusion that was missing

intron 1. This result indicates that the 4106-bp hFP fragment

is able to promote basal gene transcription.

In order to identify basal transcription regulatory regions

in the 5V-flanking sequence of the hFP gene, we assessed the

ability of constructs containing successive 5V deletions of

hFP promoter to direct transcription of the FP-GFP fusion

mRNA by real-time PCR. Because we are detecting actual

fusion FP-GFP transcripts (sense primer-FP; antisense prim-

er-GFP), an empty control vector is not shown since no FP-

GFP would be detected in this case. Deletion of the � 2437-

bp to � 1946-bp region resulted in a 2.6-fold increase in the

levels of FP-GFP mRNA, suggesting the presence of a

repressor element (Fig. 4). Successive deletions to � 1118

bp resulted in no significant changes in the levels of FP-GFP

mRNA; however, deleting to � 582 bp resulted in a fivefold

decrease, suggesting the presence of an enhancer element in

the � 1118/� 582 region (Fig. 4).

The existence of repressor and enhancer regions in the

FP promoter has important implications for the regulation

of pregnancy. The repressor regions may be important for

the down-regulation of FP expression observed during

pregnancy resulting in relaxation of the myometrium [4].

La cells were transfected with GFP reporter constructs containing successive

x-well culture plates and grown in D-MEM containing 10% FBS and 1�th the appropriate hFP-GFP construct and pSV-hgalactosidase control vectorfection Reagent (Qiagen Inc., Canada). Each plasmid (0.75 Ag) was dilutedmicroliters of enhancer (Qiagen proprietary compound mixture) was added

effectene was added (1:10 DNA to effectene ratio) followed by a 10-min

, 10% FBS, and 1� antimycotic), DNA–effectene complexes were added

harvested and hFP-GFP, hGAPDH, and h-galactosidase mRNA levels were

levels were first normalized to GAPDH. GAPDH normalized FP-GFP levels

normalized h-galactosidase values. Values are meansF S.E.M. from six

ean were discarded from the analysis resulting in N = 5 for some groups.


Further, the enhancer region may be important for up-

regulating FP expression at the time of birth, resulting in

the contraction of the uterus and termination of pregnancy

[5].

There are several candidate repressors that may bind this

repressor region (� 2436/� 1946). Within this region, there

are three AP-1 sites (� 2371, � 2145, and � 1969), one

CRE site (� 2124), and two GATA-like sites (� 2400,

� 2030). The presence of a CRE site with upstream AP-1

sites may be relevant for transcriptional repression. AP-1

sites in this configuration, through binding CBP/AP-1

complexes, may sequester CBP away from transcription

preinitiation complexes formed on CRE sites [16]. Several

Fig. 5. DNase I footprinting analysis of the � 2436/� 1946 repressor region. phF

Labeled linearized plasmid was then digested with NcoI. The labeled SpeI/Nc

chromatography. Binding reactions were performed for 30 min on ice and containe

(pH 8.0), 20% glycerol, 1 mM DTT, 0.02 Ag/Al poly (dI–dC), 10000 cpm probe

reactions, DNase I treatments were carried out for each reaction in a staggered fas

CaCl2, 10 mM MgCl2), reactions were incubated at room temperature for 1.5 mi

temperature for 1.5 min. Samples were phenol/chloroform-extracted, ethanol-prec

Samples were run against a Maxam and Gilbert G +A sequencing reaction, and a

Full-length undigested probe was also included in the run. Four identical reaction

regions in the + nuclear extract lanes are shown with a bracket ( ] ).

factors have also been shown to bind to GATA-like

sequences (GATA/G) and mediate transcriptional repression

[17–19].

In an effort to determine whether the � 2436/� 1946

contains functional DNA binding sites, DNase I footprinting

was performed on this region (Fig. 5). Incubating a 32P-end-

labeled probe (corresponding to the � 2436/� 1946 region)

with nuclear extracts resulted in a DNase I protected region

at f� 2400 as determined by a G+A sequencing reaction

and the fX174RF marker (Fig. 5, + nuclear extract lanes).

This region corresponds to a GATA-like motif. This site may

also be capable of mediating suppression of gene transcrip-

tion through binding of the GAP-12 repressor, Ash1, or

Pprom-GFP was digested with SpeI and end-labeled with Klenow fragment.

oI fragement was purified by agarose electrophoresis and reverse-phase

d 50 mM Tris HCl (pH 8.0), 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA

and either 40 Ag of nuclear extract or extraction buffer. Following binding

hion, 30 s apart. Following addition of 50 Al of Ca2 +/Mg2 + solution (5 mM

n. Reactions were either treated with water or 0.6 U RQ DNase I at room

ipitated and loaded onto a 6% PAGE gel and analyzed by autoradiography.

n fX174 RF DNA/HaeIII ladder in order to identify the protected regions.

s are shown comparing � nuclear extact with + nuclear extract. Protected


TRPS1 [17–20]. These proteins have all been demonstrated

to repress transcription through binding to GATA-like ele-

ments. While the site found at � 2400 lacks a conventional

GATA core sequence, it does contain the GATA-like motif

YTGAT (-strand) which has been shown to bind the Ash1

repressor [19]. Mutational analysis of this site will reveal

whether it has any functional role in the repressive activity of

this region of the hFP promoter. In addition, gel shift and

supershift assays will identify which DNA binding proteins

bind to the GATA-like site at position � 2400 of the FP

promoter.

We also analyzed the enhancer region (� 1118/� 582)

for nuclear extract binding activity; however, to date, we

have not identified any DNase I protected regions (data not

shown). It is possible that protein binding to these sites

may require additional downstream sequences that are not

present in the 32P-labeled probe. A thorough mutational

analysis may be necessary to identify specific sites respon-

sible for the enhanced transcription observed in our experi-

ments. Oddly, this enhancer region of the hFP promoter

(Fig. 4, � 1118/� 582) also contains a GATA element

(� 984) and an AP-1 site (� 833), in addition to a

STAT-1 site (� 950). Whether these sites are required for

basal transcription of the hFP gene remains to be deter-

mined. This region also contains the portion of the hFP

promoter that is found in the mouse, rat, and bovine FP

promoters (� 1013 and � 920). This finding suggests the

existence of a common regulatory element of FP gene

transcription among different species. In fact, 5V deletions

of the rat FP promoter from � 1418 to � 608 which

contain this common region (starting at � 1154 of the rat

FP promoter) resulted in a f 5-fold decrease in luciferase

reporter activity [9]. To date, no analysis of the homolo-

gous region has been performed on the mouse or bovine FP

promoters. Nevertheless, our data and the rat data indicate

an important role for this homologous region in regulating

FP gene transcription. A more detailed analysis within this

100-bp region will be crucial to understanding the regula-

tion of FP gene transcription.

To date, the transcription regulatory mechanisms of the

human FP receptor are largely unknown. In this study we

have taken the initial steps to understanding how this

important molecule is regulated at the transcriptional level.

The characterization of the promoter presented here will be

important for future studies analyzing the transcriptional

regulation of FP and how it relates to the maintenance and

termination of human pregnancy. The identification of both

negative and positive cis-acting regions important for basal

transcription may provide clues to how this gene regulates

uterine contractions during pregnancy. Further, the identifi-

cation of a GATA-like site able to bind nuclear extracts in

the repressor region should prove beneficial to more in-

depth analyses of the control of transcription of the human

FP gene. Ultimately such studies will provide important

information on how this gene is regulated in the context

parturition.

Acknowledgements

The authors would like to thank Dr. Jacob T. Ross for

technical assistance. This work was supported by the Ca-

nadian Institutes of Health Research (CIHR), the Alberta

Heritage Foundation for Medical Research, the Faculty of

Medicine and Dentistry, University of Alberta, the University

of Alberta Perinatal Research Centre and the National

Science Foundation POWRE Program.

References

[1] J.R. Challis, D.M. Olson, in: E. Knobil, J. Neill (Eds.), The Physiol-

ogy of Reproduction, Raven Press, New York, 1988, pp. 2177–2216.

[2] J. Senior, K. Marshall, R. Sangha, J.K. Clayton, In vitro characte-

rization of prostanoid receptors on human myometrium at term preg-

nancy, Br. J. Pharmacol. 108 (1993) 501–506.

[3] J. Senior, R. Sangha, G.S. Baxter, K. Marshall, J.K. Clayton, In vitro

characterization of prostanoid FP-, DP-, IP- and TP-receptors on the

non-pregnant human myometrium, Br. J. Pharmacol. 107 (1992)

215–221.

[4] T. Matsumoto, N. Sagawa, M. Yoshida, T. Mori, I. Tanaka, M. Mu-

koyama, M. Kotani, K. Nakao, The prostaglandin E2 and F2 alpha

receptor genes are expressed in human myometrium and are down-

regulated during pregnancy, Biochem. Biophys. Res. Commun. 238

(1997) 838–841.

[5] J. Brodt-Eppley, L. Myatt, Prostaglandin receptors in lower segment

myometrium during gestation and labor, Obstet. Gynecol. 93 (1999)

89–93.

[6] R. Betz, J. Lagercrantz, D. Kedra, J.P. Dumanski, A. Nordenskjold,

Genomic structure, 5V flanking sequences, and precise localization in

1P31.1 of the human prostaglandin F receptor gene, Biochem. Bio-

phys. Res. Commun. 254 (1999) 413–416.

[7] K. Hasumoto, Y. Sugimoto, M. Gotoh, E. Segi, A. Yamasaki, M.

Yamaguchi, H. Honda, H. Hirai, M. Negishi, A. Kakizuka, A. Ichi-

kawa, Characterization of the mouse prostaglandin F receptor gene: a

transgenic mouse study of a regulatory region that controls its ex-

pression in the stomach and kidney but not in the ovary, Genes Cells 2

(1997) 571–580.

[8] T. Ezashi, K. Sakamoto, K. Miwa, E. Okuda-Ashitaka, S. Ito, O.

Hayaishi, Genomic organization and characterization of the gene en-

coding bovine prostaglandin F2alpha receptor, Gene 190 (1997)

271–278.

[9] F. Neuschafer-Rube, U. Moller, G.P. Puschel, Structure of the 5V-flanking region of the rat prostaglandin F(2alpha) receptor gene and

its transcriptional control functions in hepatocytes, Biochem. Bio-

phys. Res. Commun. 278 (2000) 278–285.

[10] K. Quandt, K. Frech, H. Karas, E. Wingender, T. Werner, MatInd and

MatInspector: new fast and versatile tools for detection of consensus

matches in nucleotide sequence data, Nucleic Acids Res. 23 (1995)

4878–4884.

[11] P.W. Rigby, Three in one and one in three: it all depends on TBP, Cell

72 (1993) 7–10.

[12] B.F. Pugh, R. Tjian, Diverse transcriptional functions of the multi-

subunit eukaryotic TFIID complex, J. Biol. Chem. 267 (1992)

679–682.

[13] B.F. Pugh, R. Tjian, Mechanism of transcriptional activation by Sp1:

evidence for coactivators, Cell 61 (1990) 1187–1197.

[14] W.S. Dynan, S. Sazer, R. Tjian, R.T. Schimke, Transcription factor

Sp1 recognizes a DNA sequence in the mouse dihydrofolate reductase

promoter, Nature 319 (1986) 246–248.

[15] K. Narko, O. Ritvos, A. Ristimaki, Induction of cyclooxygenase-2

and prostaglandin F2alpha receptor expression by interleukin-1beta in


cultured human granulosa-luteal cells, Endocrinology 138 (1997)

3638–3644.

[16] A.S. Guberman, M.E. Scassa, L.E. Giono, C.L. Varone, E.T. Canepa,

Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase

gene expression through sequestration of cAMP-response element

protein (CRE)-binding protein (CBP) coactivator, J. Biol. Chem.

278 (2003) 2317–2326.

[17] T.H. Malik, S.A. Shoichet, P. Latham, T.G. Kroll, L.L. Peters, R.A.

Shivdasani, Transcriptional repression and developmental functions

of the atypical vertebrate GATA protein TRPS1, EMBO J. 20

(2001) 1715–1725.

[18] F.J. Kaiser, K. Tavassoli, G.J. Van den Bemd, G.T. Chang, B. Hors-

themke, T. Moroy, H.J. Ludecke, Nuclear interaction of the dynein

light chain LC8a with the TRPS1 transcription factor suppresses the

transcriptional repression activity of TRPS1, Hum. Mol. Genet. 12

(2003) 1349–1358.

[19] M.E. Maxon, I. Herskowitz, Ash1p is a site-specific DNA-binding

protein that actively represses transcription, Proc. Natl. Acad. Sci.

U. S. A. 98 (2001) 1495–1500.

[20] C. Becker, S. Wirtz, X. Ma, M. Blessing, P.R. Galle, M.F. Neurath,

Regulation of IL-12 p40 promoter activity in primary human mono-

cytes: roles of NF-kappaB, CCAAT/enhancer-binding protein beta,

and PU.1 and identification of a novel repressor element (GA-12)

that responds to IL-4 and prostaglandin E(2), J. Immunol. 167

(2001) 2608–2618.

Short sequence-paper Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebra¢sh,...

Documents

Transcript of Short sequence-paper Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebra¢sh,...