Tài liệu Báo cáo khoa học: Characterization of promoter 3 of the human thromboxane A2 receptor gene A functional AP-1 and octamer motif are required for basal promoter activity docx

Hence, deletion of nucleotides between 404 and 320 removes a gene segment that has a repressive effect on Prm3 activity in both HEL and HEK293 cells whilst further deletion of nucleotide

Trang 1

A2 receptor gene

A functional AP-1 and octamer motif are required for basal

promoter activity

Adrian T Coyle and B Therese Kinsella

Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

The prostanoid thromboxane (TX)A2 induces

activa-tion and aggregaactiva-tion of platelets, constricactiva-tion of

vascu-lar (V) and bronchial smooth muscle (SM) and of

renal mesangial cells [1–4], and may induce other

diverse cellular responses including mitogenic and⁄ or

hypertrophic growth of VSM [5,6], inhibition of

angio-genesis⁄ neo-vascularization [7] and apoptosis of

CD4⁄ CD8++⁄ – thymocytes [8] Alterations in the level

of this potent autocoid, or of its speciﬁc synthase or its receptor (TP) are widely implicated in a variety of vascular diseases including thrombosis, unstable angina, asthma, systemic and pregnancy-induced hyperten-sion, and glomerulonephritis [9–12] Moreover, mice deﬁcient in the TXA2 receptor (TP–⁄ –) display increased bleeding and altered hemodynamic proper-ties, highlighting the essential role of TXA2 and its

Keywords

AP-1; gene expression; isoforms; Oct;

promoter; splicing; thromboxane receptor

Correspondence

B T Kinsella, Department of Biochemistry,

Conway Institute of Biomolecular and

Biomedical Research, University College

Dublin, Belfield, Dublin 4, Ireland

Fax: +353 1 2837211

Tel: +353 1 7166727

E-mail: Therese.Kinsella@ucd.ie

(Received 18 October 2004, revised 8

December 2004, accepted 20 December

2004)

doi:10.1111/j.1742-4658.2004.04538.x

The TPa and TPb isoforms of the human thromboxane A2 receptor (TP) arise by differential splicing but are under the transcriptional control of two distinct promoters, termed Prm1 and Prm3, respectively (Coyle et al

2002 Eur J Biochem 269, 4058–4073) The aim of the current study was to determine the key factors regulating TPb expression by functionally charac-terizing Prm3, identifying the core promoter and the cis-acting elements regulating basal Prm3 activity Hence, the ability of Prm3 and a series of Prm3 deleted⁄ mutated subfragments to direct reporter gene expression in human erythroleukemia 92.1.7 and human embryonic kidney 293 cells was investigated It was established that nucleotides )118 to +1 are critical for core Prm3 activity in both cell types Furthermore, three distinct regulatory regions comprising of an upstream repressor sequence, located between )404 to )320, and two positive regulatory regions required for efficient basal gene expression, located between )154 to )106 and )50 to +1, were identified within the core Prm3 Deletion and site-directed mutagenesis of consensus Oct-1⁄ 2 and AP-1 elements within the latter )154 to )106 and )50 to +1 regions, respectively, substantially reduced Prm3 activity while mutation of both elements abolished Prm3 activity Electromobility shift and supershift assays confirmed the specificity of nuclear factor binding to the latter Oct-1⁄ 2 and AP-1 elements Moreover, herein it was established that the core AP-1 element mediates phorbol myristic acid-induction of Prm3 activity hence providing a mechanistic explanation of phorbol ester up-regulation of TPb mRNA expression

Abbreviations

AP-1, activator protein-1; EMSA, electromobility shift assay; FBS, fetal bovine serum; HEK, human embryonic kidney; HEL, human

erythroleukemia; I, intron; NT, nucleotide; PMA, phorbol myristic acid; Prm, promoter; RLU, relative luciferase units; TP, thromboxane receptor; TI, transcription initiation; TXA2, thromboxane A2; UAS, upstream activation sequence; URS, upstream repressor sequence; UTR, untranslated region.

Trang 2

receptor in the dynamic regulation of haemostasis

[13,14]

As a member of the G protein coupled receptor

(GPCR) superfamily, the TXA2 receptor or TP is

pri-marily coupled to Gq-dependent activation of

phos-pholipase (PLC) Cb isoforms [1,3] In humans, but not

in nonprimates, TXA2signals through two TP isoforms

referred to as TPa and TPb that are encoded by a single

TP gene located on chromosome 19p13.3 and that arise

through a novel differential splicing mechanism

invol-ving retention of bifunctional intronic⁄ exonic sequences

within the TPa mRNA [15–17] TPa and TPb are

iden-tical for their N-terminal 328 amino acids but differ

exclusively in their C-terminal domains [16,17] Whilst

TPa and TPb mediate almost identical PLCb effector

activation, they differentially couple to adenylyl cyclase

via Gas and Gai, respectively [18], and TPa, but not

TPb, couples to the novel high molecular weight

G-pro-tein Gh [19] TPa and TPb also undergo differential

homologous (agonist-dependent) and heterologous

desensitization For example, TPb, as opposed to TPa,

undergoes tonic and agonist induced-internalization

[20,21] On the other hand, TPa, but not TPb,

undergoes inhibitory cross-talk or heterologous

desensitization of its signaling in response to the potent

anti-aggregatory⁄ vasodilatory autocoids prostaglandin

(PG) I2 (prostacyclin) and nitric oxide (NO) through

mechanisms involving direct cAMP- and

cGMP-dependent protein kinase phosphorylation, respectively,

of TPa within its unique C-tail domain [22,23]

Hence, whilst the biological signiﬁcance for the

existence of two TP receptors in humans is indeed

unclear, there is mounting evidence that they undergo

differential signaling and regulation, strengthening the

viewpoint that TPa and TPb may have distinct

physio-logic⁄ pathophysiologic roles Consistent with this, TPa

and TPb are also subject to differential expression and

gene regulation [24,25] Whilst TPa and TPb mRNAs

are coexpressed in a range of cell⁄ tissue types of

rele-vance to TXA2 biology, there are extensive differences

in the relative levels of expression of TPa: TPb mRNA

in several tissues [24] Moreover, recent studies have

conﬁrmed that TPa and TPb expression are under the

genetic control of distinct promoters within the single

human TP gene located on chromosome 19 [16,25]

Whilst the originally identiﬁed promoter (Prm) 1

directs TPa expression, a novel promoter (Prm3) was

identiﬁed within the human TP gene that exclusively

directs TPb expression [25] Similar to that of the

pre-viously characterized Prm1 and Prm2, Prm3 lacks a

consensus TATA box or initiator element and, hence,

the transcription factor elements directing basal

Prm3-activity remain to be identiﬁed [25]

The aim of the current study was to define the core promoter and to identify the cis-acting elements regu-lating basal Prm3 activity with the view to determining the key factors that regulate TPb expression in human subjects Through 5¢- and 3¢-deletion analyses it was found that the nucleotides between )118 to +1 were required for core Prm3 activity, where +1 represents the translational ATG start codon and the transcrip-tion initiatranscrip-tion of the TPb mRNA was previously identified at)12 [25] Furthermore, three distinct regu-latory regions were identified, the first of which was an upstream repressor sequence (URS) located between )404 and )320 and was found to have a repressive effect on the basal Prm3 activity in both cell types Two additional regions that positively regulate or are required for efficient basal Prm3-directed gene expres-sion were also identified within the core Prm3 Detailed characterization of the consensus transcrip-tion factor elements within these latter sites revealed a crucial role for both an Oct-1⁄ 2 and an activator pro-tein-1 (AP-1) element in the regulation of basal Prm3 activity It is anticipated that the functional characteri-zation of Prm3 reported herein should provide critical knowledge of the modes of regulation of TPb expression and hence may shed further insights as to the physiologic requirement for two TP receptors, namely TPa and TPb, in humans

Results

Functional analysis of promoter 3 of the human TXA2receptor (TP) gene

We have previously identiﬁed a novel promoter (Prm)3 within the human TXA2 receptor (TP) gene that directs expression of TPb in HEL92.1.7 and HEK293 cells [25] A schematic of the human TP gene highlight-ing the positions of the previously identiﬁed Prm1, Prm2 [16,25] and the novel Prm3 [25] relative to its translational start site (ATG, designated +1) is presented in Fig 1 In order to gain further insights into the modes of regulation of TPb, the aim of the current study was to map the minimal transcriptional unit and to identify the key regulatory elements within Prm3 directing basal gene expression

The recombinant pGL3Basic encoding Prm3 direc-ted 3.65 ± 0.23 RLU and 3.0 ± 0.25 RLU of luci-ferase activity in HEL (Fig 1A) and HEK293 (Fig 1C) cells, respectively, whilst the empty pGL3 Basic vector directed minimal activity in either cell type (Fig 1A,C) Progressive 5¢ deletion of Prm3 sequences in pGL3Basic to generate )975 and )404 subfragments did not signiﬁcantly affect luciferase

Trang 3

expression in either HEL (Fig 1A) or HEK293

(Fig 1C) cells Similarly, whilst the overall levels of

Prm3-luciferase activity directed by the pGL3Enhancer

plasmids, containing an SV40 enhancer element

down-stream of the luciferase gene, were generally two- to

three-fold higher than by the equivalent pGL3Basic

plasmids in both HEL (7.57 ± 0.53 RLU; Fig 1B)

and HEK (9.60 ± 0.41 RLU; Fig 1D) cells, there was

no signiﬁcant difference in luciferase expression

direc-ted by the corresponding )1394, )975 or )404

frag-ments cloned into pGL3Enhancer in either HEL

(Fig 1C) or HEK293 (Fig 1D) cells Moreover, the

empty pGL3Enhancer vector yielded minimal

luci-ferase activity in either cell type (Fig 1B,D) Hence,

deletion of sequences between )1394 and )404 of

Prm3 does not affect Prm3-directed basal gene

expres-sion

However, further 5¢ deletion of Prm3 from a)404 bp

to a )320 bp fragment expressed in either pGL3Basic

or pGL3Enhancer yielded approximately two-fold increases in luciferase activity in both HEL (Fig 1A,B) and HEK293 (Fig 1C,D) cells Moreover, further 5¢ deletion of the)320 bp to a )154 bp fragment did not signiﬁcantly affect the level of luciferase expression in either pGL3Basic or pGL3Enhancer vectors expressed

in either HEL (Fig 1A,B) or in HEK293 (Fig 1C,D) cells Hence, deletion of nucleotides between )404 and )320 removes a gene segment that has a repressive effect on Prm3 activity in both HEL and HEK293 cells whilst further deletion of nucleotides between)320 and )154 had no additional effect on luciferase expression

in either cell type

Further 5¢ deletion of nucleotides )154 to )106 resulted in between two- and eight-fold decreases in

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

-8500

-3308

+786

8

A

B

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

-8500

-3308

+786 -8500

-3308

+786

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

-8500

-3308

+786

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

+1Luc

-975

pGL3Basic +1Luc

-50 +1Luc

-106 +1Luc

-8500

-3308

+786 -8500

-3308

+786

+1

+1Luc

-975

pGL3Enhancer +1Luc

-50 +1Luc

-106 +1Luc

-8500

-3308

+786

+1

+1Luc

-975

-50 +1Luc

-106 +1Luc

-8500

-3308

+786

+1

+1Luc

-975

-50 +1Luc

-106 +1Luc

-8500

-3308

+786 -8500

-3308

+786

+1Luc

-975

-50 +1Luc

-8500

-3308

+786

+1Luc

-975

-50 +1Luc

-8500

-3308

+786

+1Luc

-975

-50 +1Luc

-8500

-3308

+786

+1Luc

-975

-50 +1Luc

-8500

-3308

+786 -8500

-3308

+786

Fig 1 Effect of 5¢-deletion mutagenesis on Prm3-directed luciferase expression (A–D) A schematic figure of the human TP genomic region

above each panel Nucleotide +1 corresponds to the translational start site (ATG) and nucleotides 5¢ of that site are given a – designation.

pRL-TK into HEL92.1.7 (A and B) and HEK293 (C and D) cells Firefly and renilla luciferase activity was assayed 48 h post-transfection; results are presented as mean firefly relative to renilla luciferase activity, expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5).

Trang 4

luciferase expression in HEL (Fig 1A,B) and HEK293

(Fig 1C,D) cells indicating that the )154 to )106

sequence contains positive regulatory element(s)

required for efﬁcient basal Prm3 activity Moreover, 5¢

deletion of the )106 bp to a )50 bp fragment did not

further reduce the level of luciferase expression in

either pGL3Basic or pGL3Enhancer plasmids in either

HEL (Fig 1A,B) or in HEK293 (Fig 1C,D) cells It

was noteworthy that plasmids containing the )50 to

+1 bp subfragment of Prm3 retained low though

signiﬁcant promoter activity relative to the empty

pGL3Basic (P£ 0.05) and pGL3Enhancer (P £ 0.05)

vectors, respectively, when expressed in both HEL

(Fig 1A,B) and HEK293 (Fig 1C,D) cells Hence,

these data suggest that Prm3 contains positive

regula-tory DNA sequences between)50 and +1 in addition

to sequences between)154 and )106 required for

efﬁ-cient basal Prm3 activity

To test this hypothesis, 3¢ deletion of nucleotides

)118 to +1 abolished Prm3-directed luciferase activity

when expressed in HEL cells (Fig 2A,B) such that the

level of luciferase activity directed by the respective

recombinant plasmids was not substantially different

from that of the corresponding empty pGL3Basic

(compare 0.35 ± 0.08 RLU vs 0.11 ± 0.03 RLU)

or pGL3Enhancer (compare 0.84 ± 0.11 RLU vs

0.32 ± 0.01 RLU) vectors Similar data were observed

in HEK293 cells (data not shown) Moreover, the

possible requirement for regulatory DNA sequences 3¢

of the +1 translational start site was investigated by

comparing luciferase activity of the previously charac-terized )404 to +1 fragment to that of a )404 to +119 fragment, containing an additional 119 bp of TP genomic sequence downstream of the translational start site (Fig 2A,B) However, the level of )404 to +119 directed luciferase activity was not signiﬁcantly different from that of the Prm3-directed luciferase activity (e.g.)404 to +1) expressed in HEL cells irres-pective of whether recombinant pGL3Basic (Fig 2A)

or pGL3Enhancer (Fig 2B) based-vectors were used Similar data were observed in HEK293 cells (data not shown) In summary, we have identiﬁed three regula-tory regions within Prm3 that contribute to basal pro-moter activity, one that negatively ()404 to )320) regulates the action of Prm3 while two of which posi-tively ()154 to )106, )50 to +1) regulate basal Prm3 activity Moreover, we have conﬁrmed that nucleotides )118 to +1 are essential for the core Prm3

Identification of a functional Oct-1⁄ 2 element within promoter 3

In order to further localize and identify the positive regulatory element(s) positioned between )154 to )106

of Prm3, additional 5¢ deletions were generated in pGL3Basic Successive 5¢ deletion of nucleotides between )154 and )119 did not signiﬁcantly affect the level of luciferase activity in HEL cells (Fig 3A) sug-gesting that the latter gene segment is not required for efﬁcient basal Prm3 activity In contrast, further 5¢

+1 Luc

-118 Luc

+119 Luc

-118

-1394

E2

+1 Luc

-118 Luc

+119 Luc

-118

-1394

E2

Luciferase Activity (RLU)

+1 Luc +1 Luc

-118 Luc -118 Luc

+119 Luc +119 Luc

-118

-1394

E2

-118

-1394

E2

-1394

E2

-1394

E2

+1 Luc

-118 Luc

+119 Luc

-118

+1 Luc

-118 Luc

+119 Luc

-118

+1 Luc +1 Luc

-118 Luc -118 Luc

+119 Luc +119 Luc

-118

A

B

Fig 2 Localization of the core Prm3 by

5¢- and 3¢-deletion analysis (A and B) The

TP genomic region spanning nucleotides

)1394 to +786, and encoding Prm3 ()1394

to +1) in addition to exon (E) 2, is illustrated

above each panel Recombinant pGL3Basic

(A) or pGL3Enhancer (B) plasmids encoding

Prm3a ( )404 to +1), Prm3f ()404 to )118)

and Prm3e ( )404 to +119) were

cotransfect-ed with pRL-TK into HEL92.1.7 cells Firefly

and renilla luciferase activity was assayed

48 h post-transfection; mean firefly relative

to renilla luciferase activity are expressed in

arbitrary relative luciferase units (RLU ±

SEM; n ¼ 5) The asterisks (*) indicate that

the level of Prm3f-directed luciferase activity

was significantly reduced relative to

Prm3a-directed luciferase expression, where

Trang 5

deletion of a 13 bp gene segment between )119 and

)106 led to a 2.5-fold decrease in luciferase expression

(Fig 3A), conﬁrming that this sequence contains

positive regulatory element(s) required for basal Prm3

activity

Bioinformatic analysis of Prm3, using the

mat-inspectorTMprogram [26], for transcription factor

ele-ments between )154 and )106 identiﬁed three

consensus transcription factor binding sites including a

putative Oct-1 site centered at )123, a Oct-1 ⁄ 2 site at

)105 and an adjacent AP-1 element at )27 (Fig 3)

Hence, to investigate the role of these elements in

regu-lating basal Prm3 activity, site-directed mutagenesis

was used to disrupt the putative Oct elements located

between )154 and )106 (Fig 3B) Mutation of the

consensus Oct-1 site (GCATTTCA to GCTTCCCA)

had no effect on luciferase activity directed by the

)404 or )320 subfragments of Prm3 suggesting that

the putative Oct-1 site centered at)123 is not required

for basal Prm3 activity (Fig 3B) Conversely, mutation

of the consensus Oct-1⁄ 2 site (AAGCAAAT to

AAGCAAGT) centered at )105 signiﬁcantly reduced

luciferase activity directed by the )404 and )320 sub-fragments of Prm3 approximately 2.5- to threefold (Fig 3B) Hence, these data suggest that the latter putative Oct-1⁄ 2 element at )105 may be critical for basal Prm3 activity in HEL cells Similar data were observed in HEK293 cells (data not shown)

To conﬁrm the presence of nuclear⁄ transcription factors capable of binding to the latter Oct-1⁄ 2 site centered at)105, electromobility shift assays (EMSAs) were carried out using a radiolabeled double-stranded DNA probe spanning nucleotides )115 to )92

(Oct-1⁄ 2WT; Kin195) and nuclear extract prepared from HEL cells Incubation of the radiolabeled Oct-1⁄ 2WT probe with HEL nuclear extract resulted in the appear-ance of a single-labeled DNA–protein band (Fig 4A, lane 2) that was efﬁciently inhibited by an excess of the corresponding nonlabeled double-stranded

Oct-1⁄ 2WT oligonucleotide (Fig 4A, lane 3) or by a dou-ble-stranded oligonucleotide containing a recognized consensus Oct-1⁄ 2 (Fig 4A, lane 5) The speciﬁcity of nuclear factor binding to the latter Oct-1⁄ 2 site was also veriﬁed by the failure of excess double-stranded

AP-1 (-27)

Oct1

(-123)

Oct1 /2 (-105)

AP-1 (-27)

Oct1

(-123)

Oct1 /2 (-105)

AP-1 (-27)

Oct1

(-123)

Oct1 /2 (-105)

+1 Luc

-404

+1 Luc

-404

+1 Luc

-320

+1 Luc

-320

+1 Luc

-320

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105)

+1 Luc

-404

+1 Luc

-404

+1 Luc

-320

+1 Luc

-320

+1 Luc

-320

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105)

+1 Luc

-404

+1 Luc

-404

+1 Luc

-320

+1 Luc

-320

+1 Luc

-320

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105)

+1 Luc

-404

+1 Luc

-404

+1 Luc

-320

+1 Luc

-320

+1 Luc

-320

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105)

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105) -1394

AP-1 (-27)

Oct1 (-123) Oct1 /2 (-105) -1394

A

B

+1

Fig 3 Identification of a functional Oct-1 ⁄ 2 site within Prm3 (A and B) Scheme of the

+1) in addition to the relative positions of putative Oct-1, Oct-1 ⁄ 2 and AP-1 elements

is illustrated above each panel Recombinant

to +1), Prm3ax ( )140 to +1), Prm3ac ()119

to +1) and Prm3aab ( )330 to +1) (A) or Prm3a ( )404 to +1) or Prm3ab ()320 to +1) and their site-directed variants Prm3a Oct-1 * and Prm3ab Oct-1 *, Prm3aOct-1⁄ 2* and Prm3aOct-1⁄ 2* (B) were cotransfected with pRL-TK into HEL92.1.7 Firefly and renilla luciferase activity was assayed 48 h post-transfection; mean firefly relative to renilla luciferase activity are expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The star symbol indicates mutated transcrip-tion factor elements The asterisks (*) indicate that either deletion or site-directed mutagenesis of Prm3 sequences signifi-cantly reduced luciferase expression in HEL cells, where **** indicates P £ 0.0001.

Trang 6

oligonucleotides containing a mutated Oct-1⁄ 2*

sequence (Kin193) or an AP-1 consensus sequence to

effectively inhibit nuclear factor- DNA complex

formation (Fig 4A, lanes 4 and 6, respectively) Western blot analysis of whole cell lysates conﬁrmed the presence of Oct-2, but not Oct-1, in HEL cells

A

E

nuclear extracts from HEL92.1.7 cells (A) 32 P-labeled Oct-1 ⁄ 2 WT probe was incubated: without nuclear extract (lane 1); with nuclear extract alone (lane 2); with nuclear extract in the presence of excess nonlabeled double-stranded specific competitor Oct-1 ⁄ 2 WT oligonucleotide

oligonucleotide (Kin193 and its complement, lane 4); with nuclear extract in the presence of excess nonlabeled double-stranded consensus

extract (lane 1); with nuclear extract alone (lane 2); with nuclear extract preincubated for 30 min with anti-(Oct-1) IgG (sc-232x; lane 3); with

oligonucleotide (Kin340 and its complement, lane 4); with nuclear extract preincubated for 30 min with anti-(Oct-2) IgG (sc-233x; lane 5); with

oligonucleotide (Kin340 and its complement, lane 6) The arrow indicates the supershifted transcription factor: DNA complex detected in the presence of the anti-(Oct-2) IgG (lane 5) DNA–protein complexes were subject to PAGE followed by autoradiography, as outlined (C and D) Western blot analysis of Oct-1 (C) and Oct-2 (D) expression in whole cell protein (60 lgÆ lane)1) prepared from HEL (C and D; lane 1) and HEK293 (C and D; lane 2) cells The positions of the molecular size markers (kDa) are indicated to the left and right of the (C) and (D), respectively, whilst the position of the Oct-1 (98 kDa approximately) and the two major forms of Oct-2 (75–80 and 55–60 kDa) detected in HEK293 and HEL cells, respectively, are indicated by arrows in (C) and (D) (E) To investigate the effect of Oct-1 or Oct-2 on Prm3-directed luciferase gene expression, HEK293 cells were transiently cotransfected with pGL3b:Prm3ab plus pRL TK in the presence of pcDNA3: HaOct-1 (Oct-1), pcDNA3:HaOct-2 (Oct-2) or, as a control, with pcDNA3 (Control) Firefly and renilla luciferase activity was assayed 48 h post-transfection; mean firefly relative to renilla luciferase activity are expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 6) The asterisks (*) indicate that over-expression of Oct-1 and Oct-2 significantly increased Prm3ab-directed luciferase expression in HEK293 cells, where * and ** indicate P £ 0.05 and 0.01, respectively.

Trang 7

(Fig 4C,D, lane 1) while Oct-1, but not Oct-2,

expression was readily detected in HEK293 cells

(Fig 4C,D, lane 2) Moreover, electromobility

super-shift assays employing anti-Oct selective antibodies

demonstrated the direct binding of Oct-2, but not

Oct-1, to the Oct-1⁄ 2 element of Prm3 within HEL

cells (Fig 4B, lane 5) Oct nuclear factor–DNA

com-plexes (Fig 4B, lanes 4 and 6) and anti-Oct-2

super-shifted complexes (Fig 4B, lane 6) were efﬁciently

competed by an excess of a non-labeled

double-stranded oligonucleotide containing a consensus

Oct-1⁄ 2 site Failure to observe an anti Oct-1 supershift

is consistent with the absence of Oct-1 in HEL cells

but did not exclude the possibility that Oct-1 may

regulate Prm3 activity in cell types where Oct-1 is

abundantly expressed Consistent with this,

heterolo-gous over-expression of both Oct-1 and Oct-2

signiﬁ-cantly increased Prm3-directed luciferase activity in

HEK293 (Fig 4E) and HEL (data not shown)

cells Hence, we have identiﬁed a consensus Oct-1⁄ 2

transcription factor site centered at )105 that is

crit-ical for efﬁcient basal Prm3-directed gene expression

and have conﬁrmed the ability of both Oct-1 and

Oct-2 to bind and regulate Prm3-directed gene

expression

Identification of a functional AP-1 element within

promoter 3

To further investigate the positive regulatory element(s)

located between )50 to +1 of Prm3 that directs low,

though signiﬁcant, luciferase activity in both HEL

and HEK293 cells (Fig 1), matinspectorTM analysis

[26] of Prm3 revealed the presence of a high

consen-sus AP-1 element centered at )27 (Fig 4) located

some 15 bp 5¢ of the previously identiﬁed

transcrip-tion initiatranscrip-tion site within the TPb mRNA [25] Hence,

to ascertain the functional role of this AP-1 site in

mediating basal Prm3 activity, its consensus core

sequence was disrupted by site directed mutagenesis

(GTGACT to GATCCT) in a range of 5¢-deletion

subfragments and the ability of the mutated AP-1

(AP-1*) relative to the AP-1WTPrm3 subfragments to

direct luciferase activity in HEL (Fig 5A,B) and

HEK293 (Fig 5C,D) cells was investigated Following

transfection into HEL cells, in general the 5¢-deletion

fragments containing the mutated AP-1* site yielded

approximately 2.5-fold reductions in luciferase activity

relative to that of the corresponding subfragments

containing an intact AP-1 site in either pGL3Basic

(Fig 5A) or pGL3Enhancer (Fig 5B) Consistent

with this, mutation of the AP-1* element within the

smallest )50 bp fragment almost abolished luciferase

activity indicating an essential role for the AP-1 element in mediating basal Prm3 gene expression Similarly, transfection of HEK293 cells with the var-ious 5¢-deletion fragments containing the mutated AP-1* site yielded between four- and ﬁve-fold reduc-tions in luciferase activity relative to that of the cor-responding subfragments containing an intact AP-1 site in either pGL3Basic (Fig 5C) or pGL3Enhancer (Fig 5D) whilst mutation of the AP-1* element within the smallest )50 bp fragment almost com-pletely abolished luciferase activity also, similar to that observed in HEL cells Hence, whilst disruption

of the AP-1 element centered at )27 signiﬁcantly reduces basal Prm3 activity and this effect appears to

be independent of the presence or absence of the pre-viously identiﬁed negative regulatory element located between )404 and )320 in either cell type, Prm3-directed gene expression shows a greater sensitivity to AP-1 disruption in HEK293 cells than in HEL cells Moreover, the absence of any discernable Prm3 activ-ity directed by the )50 bp subfragment containing the AP-1* mutation conﬁrms that there are no other reg-ulatory elements within the )50 to +1 bp region required for basal Prm3 activity

To conﬁrm the presence of nuclear⁄ transcription factors capable of binding to the latter AP-1 site centered at )27, EMSAs were carried out using a radiolabeled double-stranded oligonucleotide probe spanning nucleotides )32 to )10 (AP-1WT) of Prm3 and nuclear extracts prepared from HEL92.1.7 cells Incubation of the radiolabeled AP-1WTprobe (Kin189; Fig 6) with HEL nuclear extract resulted in the forma-tion of a single radiolabeled nuclear factor–DNA com-plex (Fig 6, lane 2) that was efﬁciently competed by

an excess of the corresponding nonlabeled double-stranded AP-1WT oligonucleotide (Fig 6, lane 3) or

by a double-stranded oligonucleotide containing a recognized consensus AP-1 sequence (Fig 6, lane 5) The specificity of nuclear factor binding to the radio-labeled AP-1WT probe was further confirmed by the failure of both a double-stranded oligonucleotide span-ning nucleotides )32 to )10 but containing a mutated AP-1* site (Kin162; Fig 6, lane 4) and a double-stran-ded oligonucleotide based on the previously identified Oct-1⁄ 2 (Oct-1 ⁄ 2WT, Kin195, Fig 6, lane 6) to inter-fere with nuclear factor: DNA complex formation Similar data were generated in HEK293 cells (data not shown) Hence, we have identified a consensus AP-1 transcription factor site centered at )27 that is critical for efficient basal Prm3-directed gene expression and have confirmed the presence and specificity of nuclear factors in HEL and HEK293 cells that specifically bind

to the latter AP-1 site

Trang 8

Examination of the coordinate regulation of

pro-moter 3 basal activity by the AP-1 and Oct-1/2

transcription factors

To determine the combined contribution of the AP-1

and Oct-1⁄ 2 cis-acting elements in directing basal

Prm3 activity, the effect of collectively mutating the

latter sites (AP-1*, Oct-1⁄ 2*) within the )320 bp Prm3 subfragment subcloned into pGL3Basic (Fig 7A) or pGL3Enhancer (Fig 7B) on luciferase activity was compared to the corresponding subfrag-ment containing the wild-type AP-1 and Oct-1⁄ 2 ele-ments Following transfection into HEL92.1.7 cells, recombinant pGL3Basic (Fig 7A) or pGL3Enhancer

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

AP-1 +1

E2

+786 +1

E2

+786

Luc

+1

-320

Luc

+1 -50 +1Luc

-50

Luc

+1 -50 +1Luc

-50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

D C

3

AP-1 +1

E2

+786 +1

E2

+786

Luc

+1

-320

Luc

+1 -50 +1Luc

-50

Luc

+1 -50 +1Luc

-50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

A - 1

+1

E2

+786 +1

E2

+786

Luc

+1

-320

Luc

+1 -50 +1Luc

-50

Luc

+1 -50 +1Luc

-50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

Luc

+1 -320

Luc

+1 -50

Luc

+1 -50

+1

AP-1 +1

E2

+786

AP-1 +1

E2

+786 +1

E2

+786

Luc

+1

-320

Luc

+1 -50 +1Luc

-50

Luc

+1 -50 +1Luc

-50

Prm3 ( )1394 to +1), a putative AP-1 element in addition to exon (E) 2 are illustrated above each panel Recombinant pGL3Basic (A and C) or

( )50 to +1) or their respective site-directed variants Prm3 AP)1*, Prm3aAP)1*, Prm3abAP)1*, Prm3aaAP)1* and Prm3aaaAP)1*, where the AP-1

renilla luciferase activity was assayed 48 h post-transfection; results are presented as mean firefly relative to renilla luciferase activity, expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The asterisks (*) indicate that mutation of the AP-1 element significantly

P £ 0.0001, respectively.

Trang 9

(Fig 7B) plasmids containing these mutations resulted

in a complete loss in Prm3-directed luciferase

expres-sion More speciﬁcally, there was a 10- to 16-fold

reduction in luciferase activity directed by the Prm3

subfragment containing the mutated AP-1*, Oct-1⁄ 2*

sites relative to the corresponding subfragments

con-taining the wild-type sequences in either pGL3Basic

(Fig 7A) or pGL3Enhancer (Fig 7B) In fact, the

level of luciferase activity directed by the AP-1*, Oct-1⁄ 2* subfragment in either pGL3Basic or pGL3 Enhancer was not substantially greater than that level found in cells transfected with the equival-ent promoter-less empty vectors (Fig 7A,B) Similar data were generated in HEK293 cells (data not shown) These data strongly indicate that the Oct-1⁄ 2 and AP-1 elements independently regulate Prm3 activ-ity and that disruption of both sites obliterates basal Prm3 activity

Investigation of the role of the AP-1 site at -27

in phorbol myristic acid (PMA) induction of promoter 3

Previous studies have shown that TPb mRNA and Prm3-directed luciferase activity in HEL92.1.7 cells is up-regulated in response to phorbol myristic acid (PMA) [25] Moreover, AP-1 elements have a well-established role in the transduction of PMA-mediated gene expression and mitotic signaling in a number of cell models [27] Hence, in the current study, we examined the effect of PMA on luciferase expression directed by Prm3 containing either the wild-type AP-1 element or its mutated AP-1* equiv-alent Following transfection into HEL92.1.7 cells, consistent with previous data, Prm3 containing the mutated AP-1* site yielded approximately 2.0-fold reductions in basal luciferase activity relative to that

of the corresponding Prm3 subfragments containing the wild-type AP-1 sequence in pGL3Basic (Fig 8A) Whilst preincubation of cells with PMA (100 nm,

16 h) yielded a 1.5-fold increase in Prm3 directed-luciferase activity, PMA did not signiﬁcantly increase luciferase activity directed by Prm3 subfragments containing the AP-1* mutation Moreover, preincu-bation of HEL cells with PMA resulted in a 2.3-fold increase in nuclear factor binding to the consensus AP-1 element centered at )27 within Prm3 relative

to vehicle-treated cells, as determined in EMSAs (Fig 8B) Nuclear factor–DNA complex formation was efﬁciently competed by an excess of a double-stranded oligonucleotide containing a consensus AP-1 site, regardless of preincubation of cells with PMA

or not (Fig 8B) Hence, to conclude, Prm3 contains

an AP-1 and Oct-1⁄ 2 element centered at )27 and )105 within the core promoter, respectively, that are critical for basal Prm3 activity and the AP-1 element mediates PMA-induction of Prm3 expression In addition, we have identiﬁed the presence of a negat-ive regulatory region between )404 and )320 upstream of the core promoter that acts as an upstream repressor sequence

Fig 6 Demonstration of nuclear factor binding to a putative AP-1

element within Prm3 by electromobility shift assay A 32 P-labeled

was used in electromobility shift assays (EMSAs) using nuclear

extracts prepared from HEL92.1.7 cells 32 P-labeled AP-1 WT probe

was incubated: without nuclear extract (lane 1); with nuclear extract

(lane 2); with nuclear extract in the presence of excess of

(Kin189 and its complement; lane 3); with nuclear extract in the

presence of excess nonlabeled double-stranded AP-1*

noncompeti-tor oligonucleotide (Kin162 and its complement where the putative

extract in the presence of excess nonlabeled consensus

double-stranded AP-1 oligonucleotide (Kin338 and its complement, lane 5);

with nuclear extract in the presence of excess nonlabeled

complement, lane 6) DNA–protein complexes were subject to

PAGE followed by autoradiography, as outlined in Experimental

procedures.

Trang 10

In humans, TXA2 signals through two receptor

iso-forms termed TPa and TPb Although the physiologic

requirement for two TXA2 receptors in humans is

unclear, alterations in TP expression are implicated in

a range of vascular diseases [9–12] Whether TPa

and⁄ or TPb independently or differentially contribute

to those disease processes in human subjects is

cur-rently unknown but in view of the extensive differences

between the TP isoforms in terms of their mechanisms

of signaling [18,19], modes of regulation⁄

desensitiza-tion [20–23] and patterns of expression [24], this

repre-sents a question of potentially immense importance

The fact that TPa and TPb are differentially expressed

and are under the transcriptional control of two

dis-tinct promoters, Prm1 and Prm3, respectively [25],

greatly adds to the complexity of TXA2 signaling and

provides an additional critical mechanism whereby the

effects of TXA2 can be modulated in an isoform

and⁄ or cell ⁄ tissue speciﬁc manner The overall aim of

the current study was to carry out a detailed functional

characterization of Prm3, identifying the cis-acting

ele-ments regulating basal Prm3 activity with a view to

deﬁning the key factors that direct TPb expression

under normal cellular conditions

Similar to that of the previously characterized Prm1,

Prm3 belongs to the class of TATA-less promoters

[16,25] In TATA-less promoters, assembly of the pre-initiation complex relies on binding of multiple general transcription factors, such as SP-1, in proximity to the transcription initiation site [28] Herein, successive 5¢-deletion of Prm3 to either 106 bp ()106 to +1) or

50 bp ()50 to +1) yielded a subfragment that retained

a signiﬁcant, albeit reduced, ability to direct reporter gene expression in both HEL and HEK293 cells whilst deletion of the 3¢-terminal 118 bp of Prm3 ()118

to +1) led to a complete loss of promoter activity in both cell types Collectively these data established that the critical core element(s) are located within the )118

to +1 region of Prm3

Upstream activation sequences (UAS) and upstream repressor sequences (URS) are gene-speciﬁc sequences controlling the rate of transcription initiation [29] Negative regulatory elements in particular have been identiﬁed in a number of TATA-less promoters [30,31] Consistent with this, successive 5¢-deletion of nucleo-tides between )404 and )320 removed a URS that has

a repressive effect (two-fold) on Prm3 activity in both HEL and HEK293 cells Further deletion of nucleo-tides between )320 and )154 had no additional effect

in either cell type suggesting that )320 to )154 region does not contribute to basal Prm3 activity The iden-tity of the transcription factors element(s) regulating the URS is unknown but will be a subject of further characterization of Prm3

+1Luc

-320

+1 Luc

-320

pGL3Basic +1 Luc

-1394

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

+1Luc

-320 +1 +1Luc Luc

-320

+1 Luc

-320 +1 +1 LucLuc -320

pGL3Basic +1 Luc

-1394

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

-1394

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105) -404 -320

A

B

+1

+1Luc

-320

+1Luc

-320

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

+1Luc

-320

+1Luc

-320

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

+1Luc

-320

+1Luc

-320

+1Luc

-320 +1 +1Luc Luc

-320

+1Luc

-320 +1 +1Luc Luc

-320

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105) -1394 -404 -320

AP-1 (-27)

Oct 1 (-123) Oct 1/2 (-105)

Fig 7 Effect of mutation of the Oct-1 ⁄ 2

and AP-1 sites on Prm3-directed luciferase

expression (A and B) The TP genomic

region spanning Prm3 ( )1394 to +1) in

addi-tion to the relative posiaddi-tions of putative

Oct-1, Oct-1⁄ 2 and AP-1 elements are illustrated

above each panel Recombinant pGL3Basic

(A) or pGL3Enhancer (B) plasmids encoding

Prm3ab ( )320 to +1) or its respective

site-directed variant Prm3ab Oct1 ⁄ 2 *,AP)1*, where

both the Oct-1 ⁄ 2 and AP-1 elements

respect-ively, was disrupted by site-directed

mutagenesis were cotransfected with

pRL-TK into HEL92.1.7 Firefly and renilla

luciferase activity was assayed 48 h

post-transfection; results are presented as mean

firefly relative to renilla luciferase activity,

expressed in arbitrary relative luciferase

units (RLU ± SEM; n ¼ 5) The asterisks (*)

indicate that either deletion or site-directed

mutagenesis of Prm3 sequences

signifi-cantly reduced luciferase expression in HEL

cells, where **** indicates P £ 0.0001.

Định dạng
Số trang	18
Dung lượng	464,27 KB