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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):64-67 CN 31-1300/Q

Short
Communication

Molecular Cloning and
Characterization of Human Homeobox Gene Nkx3.1 Promoter
An-Li JIANG, Jian-Ye ZHANG*, Charles YOUNG1, Xiao-Yan HU, Yong-Mei WANG#, Zhi-Fang LIU, and
Mei-Lan HAO
( Department of Biochemistry, School of medicine, Shandong University,
Ji’nan 250012, China; 1 Department of Urology Research, Mayo Clinic )

Abstract Nkx3.1 is a prostate-specific homeobox
gene related strongly to prostate development and prostate cancer. To study its
regulation of transcription, 1.06 kb 5′flanking region of Nkx3.1 gene
and its 5′deletion
mutants (861, 617, 417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, a promoterless luciferase
reporter vector, to examine their promoter activities driving the reporter gene
transcription. pRL-TK, a Renilla luciferase reporter vector was used as
internal control, and pGL3-control
and pGL3-basic were used as positive and
negative control respectively. The promoter activities were determined by
dualluciferase reporter assay 48 h after pGL3 constructs were cotransfected with pRL-TK into
prostate cancer cell LNCaP. The results showed that dual-luciferase reporter
assay (M1/M2) of pGL3-1.06 kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold
higher than that of pGL3-control
cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The
results also showed that the relative activities (M1/M2)
were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861 bp, pGL3-617 bp, pGL3-417 bp, pGL3-238 bp, the last one still had 80% promoter
activity compared with pGL3-1.06
kb, which showed that deletion from 1.06 kb to 238 bp had small effects on
promoter activity. The conclusion was that the 238 bp fragment containing a
TATA box and two CAAT boxes had strong promoter activity. However, the deletion
from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417 bp
to 238 bp enhanced activity 4.7-fold, which indicated that these deleted
sequences might contain some important positive or negative regulatory elements.
It will be important to identify the elements within the Nkx3.1 promoter
that contribute to regulation of the gene transcription in the future studies.

Key
words homeobox gene Nkx3.1;
promoter; deletion mutagenesis; LNCaP cell line

Nkx3.1
is an androgen
regulated prostate-specific homeobox gene that is thought to play important
roles in normal prostate development. In mice Nkx3.1 is exclusively
expressed in prostate epithelium [1,2] and its targeted disruption leads to
aberrations in prostate ductal morphogenesis and secretory protein production,
and epithelial hyperplasia and dysplasia [3]. Notably Nkx3.1 mutant mice
display the pathologic changes of prostatic intraepithelial neoplasia (PIN) [4]
that is the presumed precursor to prostate cancer in human, which implies that
loss of Nkx3.1 expression correlates with the initiation of prostate
carcinogenesis. In human Nkx3.1 expression is generally restricted to
the prostate and it is androgen regulated [5,6]. Since Nkx.3.1 gene maps
to the chromosomal region 8p21, a region with high loss of heterozygosity in
human prostate cancer [7], the gene has been proposed to have tumor suppressor
function. Loss of Nkx3.1 protein expression has been observed in about
40% of human prostate cancers and in about 20% of PIN lesions[8]. Furthermore,
loss of Nkx3.1 protein expression correlates well with prostate tumor
progression [8]. But no mutations in the Nkx3.1 gene have been found in
prostate tumor specimens [7]. Its second allele is inactivated by some
mechanisms other than mutations in the coding region. So far, it is not well
known that how the Nkx3.1 gene expression is regulated and what
cis-acting elements and transacting factors are involved in the regulation. The
importance and specificity of Nkx3.1 gene expression in prostate prompts
us to study its promoter and regulation. We have firstly cloned the 1.06 kb 5′flanking region of Nkx3.1
gene and its 5′deletion
mutants to determine their promoter activity. It will provide an insight into
the regulatory mechanisms of Nkx3.1 gene expression in further study.

Materials and Methods

Materials

Human
genomic DNA was extracted from human white blood cells. LNCap cell line was
from ATCC. Taq DNA pol, SacI, XhoI, KpnI, HindIII
and pMD18-T vector were purchased from TaKaRa Biotech Co.. PCR primers were
synthesized by Shanghai Sangon Biological Engineering technology and Service
Co., Ltd.. pGL3-control, pGL3– basic, lipofectimineTM 2000 reagent and dual-luciferase assay
kit were purchased from Promega Co. RPMI 1640 media and fetal bovine serum were
obtained from Gibco. Other reagents were commercially available in China.

Amplification and subcloning of 1.06 kb 5′flanking
region of Nkx3.1 gene

Human
genomic DNA was extracted from blood white cells by using the method of rapid
isolation of mammalian DNA [9]. The primer pair PF (5′-CGCGAGCTCAAGGCAGGATCACTTG-
3′)
with SacI site at 5′ end and PR (5′-GGCCTCGAGCGCACCGCTTTCACTTC-3′) with XhoI
site at 5′end
were used to amplify the 5′ flanking region of Nkx3.1 gene from the extracted
human genomic DNA. The primer pair PF and PR were designed using Primer3
software according to the sequence in Genbank (accession number AC012574). The
PCR was conducted at 94 ℃ for 5 min followed by 35 cycles at 94 ℃ for 45 s, 60 ℃ for 30 s, and 72 ℃ for 1.5 min.
PCRamplified fragment was about 1.06 kb (+8 bp to –1032 bp of Nkx3.1
gene plus 18 bp of the two restriction sites for XhoI and SacI)
that was subcloned into a T/A clone vector of pMD18-T to form T/A cloning
recombinant.

Construction
of luciferase reporter plasmid

The
1.06 kb fragment was excised from T/A cloning recombinant with XhoI and SacI
and ligated into the equivalent site of pGL3-basic vector to form pGL3-1.06 kb construct. The resultant construct was
confirmed by KpnI digestion and sequence analysis using the general
primers Rvprimer3 and Rvprimer2.

Deletion
mutagenesis of the 1.06 kb promoter

5′deletion mutants of
different sizes (238, 417, 617 and 861 bp) that all begin at +8 bp of Nkx3.1
gene were obtained from pGL3-1.06
kb by PCR using one primer PR spanning +8 bp and four different primers with SacI
site at 5′end,
PFD1 (5′-CGCGAGCTCGGGAAGGAGAGGGAATTG-
3′),
PFD2 (5′-CGCGAGCTCAATTGGCTCTGACGGTCC-3′), PFD3 (5′-CGCGAGCTCACCACTTCTGCAACGGGA-
3′),
PFD4(5′-CGCGAGCTCACCGGTTGGACCAATCTG-3′). All of the
amplified fragments were separated by 1.5% agarose gel electrophoresis, excised
and purified with QIAquick gel extraction kit. Then they were cut with XhoI
and SacI and cloned into the equivalent site of pGL3-basic vector to construct 4 kinds of
deletion mutants, pGL3-238 bp, pGL3-417 bp, pGL3-617 bp and pGL3-861 bp. All of them were sequenced.

Cell
culture and transient transfection

LNCaP
cells were grown at 37 ℃ in 5% CO2 with RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and
ampicillin 100 u/ml and streptomycin 100 u/ml. Within 60 h of passage and more
than 90% confluent, LNCaP cells were transfected with lipofectimineTM 2000 in 24-well plates. Each well included
1.5×105 cells, 1 mg pGL3 construct, 0.04 mg internal control vector pRL-TK,
2 ml lipofectimineTM 2000 and 500 ml
RPMI 1640 media without serum and antibiotics. All cells were analyzed for
dual-luciferase reporter gene expression 48 h after completion of the
transfection procedure.

Dual-luciferase
reporter assay

The
activities of firefly luciferase in pGL3 and Renilla luciferase in pRL-TK were
determined following the dualluciferase reporter assay protocol recommended by
Promega. The cells were rinsed with PBS after harvest and cell lysates were
prepared by manually scraping the cells from culture plates in the presence of
1×PLB (passive lysis buffer). 20 ml of cell lysate was transferred into the
luminometer tube containing 100 ml LAR II, and firefly luciferase activity (M1) was firstly measured then Renilla
luciferase activity (M2)
was measured after adding 100 ml of Stop & Glo Reagent. The program of
luminometer was a 2 s premeasurement delay followed by a 10 s measurement
period for each assay.

Results

Amplification
and Cloning of 1.06 kb 5′flanking region of Nkx3.1 gene and its
deletion mutants

Fig.
1(A) showed the 1.06 kb fragment amplified by PCR in electrophoresis. The
fragments amplified by PCR were all cloned into pGL3-basic at the XhoI site and SacI
site to form pGL3-1.06 kb, pGL3-861 bp, pGL3-617 bp, pGL3-417 bp and pGL3-238 bp. All of the pGL3 constructs were confirmed by restriction enzyme
digestion [Fig.1 (B)] and DNA sequencing (Fig. 2).

Transient
transfection and promoter activity assay of the cloned 1.06 kb fragment

The
firefly luciferase expression driven by the 1.06 kb promoter of Nkx3.1
was examined to evaluate its promoter activity. To normalize all of the
transfection experiment an internal control plasmid pRL-TK that contain Renilla
luciferase was used to cotransfect with pGL3-1.06 kb into prostate cancer cell line LNCaP. The
relative luciferase activity of experimental sample is presented by the ratio
of the activities of firefly luciferase and Renilla luciferase (M1/M2). For the initial experiment two primary control
transfections also were conducted. One control was a transfection with pGL3-basic (promoter-less). Another control
transfection was performed with pGL3-control containing SV40 promoter and enhancer. Our result showed that
dual-luciferase reporter assay (M1/M2) was 2.7, 48 hours after pGL3-1.06 kb were cotransfected with pRL-TK
into LNCaP cells, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and
50-fold higher than that of pGL3-basic
cotransfection with pRL-TK. The cloned 1.06 kb fragment of 5′flanking region of Nkx3.1
gene presented a strong promoter activity.

Deletion
mutagenesis analysis

To
define the boundary of the functional promoter and its minimal sequence, four
of 5′deletion
mutants of the 1.06 kb were produced by PCR and analyzed by dual-luciferase
reporter assay. The results in Fig.3 showed that the relative activities (M1/M2) were 2.7, 0.71, 0.84, 0.44 and 2.07 respectively
for pGL3-1.06 kb, pGL3-861 bp, pGL3-617 bp, pGL3-417 bp and pGL3-238 bp, the last one still had 80% promoter
activity compared with pGL3-1.06
kb, which showed that deletion from 1.06 kb to 238 bp had small effects on
promoter activity. But the deletion from 1.06 kb to 861 bp reduced activity
3.8-fold while the deletion from 417 bp to 238 bp enhanced activity 4.7-fold.

Fig. 1 1.06 kb fragment of 5′flanking region of Nkx3.1 gene amplified by PCR
and identification of the cloned deletion mutants by restriction enzyme
digestion

(A)     
Amplified 1.06 kb fragment with SacI site at 5′end and XhoI site at 3′end spanning from +8 bp
to –1032 bp of Nkx3.1 gene; (B) Identification of the cloned deletion mutants
by restriction digestion. 1, DNA marker (from 100 bp to 10 kb); 2, pGL3-basic was opened by XhoI
producing 4.818 kb fragment; 3, pGL3-861 bp was cut by XhoI and SacI to excise
861 bp insert; 4, pGL3-617
bp was cut by XhoI and SacI to excise 617 bp insert; 5, pGL3-417 bp was cut by XhoI
and SacI to excise 417 bp insert; 6, pGL3-238 bp was cut by XhoI and
SacI to excise 238 bp insert;

Fig. 2 Sequence of cloned 1.06 kb fragment of 5′flanking region of Nkx3.1 gene

The cloned 1.06 kb Nkx3.1 promoter was sequenced using the general
primers Rvprimer3 and Rvprimer2. It has consensus with the sequence in Genbank.
There is a TATA box at –30 bp and two putative CAAT boxes at –64 bp and –92 bp
relative to the transcription start site. The transcription start site is G in
italic that is reported by Korkmaz KS et al. in cloning of full-length
cDNA sequence of human Nkx3.1 gene[10]. The position of the primers used
for PCR to produce 1.06 kb and its deletion mutants are double underlined.

Fig. 3 Dual-luciferase assay for deletion mutagenesis

1.06 kb fragment and its 5′deletion mutants were
obtained by PCR and cloned into pGL3-basic. Dual-luciferase assay was conducted 48 h after
prostate cancer cell LNCaP was transfected. The relative activities (M1/M2) were 2.7, 0.71, 0.84,
0.44 and 2.07 respectively for pGL3-1.06 kb, pGL3-861 bp, pGL3-617 bp, pGL3-417 bp and pGL3-238 bp. The results are presented as x ± s. ˉ

Discussions

There
is a strong association of Nkx3.1 with prostate development and prostate
cancer, making this gene an attractive molecular target for further study.
Little is known about the regulatory mechanisms of Nkx3.1 gene
expression as well as relevant regulatory elements and factors. Our research on
the Nkx3.1 gene has been initiated to define the promoter and to determine
the degree to which this gene is regulated at the transcriptional level.

In this
study, 1.06 kb 5′flanking
region of Nkx3.1 gene was amplified by PCR using human genomic DNA as
the template. To evaluate its promoter activity, the 1.06 kb fragment was
cloned into pGL3-basic vector that
contains a firefly luciferase reporter gene. Our result has shown that pGL3-1.06 kb provided a much higher level
of luciferase transcription in LNCaP cell line compared with promoterless pGL3-basic vector and also provided a
significantly higher transcription level than did pGL3-control that contained a putative strong promoter
of SV40, which indicated that cloned 1.06 kb 5′flanking region of Nkx3.1 gene
presented a strong promoter activity. To define the boundary of the functional
promoter and its minimal sequence we constructed four deletion mutants to
dissect the 1.06 kb promoter. The results showed that deletion from 1.06 kb to
238 bp had small effects on promoter activity. pGL3-238 bp still had 80% promoter activity compared
with pGL3-1.06 kb. So we concluded that 238 bp
fragment (+8 to –230 bp) containing a TATA box and two CAAT boxes was
functional in promoter activity. The deletion from 1.06 kb to 861 bp reduced
promoter activity 3.8-fold while the deletion from 417 bp to 238 bp enhanced
promoter activity 4.7-fold, which implied that these deleted sequences might
contain some important positive or negative regulatory elements. Therefore,
future experiments will be directed toward the identification of these elements.

Nkx3.1 is a newly identified
androgen-regulated homeobox gene. Previous studies [10] showed that Nkx3.1
gene is regulated by androgen and estrogen in LNCaP cells. In our experiments
we also tested the effects of androgen and estrogen on 1.06 kb promoter
activity. R1881, testosterone (10–6–10–8 mol/L), 17 â estradiol (10–7–10–9 mol/L)
and charcoal treated medium were used to treat the LNCaP cells that were
cotransfected with pGL3-1.06
kb promoter and pRL-TK. They did not appear to affect the 1.06 kb promoter
activity obviously (the results were not shown here). Androgen response element
(ARE) and estrogen response element (ERE) were not found within this 1.06 kb
fragment using software program (TRANSFAC). Maybe they are located farther
upstream of Nkx3.1 gene. There is a TATA box at –30 bp and two putative
CAAT boxes at –64 bp and –92 bp relative to the transcription start site. In
addition, multiple consensus sequence elements have been identified in the 1.06
kb  fragment using the software,
such as Nkx2.5 consensus, SRY, M Z F 1 , NF-Y and Sox-5. However, there
is no evidence now indicating which, if any, of these sequence elements are
functional. It will be important to further identify the ciselements within the
Nkx3.1 promoter that contribute to regulation of the gene transcription
functionally. Since Nkx3.1 is specifically expressed in prostate epithelial
cells, there may be a tissue-specific enhancer in the upstream regulatory regions
or in the intron of the gene need to be identified.

References

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Received: September 8, 2003 Accepted: November 5, 2003

This work was supported by a grant from the National Natural Science
Foundation of China (No. 30171026)

# Present
address: Department of biochemistry, Health School of Ji’nan

*Corresponding author: Tel, 86-531-8382092; E-mail, [email protected]