Research Paper
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Acta Biochim Biophys Sin
2005,37: 773–778
doi:10.1111/j.1745-7270.2005.00104.x
Identification of a Positive Cis-Element
Upstream of Human NKX3.1 Gene
An-Li JIANG, Peng-Ju ZHANG, Xiao-Yan
HU, Wei-Wen CHEN, Feng KONG, Zhi-Fang LIU, Hui-Qing YUAN, and Jian-Ye ZHANG*
Department
of Biochemistry, Medical School of Shandong University, Jinan 250012, China
Received: June 27,
2005
Accepted: August
11, 2005
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30470952) and the Natural Science Foundation of Shandong Province (No.
Y2004C26)
*Corresponding
author: Tel, 86-531-88382092; E-mail, [email protected]
Abstract NKX3.1 is a
prostate-specific homeobox gene related to prostate development and prostate
cancer. In this work, we aimed to identify precisely the functional cis-element
in the 197 bp region (from –1032 to –836 bp) of the NKX3.1 promoter
(from –1032 to +8 bp), which was
previously identified to present positive regulatory activity on NKX3.1 expression,
by deletion mutagenesis analysis and electrophoretic mobility shift assay
(EMSA). A 16 bp positive cis-element located between –920 and –905
bp upstream of the NKX3.1 gene was identified by deletion mutation
analysis and proved to be a functional positive cis-element by EMSA. It
will be important to further study the functions and regulatory mechanisms of
this positive cis-element in NKX3.1 gene expression.
Key words human NKX3.1; cis-acting
element; positive regulation; mutagenesis
NKX3.1 is an androgen regulated
prostate-specific homeobox gene [1,2] that is thought to play important roles
in prostate development and carcinogenesis [3–5]. The strong association
between NKX3.1 and prostate development and prostate cancer makes this
gene an attractive molecular target for further study. It provides an
excellent model to explore the relationship between embryogenesis and
oncogenesis. So far, little is known about the regulatory mechanisms of
NKX3.1 gene expression or the relevant regulatory elements and factors. To
study its transcriptional regulation, a 1040 bp promoter (from –1032 to +8 bp)
of the human NKX3.1 gene was cloned upstream of the luciferase reporter
gene in pGL3-basic plasmid and a 197 bp region extending
from –1032 to –836 bp upstream of the NKX3.1 gene was identified presenting
positive regulatory activity for luciferase reporter expression in our previous
experiments [6].In our present study, we have identified a functional positive cis-element
between –1032 and –836 bp upstream of the NKX3.1 gene; it plays an important
role in upregulating NKX3.1 gene transcription. It will be important to further
study the functions and regulatory mechanisms of this positive element in NKX3.1
gene expression.
Materials and Methods
Construction of luciferase
reporter plasmids
pGL3-1040 containing the 1040 bp NKX3.1
promoter was constructed as previously described [6] and its 5‘ deletion
mutants were generated by the polymerase chain reaction (PCR) method using pGL3-1040 as the template. The primers used in PCR were one lower primer
PF+8 and 10 upper primers, PR-1032, PD-999, PD-966, PD-945, PD-936, PD-920,
PD-904, PD-883, PD-869 and PD-835 (Table 1). The PCR was conducted at 94
?C for 2 min
followed by 35 cycles at 94 ?C for 30 s, 60 ?C for 30 s, and 72 ?C for 1 min. The PCR products were separated by 1% agarose gel electrophoresis
and purified with QIAquick gel extraction kit (QIAgene, Ontario, Canada) cut
with XhoI and SacI, and inserted into the pGL3-basic vector digested with XhoI and SacI to generate
10 constructs that were designated pGL3-1040, pGL3-1007, pGL3-974, pGL3-953, pGL3-944, pGL3-928, pGL3-912, pGL3-891, pGL3-877 and pGL3-843. All were confirmed by restriction enzyme digestion and DNA
sequencing and tested by reporter assay.A 16 bp positive cis-element from –920 to –905 bp was
identified by 5‘ deletion mutation analysis, as described above. To
confirm its positive regulatory activity, the 16 bp cis-element was
inserted upstream of the heterogeneous promoters to generate the plasmid of the
16 bp cis-element heterogeneous promoter-luciferase reporter gene. The
sequence of the synthetic 16 bp cis-element was 5‘–TCGAGTTTCCTTGTCTTTTCTGAGCT-3‘
(sense strand), and 3‘-CAAAGGAACAGAAAAGAC-5‘ (antisense strand).
In the sense strand, a 5‘ overhang XhoI and a 3‘ overhang SacI
sites (underlined) at the 5‘ and 3‘ ends, respectively, were
produced when the two strands were annealed.The double-stranded 16 bp fragment was generated by annealing the
equimolar complementary oligonucleotides in sterilized water at 95 ?C for 10 min, then the reaction
mixture was slowly cooled to room temperature. The product was inserted
upstream of the maspin gene promoter in pGL3-maspin (a gift from Dr. Charles Y. F. YOUNG, Mayo Clinic,
Rochester, USA), upstream of the SV40 gene promoter in pGL3-promoter plasmid (Promega, Madison, USA), or upstream of the
luciferase gene reporter in pGL3-basic plasmid, which was
used as a promoter-less control. The constructs were designated pGL3-M-A, pGL3-P-A and pGL3-B-A, respectively. All constructs were confirmed by DNA sequencing
analysis, then were tested by transient transfection and dual-luciferase
reporter assay described following.
Cell culture and transient
transfection
The human prostate cancer cell line LNCaP was obtained from the American
Type Culture Collection (ATCC, Manassas, USA). The cell line was established
from a lymph node metastasis of a prostate cancer patient. It expresses the
androgen receptor gene and the NKX3.1 gene. The cells were routinely
grown at 37 ?C in a 5% CO2 incubator with RPMI 1640 medium supplemented with 10% fetal bovine
serum, 100 U/ml ampicillin and 100 U/ml streptomycin.LNCaP cells were seeded in 24-well plates to approximately 90%
confluence and transiently transfected by pGL3-construct using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).
Each well included 2 ml Lipofectamine 2000, 1.0 mg pGL3-construct (10 constructs
of pGL3–NKX3.1 promoter, pGL3-M-A, pGL3-P-A, or pGL3-B-A), 0.04 mg internal control vector pRL-TK and 500 ml RPMI 1640 medium without
serum or antibiotics. Cells were harvested for dual-luciferase activity assay
after 48 h of transfection.
Dual-luciferase reporter assay
Dual-luciferase reporter assay
The activities of firefly luciferase in pGL3 and Renilla luciferase in pRL-TK were determined by the dual-luciferase
reporter assay following the protocol supplied by Promega. The cells were
rinsed with phosphate-buffered saline, then harvested using 1?passive lysis buffer. Twenty
microliters of cell lysate was transferred into the luminometer tube containing
100 ml luciferase assay reagent II. The firefly luciferase activity (M1)
was measured first, then the Renilla luciferase activity (M2) was determined
after the addition of 100 ml Stop & Glo reagent (Promega). M1/M2 was taken as the relative
activity of the pGL3-constructs.
Electrophoretic mobility shift
assay (EMSA)
Nuclear extracts were prepared from LNCaP cells using a nuclear
extraction kit (Active Motif, Carlsbad, USA) according to the manufacturer?s instructions. The sense strand of the
16 bp positive cis-element and its mutants with four-base substitution
are shown in Table 2. Equal amounts of sense and antisense
oligonucleotides were mixed and annealed in a buffer (10 mM Tris-HCl, pH 8.0,
200 mM NaCl, 1 mM EDTA) by heating to 95 ?C for 5 min and cooling slowly to room temperature. The double-stranded 16 bp cis-element was labeled with
digoxigenin (DIG). Binding reactions were performed for 20 min on ice in a 20 ml mixture
containing 0.2% (W/V) Tween-20, 1 mM EDTA, 1 mM dithiothreitol,
30 mM KCl, 20 mM HEPES (pH 7.6), 1 mg of poly(dI-dC), 0.1 mg of poly(L-Lys), 10
mg
of nuclear extract and 0.8 ng of DIG-labeled 16 bp cis-element. For the
competition experiment, unlabeled 16 bp cis-element or its mutants in
150-fold excess were added to the binding reaction mixture and co-incubated.
DNA-protein binding complexes were separated by 5% nondenaturing polyacrylamide
gel electrophoresis in 0.25?Tris-Boric acid (TBE) buffer. Electroblotting and chemiluminescence
detection were performed based on the instructions of the manufacturer of the
DIG gel shift kit (Roche, Penzberg, Germany).
Results
Construction and
identification of luciferase reporter constructs
PCR methods were used in the construction of NKX3.1
promoter-luciferase reporter plasmids and its 5‘ deletion mutants. The
lengths of PCR products were 1040 bp, 1007 bp, 974 bp, 953 bp, 944 bp, 928 bp,
912 bp, 891 bp, 877 bp and 843 bp. The related primers used in PCR are listed
in Table 1. All the constructs were confirmed to be correct by
restriction enzyme digestion (Fig. 1) and sequence analysis.
Deletion mutagenesis analysis
of NKX3.1 promoter
To identify precisely the functional cis-element within the
197 bp region between –1032 and –836 bp, we dissected the 1040 bp promoter by deletion mutagenesis
and tested their activities by using transient transfection and dual-luciferase
reporter assay. The schematic depiction of the construction is shown in Fig.
2. The results in Fig. 2 show that the deletion from –1032 to –921 bp presented
no significant effects on promoter activity while the deletion from –920 to –905 bp showed a
3-fold reduction in the promoter activity, which suggested that the 16 bp
sequence from –920 to –905 bp was important in the positive regulation of NKX3.1.
Effects of the 16 bp cis-element
on heterogeneous promoters
To confirm the positive regulatory activity of the 16 bp cis-element
and to assess whether it alone possesses positive regulatory activity, the 16
bp cis-element sequence was synthesized in vitro and inserted
upstream of the SV40 gene promoter in the pGL3-promoter, and the maspin gene promoter in pGL3-maspin. By transient transfection assay, the effects of the 16 bp cis-element
on heterologous promoter activities were tested. The results in Fig. 3
show that the 16 bp cis-element presented significant positive
regulatory effects on heterogeneous promoters and it enhanced the promoter
activity to 4.3-fold for the SV40 gene promoter, and 2-fold for the maspin gene
promoter.
Binding ability of the 16 bp
cis-element to nuclear extracts
To confirm whether the 16 bp cis-element we identified is
functional, its binding ability to nuclear extracts was determined by EMSA. The
16 bp cis-element sequence (16bp-A) was synthesized, DIG-labeled, and
reacted with nuclear extracts from LNCaP cells. A specific DNA-protein binding
complex was identified from LNCaP nuclear extracts (Fig. 4, lane 2). The
competitors used in EMSA were 16bp-m1, 16bp-m2, 16bp-m3, 16bp-m4 and unlabeled
random sequence (ARE) as shown in Table 2. The results are shown in Fig.
4. The binding of labeled 16bp-A to nuclear extract can be blocked by a
150-fold excess amount of unlabeled 16bp-A (lane 3), 16bp-m1 (lane 4) and 16bp-m4
(lane 7), but not unlabeled 16bp-m2 (lane 5), 16bp-m3 (lane 6) or ARE (lane 8).
The results indicated that the 16 bp cis-element presented a binding
ability to a specific protein in nuclear extracts from LNCaP cells. The
sequence CTTGTCTT is very important for the binding activity of the 16 bp cis-element.
Discussion
Recent studies of human cancers [7] and a mutant mouse model [8]
have implicated that the NKX3.1 homeobox gene plays a key role in
prostate carcinogenesis. In mice, NKX3.1 is a key regulator of prostatic
epithelial differentiation. NKX3.1 null mutant mice display abnormal
prostatic differentiation as well as epithelial hyperplasia and dysplasia [9].
Notably, NKX3.1 mutant mice display the pathologic changes of prostatic
intraepithelial neoplasia [10] that is the presumed
precursor to prostate cancer in humans. The NKX3.1 gene maps to the
chromosomal region 8p21 [11], a region with high loss of heterozygosity in
about 80% of human prostate cancers [12–14]. Loss of NKX3.1
protein expression is closely related with the initiation of prostate
carcinogenesis and with prostate tumor progression [7]. No
mutations in the NKX3.1 gene have been found in prostate tumor specimens [15] and its second allele is inactivated by mechanisms other than
mutations in the coding region.In this report, a 16 bp
positive cis-element was identified in the 197 bp region of NKX3.1
gene promoter. This 16 bp positive cis-element proved to be functional
by the assay of its binding ability to nuclear extracts in EMSA, and its
positive regulatory effects on heterogeneous promoters. In EMSA experiments,
the DNA-protein complex is specific because the binding complex can be blocked
by competition from the excess amount of unlabeled 16 bp cis-element and
cannot be blocked by competition from the excess amount of ARE. The results
indicated that this cis-element presented a binding ability to a
specific protein in nuclear extracts from LNCaP cells. We also synthesized four
mutants of 16 bp cis-element with four-base substitution that were used
as competitors in the EMSA. The results showed that the binding can be blocked
by competition from a 150-fold excess amount of unlabeled 16bp-m1 and 16bp-m4
and can not be blocked by competition from a 150-fold excess amount of
unlabeled 16bp-m2 and 16bp-m3, which suggested that the CTTGTCTT is the key
sequence for the binding activity of the 16 bp cis-element.In summary, we have identified a 16 bp potent positive cis-element
between –920 and –905 bp upstream of the NKX3.1 gene. Its activity is promoter
type-independent and it is likely to play an important role in regulating NKX3.1 gene transcription. It will
provide an insight into the regulatory mechanisms of NKX3.1 gene
expression in further study.
References
1 Bieberich CJ, Fujita K, He WW, Jay
G. Prostate-specific and androgen-dependent expression of a novel homeobox
gene. J Biol Chem 1996, 271: 31779–31782
2 Sciavolino PJ, Abrams EW, Yang L,
Austenberg LP, Shen MM, Abate-Shen C. Tissue-specific expression of murine Nkx3.1
in the male urogenital system. Dev Dyn 1997, 209: 127–138
3 Shen MM, Abate-Shen C. Roles
of the Nkx3.1 homeobox gene in prostate organogenesis and
carcinogenesis. Dev Dyn 2003, 228: 767–778
4 Tanaka M, Lyons GE, Izumo S.
Expression of the Nkx3.1 homeobox gene during pre and postnatal
development. Mech Dev 1999, 85: 179–182
5 Xu LL, Srikantan V, Sesterhenn IA,
Augustus M, Dean R, Moul JW, Carter KC et al. Expression profile of an
androgen regulated prostate specific homeobox gene NKX3.1 in primary
prostate cancer. J Urol 2000, 163: 972–979
6 Jiang AL, Zhang JY, Young C, Hu XY,
Wang YM, Liu ZF, Hao ML. Molecular cloning and characterization of human
homeobox gene Nkx3.1 promoter. Acta Biochim Biophys Sin 2004, 36: 64–67
7 Bowen C, Bubendorf L, Voeller HJ, Slack
R, Willi N, Sauter G, Gasser TC et al. Loss of NKX3.1 expression in
human prostate cancers correlates with tumor progression. Cancer Res 2000, 60:
6111–6115
8 Kim MJ, Bhatia-Gaur R, Banach-Petrosky
WA, Desai N, Wang Y, Hayward SW, Cunha GR et al. Nkx3.1 mutant
mice recapitulate early stages of prostate carcinogenesis. Cancer Res 2002, 62:
2999–3004
9 Bhatia-Gaur R, Donjacour AA,
Sciavolino PJ, Kim M, Desai N, Young P, Norton CR et al. Roles for Nkx3.1
in prostate development and cancer. Genes Dev 1999, 13: 966–977
10 Abdulkadir SA, Magee JA, Peters TJ,
Kaleem Z, Naughton CK, Humphrey PA, Milbrandt J. Conditional loss of Nkx3.1
in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002,
22: 1495–1503
11 Voeller HJ, Augustus M, Madike V, Bova
GS, Carter KC, Gelmann EP. Coding region of NKX3.1, a prostate-specific
homeobox gene on 8p21, is not mutated in human prostate cancers. Cancer Res
1997, 57: 4455–4459
12 Cher ML, Bova GS, Moore DH, Small EJ,
Carroll PR, Pin SS, Epstein JI et al. Genetic alterations in untreated
metastases and androgen-independent prostate cancer detected by comparative
genomic hybridization and allelotyping. Cancer Res 1996, 56: 3091–3102
13 Dong JT. Chromosomal deletions and tumor
suppressor genes in prostate cancer. Cancer Metastasis Rev 2001, 20: 173–193
14 Vocke CD, Pozzatti RO, Bostwick DG,
Florence CD, Jennings SB, Strup SE, Duray PH et al. Analysis of 99
microdissected prostate carcinomas reveals a high frequency of allelic loss on
chromosome 8p12-21. Cancer Res 1996, 56: 2411–2416
15 Ornstein DK, Cinquanta M, Weiler S, Duray
PH, Emmert-Buck MR, Vocke CD, Linehan WM et al. Expression studies and
mutational analysis of the androgen regulated homeobox gene NKX3.1 in
benign and malignant prostate epithelium. J Urol 2001, 165: 1329–1334