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Research Paper	Pdf file on Synergy Omments
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 锟紺 for 2 min followed by 35 cycles at 94 锟紺 for 30 s, 60 锟紺 for 30 s, and 72 锟紺 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 锟紺 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 锟紺 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

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锟絧assive 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锟絪 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 锟紺 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锟絋ris-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.

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