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. 2010 Jun 15;70(9):934-51.
doi: 10.1002/pros.21128.

Characterization of cis elements of the probasin promoter necessary for prostate-specific gene expression

JianFeng Zhang  1 , Nan Gao, David J DeGraff, Xiuping Yu, Qian Sun, Thomas C Case, Susan Kasper, Robert J Matusik
Affiliations

Characterization of cis elements of the probasin promoter necessary for prostate-specific gene expression

JianFeng Zhang et al. Prostate. .

Abstract

Background: The androgen-regulated probasin (PB) promoter has been used extensively to target transgenes to the prostate in transgenic mice; however, limited data exist on the mechanism that dictates prostate-specific gene expression. Tissue-specific gene expression involves synergistic effects among transcription factors associated in a complex bound to cis-acting DNA elements.

Methods: Using comprehensive linker scan mutagenesis, enzyme mobility shift and supershift assays, chromatin immunoprecipitation, and transgenic animal studies, we have extensively characterized the prostate-specific PB promoter.

Results: We identified a series of nonreceptor transcription factors that are bound to the prostate-specific rat PB promoter. These factors include several ubiquitously distributed proteins known to participate in steroid receptor-mediated transcription. In addition, we identified two tissue-specific DNA elements that are crucial in directing prostate-specific PB expression, and confirmed the functional importance of both elements in transgenic animal studies. These two elements are functionally interchangeable and can be bound by multiple protein complexes, including the forkhead transcription factor FoxA1, a "pioneer factor" that has a restricted distribution to some cells type that are ectoderm and endoderm in origin. Using transgenic mice, we further demonstrate that the minimal PB promoter region (-244/-96 bp) that encompasses these tissue-specific elements results in prostate-specific gene expression in transgenic mice, contains androgen receptor and FoxA1-binding sites, as well as ubiquitous transcription factor binding sites.

Conclusion: We propose that these sequence-specific DNA-binding proteins, including tissue-restricted and ubiquitous factors, create the first level of transcriptional control, which responds to intracellular pathways that directs prostate-specific gene expression.

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Figures

Fig. 1
Fig. 1
Identification of key cis-acting DNA elements within the –286PB promoter. Prostatic cell lines and non-prostatic cell lines were tranfected with the –286PB Luc reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (A), the wild type −286PB promoter activities on different cells are shown. The reporter gene activities of the wild type and mutant – 286PB promoters are shown in (B), LNCaP cells; (C), PC-3 cells; (D), COS-1 cells; (E), MCF-7 cells; and (F), PANC-1 cells.
Fig. 1
Fig. 1
Identification of key cis-acting DNA elements within the –286PB promoter. Prostatic cell lines and non-prostatic cell lines were tranfected with the –286PB Luc reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (A), the wild type −286PB promoter activities on different cells are shown. The reporter gene activities of the wild type and mutant – 286PB promoters are shown in (B), LNCaP cells; (C), PC-3 cells; (D), COS-1 cells; (E), MCF-7 cells; and (F), PANC-1 cells.
Fig. 1
Fig. 1
Identification of key cis-acting DNA elements within the –286PB promoter. Prostatic cell lines and non-prostatic cell lines were tranfected with the –286PB Luc reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (A), the wild type −286PB promoter activities on different cells are shown. The reporter gene activities of the wild type and mutant – 286PB promoters are shown in (B), LNCaP cells; (C), PC-3 cells; (D), COS-1 cells; (E), MCF-7 cells; and (F), PANC-1 cells.
Fig. 2
Fig. 2
The locations of the key cis-acting DNA elements within –286PB are shown. TS1, TS2, TATA box, and a putative CATT box are boxed. The potential Oct-1, C-jun, and NF-1 binding sites in –286PB promoter are marked with big arrows. The directions of the arrows indicate the sense or the antisense strand that matched the consensus sequence. ARBS-1 and ARBS-2 are up-lined. The lowercase c (small arrow) is the transcription initiation site (Tis) of the PB gene.
Fig. 3
Fig. 3
Mutational analysis of Oct-1, C-jun, and NFI binding sites within the –286PB promoter. (A), Wild type PB promoter sequences containing underlined putative Oct-1, C-jun, and NFI motifs were mutated (uppercase characters) as indicated in mtOct-1, mtC-jun, and mtNFI. The consensus Oct-1, C-jun, and NFI sequences are shown as cOct-1, cC-jun, and cNFI. (B), LNCaP cells were transiently transfected with wt –286PBLuc, mtOct-1, mtC-jun, or mtNFI reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein.
Fig. 4
Fig. 4
Characterization of the putative Oct-1, C-jun, and NFI binding sites within the –286PB promoter. (A), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated Oct-1 binding sites (−226/−197 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus Oct-1 fragment, wild type Oct-1 binding site, and mutant version of the Oct-1 binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, D, E and F were specifically eliminated or reduced; however, the complex B was enhanced when anti-Oct-1 antibody was added into reaction. Asterisk indicates a supershifted band with anti-Oct-1 antibody. (B), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated C-jun binding sites (−116/−87 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus C-jun fragment, wild type C-jun binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, B, C and D are specifically eliminated or reduced by a consensus C-jun competitor. The protein-DNA complex A was supershifted upon addition of anti-C-jun antibody. Asterisk indicates the supershifted band with anti-C-jun antibody. (C), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated NFI binding sites (−96/−67 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are unlabeled consensus NFI, consensus Oct-1, wt NFI binding site. Two specific protein-DNA complexes are marked as A and B. Both complexes A and B are inhibited upon addition of unlabeled consensus NFI probe. The complex A was supershifted when anti-NFI antibody was included in the reaction. Asterisk indicates a supershifted band with anti-NFI antibody.
Fig. 4
Fig. 4
Characterization of the putative Oct-1, C-jun, and NFI binding sites within the –286PB promoter. (A), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated Oct-1 binding sites (−226/−197 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus Oct-1 fragment, wild type Oct-1 binding site, and mutant version of the Oct-1 binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, D, E and F were specifically eliminated or reduced; however, the complex B was enhanced when anti-Oct-1 antibody was added into reaction. Asterisk indicates a supershifted band with anti-Oct-1 antibody. (B), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated C-jun binding sites (−116/−87 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus C-jun fragment, wild type C-jun binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, B, C and D are specifically eliminated or reduced by a consensus C-jun competitor. The protein-DNA complex A was supershifted upon addition of anti-C-jun antibody. Asterisk indicates the supershifted band with anti-C-jun antibody. (C), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated NFI binding sites (−96/−67 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are unlabeled consensus NFI, consensus Oct-1, wt NFI binding site. Two specific protein-DNA complexes are marked as A and B. Both complexes A and B are inhibited upon addition of unlabeled consensus NFI probe. The complex A was supershifted when anti-NFI antibody was included in the reaction. Asterisk indicates a supershifted band with anti-NFI antibody.
Fig. 4
Fig. 4
Characterization of the putative Oct-1, C-jun, and NFI binding sites within the –286PB promoter. (A), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated Oct-1 binding sites (−226/−197 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus Oct-1 fragment, wild type Oct-1 binding site, and mutant version of the Oct-1 binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, D, E and F were specifically eliminated or reduced; however, the complex B was enhanced when anti-Oct-1 antibody was added into reaction. Asterisk indicates a supershifted band with anti-Oct-1 antibody. (B), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated C-jun binding sites (−116/−87 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are consensus C-jun fragment, wild type C-jun binding site. Arrows indicate the specific protein-DNA complexes. Complexes A, B, C and D are specifically eliminated or reduced by a consensus C-jun competitor. The protein-DNA complex A was supershifted upon addition of anti-C-jun antibody. Asterisk indicates the supershifted band with anti-C-jun antibody. (C), LNCaP nuclear extracts (10 μg) were incubated with radiolabeled probes corresponding to the wild type or mutated NFI binding sites (−96/−67 bp) in the absence or presence of unlabeled competitor oligonucleotides. The competitors are unlabeled consensus NFI, consensus Oct-1, wt NFI binding site. Two specific protein-DNA complexes are marked as A and B. Both complexes A and B are inhibited upon addition of unlabeled consensus NFI probe. The complex A was supershifted when anti-NFI antibody was included in the reaction. Asterisk indicates a supershifted band with anti-NFI antibody.
Fig. 5
Fig. 5
Cis-binding factors interact with the large probasin promoter in vivo. Neo-Tag cells which contain the endogenous large probasin promoter were treated in the presence or absence of DHT for 24 hours, after which ChIP assays were performed as indicated in materials and methods.
Fig. 6
Fig. 6
TS1 and TS2 share similar biological function on −286PB promoter. (A) and (B), Similar transcription factors bind to the TS1 and TS2 in LNCaP cells. The radiolabeled PB –257 to –232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 10 μg of the LNCaP nuclear extracts in the absence or presence of 200×, 100×, 50×, 25×, 12.5× and 6.25× unlabeled TS1 or TS2 competitors. (C), Schematic representation of the wild type – 286PBLuc, TS1/TS1, TS2/TS2, and TS2/TS1 reporter constructs. (D), LNCaP cells were transiently transfected with wild type –286PBLuc, TS1/TS1, TS2/TS2, and TS2/TS1 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein.
Fig. 6
Fig. 6
TS1 and TS2 share similar biological function on −286PB promoter. (A) and (B), Similar transcription factors bind to the TS1 and TS2 in LNCaP cells. The radiolabeled PB –257 to –232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 10 μg of the LNCaP nuclear extracts in the absence or presence of 200×, 100×, 50×, 25×, 12.5× and 6.25× unlabeled TS1 or TS2 competitors. (C), Schematic representation of the wild type – 286PBLuc, TS1/TS1, TS2/TS2, and TS2/TS1 reporter constructs. (D), LNCaP cells were transiently transfected with wild type –286PBLuc, TS1/TS1, TS2/TS2, and TS2/TS1 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein.
Fig. 7
Fig. 7
Interaction between cell specific transcription factor(s) and TS1 and TS2 are required for –286PB activation. (A) and (B), Electromobility shift assays of TS1 and TS2 reveal cell specific protein-DNA complexes. The radiolabeled PB –257 to – 232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 20 μg, 10 μg, and 5 μg of the LNCaP, PC-3, Hela, and MCF-7 nuclear extracts; complexes were resolved on 5% polyacrylimide gels. (C), Radiolabeled TS1 (lanes 2-3) and TS2 probes (lanes 4-5) were used in EMSA with LNCaP nuclear extracts, in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of FoxA1 antibody. Same super shift experiments were performed for PC-3 (lanes 7-8) and Hela cells (lanes 11-12). (D), Wild type PB promoter sequences –127 to –102 bp, containing a FoxA1 motif (bolded) within the TS2 (underlined), was mutated (lowercase characters) as indicated in mt1-mt4. (E), LNCaP cells were transiently transfected with wild type –286PBLuc, and mt1-mt4 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (F), LNCaP nuclear extracts (10 μg or 20 μg) were incubated with radiolabeled probes corresponding to the wt or mutated TS2 (−127 to −102 bp) in the absence or presence of 100X unlabeled mutated TS2 competitor oligonucleotides; complexes were resolved on 5% polyacrylimide gels.
Fig. 7
Fig. 7
Interaction between cell specific transcription factor(s) and TS1 and TS2 are required for –286PB activation. (A) and (B), Electromobility shift assays of TS1 and TS2 reveal cell specific protein-DNA complexes. The radiolabeled PB –257 to – 232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 20 μg, 10 μg, and 5 μg of the LNCaP, PC-3, Hela, and MCF-7 nuclear extracts; complexes were resolved on 5% polyacrylimide gels. (C), Radiolabeled TS1 (lanes 2-3) and TS2 probes (lanes 4-5) were used in EMSA with LNCaP nuclear extracts, in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of FoxA1 antibody. Same super shift experiments were performed for PC-3 (lanes 7-8) and Hela cells (lanes 11-12). (D), Wild type PB promoter sequences –127 to –102 bp, containing a FoxA1 motif (bolded) within the TS2 (underlined), was mutated (lowercase characters) as indicated in mt1-mt4. (E), LNCaP cells were transiently transfected with wild type –286PBLuc, and mt1-mt4 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (F), LNCaP nuclear extracts (10 μg or 20 μg) were incubated with radiolabeled probes corresponding to the wt or mutated TS2 (−127 to −102 bp) in the absence or presence of 100X unlabeled mutated TS2 competitor oligonucleotides; complexes were resolved on 5% polyacrylimide gels.
Fig. 7
Fig. 7
Interaction between cell specific transcription factor(s) and TS1 and TS2 are required for –286PB activation. (A) and (B), Electromobility shift assays of TS1 and TS2 reveal cell specific protein-DNA complexes. The radiolabeled PB –257 to – 232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 20 μg, 10 μg, and 5 μg of the LNCaP, PC-3, Hela, and MCF-7 nuclear extracts; complexes were resolved on 5% polyacrylimide gels. (C), Radiolabeled TS1 (lanes 2-3) and TS2 probes (lanes 4-5) were used in EMSA with LNCaP nuclear extracts, in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of FoxA1 antibody. Same super shift experiments were performed for PC-3 (lanes 7-8) and Hela cells (lanes 11-12). (D), Wild type PB promoter sequences –127 to –102 bp, containing a FoxA1 motif (bolded) within the TS2 (underlined), was mutated (lowercase characters) as indicated in mt1-mt4. (E), LNCaP cells were transiently transfected with wild type –286PBLuc, and mt1-mt4 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (F), LNCaP nuclear extracts (10 μg or 20 μg) were incubated with radiolabeled probes corresponding to the wt or mutated TS2 (−127 to −102 bp) in the absence or presence of 100X unlabeled mutated TS2 competitor oligonucleotides; complexes were resolved on 5% polyacrylimide gels.
Fig. 7
Fig. 7
Interaction between cell specific transcription factor(s) and TS1 and TS2 are required for –286PB activation. (A) and (B), Electromobility shift assays of TS1 and TS2 reveal cell specific protein-DNA complexes. The radiolabeled PB –257 to – 232 bp probe containing the TS1 (A) and PB –127 to –102 bp probe containing the TS2 (B) were incubated with 20 μg, 10 μg, and 5 μg of the LNCaP, PC-3, Hela, and MCF-7 nuclear extracts; complexes were resolved on 5% polyacrylimide gels. (C), Radiolabeled TS1 (lanes 2-3) and TS2 probes (lanes 4-5) were used in EMSA with LNCaP nuclear extracts, in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of FoxA1 antibody. Same super shift experiments were performed for PC-3 (lanes 7-8) and Hela cells (lanes 11-12). (D), Wild type PB promoter sequences –127 to –102 bp, containing a FoxA1 motif (bolded) within the TS2 (underlined), was mutated (lowercase characters) as indicated in mt1-mt4. (E), LNCaP cells were transiently transfected with wild type –286PBLuc, and mt1-mt4 reporter constructs together with the AR and Renilla luciferase expression vectors and relative Luc activities were determined in the absence or presence of 10−8 M DHT as described in Materials and Methods. The reporter gene activities were measured in at least three determinations ± SD, and all experiments were adjusted for transfection efficiency. The luciferase activities are expressed as relative luciferase light units/min/μg protein. (F), LNCaP nuclear extracts (10 μg or 20 μg) were incubated with radiolabeled probes corresponding to the wt or mutated TS2 (−127 to −102 bp) in the absence or presence of 100X unlabeled mutated TS2 competitor oligonucleotides; complexes were resolved on 5% polyacrylimide gels.
Fig. 8
Fig. 8
Evaluation of androgen-induced activities of the wild type –286PBCAT, mtTS1/−286PBCAT and mtTS2/−286PBCAT constructs in LNCaP cells. (A) Schematic representation of the wild type –286PBCAT, mtTS/1-286PBCAT and mtTS2/−286PBCAT constructs. (B) These constructs were cotransfected with AR and Renilla luciferase expression vectors into LNCaP cells and treated with or without 10−8 M DHT for 24 hours before harvest. Background activities of cell lysates with no DNA transfection were subtracted from the data contained in the experimental group, before the normalization with Renilla luciferase activities. The activity induction in response to hormone treatment was determined by comparing receptor gene activity induced by DHT to the corresponding baseline values in the absence of hormone. Data shown here are a representative from at least three independent experiments in triplicate. Error bars indicate SD values. Results are presented as relative CAT activities dpm/min/mg protein.
Fig. 9
Fig. 9
Evaluation of androgen-induced activities of the ARR2PBCAT delARR2PBCAT, and ARR2TKCAT constructs in LNCaP cells. (A) Schematic representation of the ARR2PBCAT delARR2PBCAT, and ARR2TKCAT constructs. (B) These constructs were cotransfected with AR and Renilla luciferase expression vectors into LNCaP cells and treated with or without 10−8 M DHT for 24 hours before harvest. Background activities of cell lysates with no DNA transfection were subtracted from the data contained in the experimental group, before the normalization with Renilla luciferase activities. The activity induction in response to hormone treatment was determined by comparing receptor gene activity induced by DHT to the corresponding baseline values in the absence of hormone. Data shown here is representative for at least three independent experiments in triplicate. Error bars indicate SD values. Results are presented as relative CAT activities dpm/min/mg protein.
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