Two Inr Elements Are Important for Mediating the Activity of the Proximal Promoter of the Human Gonadotropin-Releasing Hormone Receptor Gene
Abstract|, http://www.100md.com
Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3' CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5' and 3' deletion mutant analysis. The promoter element drove a 13.1 ± 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived {alpha} T3–1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36–150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.
Introduction5i[}*r, 百拇医药
GnRH IS A DECAPEPTIDE released from the hypothalamus that plays a pivotal role in regulating human reproductive functions. In addition to the pituitary gonadotropes, GnRH receptor (GnRHR) mRNA transcripts have been detected in extrapituitary tissues including the ovary and placenta (1, 2). In human, identical GnRHR cDNAs were isolated from pituitary, ovary, and placenta. This observation suggests that the differential responsiveness of these tissues to GnRH (3, 4, 5) is the result of the difference in receptor number and/or post-translational modification of the receptors expressed on the cell surface. For these reasons, cell-specific transcriptional regulation of the human GnRHR gene has recently been a focus of interest.5i[}*r, 百拇医药
A variety of regulatory factors have been identified to be responsible for the proper expression of human GnRHR (hGnRHR) in various tissues. Previously, we have shown that a gonadotrope-specific element at -134 within the first exon of the gene mediates the gonadotrope-specific expression of the human GnRHR gene (6). More recently, a putative activator protein-1-binding motif located at -1000 of the human GnRHR 5'-flanking region has been functionally identified to be involved in the GnRH-mediated down-regulation in gonadotropes (7). In contrast to pituitary gonadotropes, down-regulation of GnRHR mRNA levels was not observed in placental cells after GnRH agonist treatment. Instead, an increase in GnRHR mRNA levels was observed in JEG-3 and IEVT cells, and this elevated expression was found to be coupled to both protein kinase C and A pathways (2). In ovarian cells (OVCAR-3), as indicated by gel shift assays, a combination of various cell-specific regulatory factors conferred different levels of hGnRHR gene expression in the ovary and pituitary (8).
Recently, we have identified an upstream promoter (-1705/-1674, relative to ATG) in the vicinity of a CAP site at -1673 found in the human pituitary (9). In this report, a proximal promoter element, containing two functional Inr motifs was located at -677/-558, which is in close proximity to a cluster of CAP sites from -635 to -578 in the pituitary. Several protein factors (36–150 kDa) were identified to interact with the core promoter to mediate its function. Interestingly, the distal and proximal promoters were found to interact with a common group of protein factors. The present report thus provides an insight for a better understanding of the transcriptional regulation of the hGnRHR gene using multiple promoter elements.'y.#&, http://www.100md.com
Materials and Methods'y.#&, http://www.100md.com
Cell lines'y.#&, http://www.100md.com
T3–1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD). All cells were incubated at 37 C with 5% CO2 in medium supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin (Life Technologies, Inc.).
Plasmids and DNA manipulations|96x}qx, 百拇医药
Escherichia coli strains JM109 and DH5{alpha} were used as the host strains for subcloning and sequencing. DNA manipulations were performed essentially as previously described (10). Deletion mutants, p1022 Luc and p2197/-771 Luc, were constructed from the clone p2200 Luc (6) by digestion with PstI, BglII, and SpeI (Life Technologies, Inc.). Deletion mutants, p677/-1 Luc, p677/-207 Luc, and p677/-407 Luc, were constructed from p2200 Luc using sequence specific primers (Table 1). Other 3' and 5' deletion mutants were obtained by exonuclease III/S1 nuclease digestion (Pharmacia LKB Biotechnology, Piscataway, NJ) of the plasmids p2200 Luc or p1022/-558 Luc. Twelve NotI block replacement mutants and two Inr site-directed mutants of p677/-558 Luc were generated by a three-step PCR mutagenesis method (11) using the mutagenic primers, PM 1–12, pInr I, or pInr II, and universal primers MP, MP-B, reverse, T7, and 5F-5I (Table 1). Mutant plasmids were identified by restriction mapping and DNA sequence analysis. Plasmid DNA used for transfection experiments was prepared by the QIAGEN Midi Preps kit (QIAGEN, Valencia, CA). Enzymes and oligoprimers were purchased from Life Technologies, Inc.
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Table 1. Oligonucleotides used in the current study2st%:), http://www.100md.com
Transient transfection assay2st%:), http://www.100md.com
One day before transfection, cells were seeded onto a 35-mm well (six-well plate, Costar, San Diego, CA). The seeding density used for {alpha} T3–1 was 5 x 105. A mixture containing 5 µg promoter-luciferase plasmid DNA, 2.5 µg pSV-ß-gal, 20 µg lipofectamine (Life Technologies, Inc.), and 200 µl serum-free medium (DMEM) was prepared, and transfection was performed following the manufacturer’s protocol. After overnight incubation, 1 ml of the medium supplemented with 20% fetal bovine serum was added. Cell lysate were prepared after 48 h by washing the cells with ice-cold PBS twice, followed by the addition of 200 µl reporter lysis buffer (Promega Corp., Madison, WI). The cell lysate was centrifuged at 12,000 x g for 2 min.2st%:), http://www.100md.com
Luciferase and ß-galactosidase measurements
To assay for the promoter activity, 100 µl luciferase substrate solution (Promega Corp.) was automatically injected into 20 µl cell lysate, and luciferase activity was measured as light emission using a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). Galactosidase activity was determined by incubating the cell lysate (100 µl) in 100 mM sodium phosphate buffer (pH 7.3), 1 mM MgCl2, 50 mM ß-mercaptoethanol, and 0.7 mg/ml O-nitrophenyl ß-D-galactopyranoside for 15 min at 37 C, and absorbance at 420 nm was measured using a spectrophotometer (UV160A, Shimadzu, Columbia, MD). For each transfection study, luciferase activity was determined and normalized on the basis of the ß-galactosidase activity.n(#q2de, http://www.100md.com
Gel mobility shift assaysn(#q2de, http://www.100md.com
Double-stranded oligonucleotides were end-labeled using the Ready-To-Go T4 polynucleotide kinase labeling kit (Amersham Pharmacia Biotech, Arlington Heights, IL) and (-32P)ATP (5000 Ci/nmol; Amersham Pharmacia Biotech). Nuclear extract was prepared from {alpha} T3–1 cells as previously described (12). Mobility shift assays were performed essentially as described earlier (12, 13) with 1 µg polydeoxyinosinic deoxycytidylic acid as a nonspecific competitor.
Southwestern blot analysis;0z\/7c, http://www.100md.com
Southwestern blot analysis was performed as previously described (14). Fifteen or 30 µg of nuclear extract from {alpha} T3–1 cells or BSA was separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. Proteins on the membrane were allowed to renature in the TNED buffer [50 mM NaCl, 10 mM Tris (pH 7.5), 0.1 mM EDTA (pH 7.5), and 1 mM diothiothreitol] containing 5% nonfat milk overnight at room temperature. The membrane was then washed three times with TNED buffer and hybridized in the same buffer containing 32P-labeled oligo PP8–11 (50 pmol) as a probe and oligo Inr II (Table 1) as a competitor (200-fold excess).;0z\/7c, http://www.100md.com
Statistical analysis;0z\/7c, http://www.100md.com
The promoter-luciferase construct was tested by three independent transfection experiments within each study, and the study was repeated three times (n = 9). When appropriate and unless otherwise stated, the transfection data were analyzed by either one-way or two-way ANOVA followed by Tukey’s test (Fig. 1) or Dunnett’s test (Figs. 2 and 3B) with the negative control (pGL2-basic in Fig. 1) or the wild-type (WT-F in Fig. 2 and p677/-558 Luc in Fig. 3B) as the independent variable (15).
fig.ommitteed;*p46, 百拇医药
Figure 1. Localization of a putative proximal promoter (-677/-558) responsible to a cluster of the 3' CAP sites from -635 to -578 (relative to ATG). Five micrograms of various 5' or 3' deletion construct was cotransfected with 2.5 µg pSV-ß-gal into {alpha} T3–1 cells using the lipofectamine reagent. At 48 h post transfection, cell lysate was prepared and used for luciferase and ß-galactosidase assays. Luciferase values are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL2-Basic). Values reported in the figure represent the mean ± SEM of at least three independent studies, each in triplicate. Bars bearing different letters (a, b, c, d) are statistically different (by ANOVA, P < 0.05).;*p46, 百拇医药
fig.ommitteed;*p46, 百拇医药
Figure 2. The sequence residing at -607 to -568 is essential for the promoter function. A family of block replacement NotI scanning mutants spanning the region -677 to -558 was constructed and used for transient transfection studies. Values are shown as the percentage changes in relative promoter activities compared with that of the positive control, p677/-558 Luc (WT). The bottom panel is the DNA sequence of the promoter, and the boldface and shaded sequences are the sites of NotI scanning mutations PM1–12. Arrows represent the previously identified CAP sites in the human pituitary. Functionally important sequences are underlined. All luciferase activities are normalized by ß-galactosidase expression, and values reported represent the mean ± SEM of three independent studies, each in triplicate. Bars bearing asterisks are statistically different (by ANOVA, **, P < 0.01; *, P < 0.05) from the wild-type constructs.
fig.ommitteedp@, http://www.100md.com
Figure 3. Two Inr motifs are essential for the proximal promoter function. A, The sequence of the synthetic oligonucleotide (sense strand). The oligo contains the functional region of the proximal promoter (PP8–11, -608/-565). Arrows represent the putative CAP sites identified in the human pituitary. B, Functional assays for the Inr (by ANOVA; **, P < 0.01) from the positive control (p677/-558 Luc). C, Competitive gel shift assays were performed with 10 µg T3–1 nuclear proteins using PP8–11 (-608/-565) as a probe and with increasing concentrations of competitor oligos containing Inr I, Inr II (50-, 100-, and 200-fold), or their mutants InrI and InrII (100- and 200-fold) as indicated. In the presence of unlabeled Inr oligos, the intensities of complex B are significantly reduced. However, an equal amount of Inr oligos had no observable effects.p@, http://www.100md.com
Resultsp@, http://www.100md.com
Identification of a proximal promoter elementp@, http://www.100md.com
The 5' flanking region (2.3 kb) of the hGnRHR gene was previously isolated (16). Eighteen transcription initiation start sites were identified in the human pituitary, and 11 of them were found to be clustered in the region -818 to -578 (-818, -808, -730, -721, -699, -683, -635, -631, -623, -602, and -578), relative to the ATG (17). To localize a putative promoter element for these CAP sites, progressive 5' and 3' deletion mutations followed by transient transfections using the gonadotrope-derived {alpha} T3–1 cells were conducted (Fig. 1). The 5' deletion mutant p1022 Luc, containing a 1022-bp DNA fragment (-1022/+1) with multiple CAP sites, was found to be transcriptionally active, which gave rise to a 3.8 ± 0.2-fold increase in the promoter activity similar to that of the 2.2-kb construct (p2200 Luc; 3.0 ± 0.1-fold; P > 0.05). Because the longest hGnRHR transcript contains a 5' untranslated region that is more than 500 bases, two 3' deletion mutants (p2197/-558 Luc, p2197/-771 Luc) were constructed to search for the 3' boundary of the promoter element. The mutant containing the region -2197/-558 retained a similar promoter activity (p2197/-558 Luc; 4.8 ± 0.3-fold; P < 0.05) as the full-length construct (p2200 Luc), whereas deletion of the region from -578 to -771 resulted in a complete loss of the promoter function (p2197/-771Luc; 0.3 ± 0.04-fold; P > 0.05). These data strongly suggested that the region from -1022 to -558 contains a functional element, and this idea is confirmed by the construct p1022/-558 Luc (Fig. 1). To define the 5' boundary of this regulatory element, a nested family of 5' deletion mutants was prepared. Deletion from -1022 (p1022/-558 Luc) to -927 (p927/-558 Luc) resulted in no significant change in promoter activity. However, further deletion from -927 to either -837 or -771 led to a marked increase in promoter activity, suggesting that a negative regulatory element is present in this region. Compared with the -771 deletion mutant, additional deletion to -677 had only a minor effect, whereas deletion to -617 caused a substantial drop in promoter activity (p617/-558 Luc; 5.4 ± 0.4-fold; P < 0.05). Several 3' deletion mutants were constructed to further confirm the 3' boundary, and these constructs had promoter activities much lower than that of the p677/-558 Luc (p677/-1 Luc, 4.45 ± 0.1-fold; p677/-207 Luc, 2.61 ± 0.1-fold; and p-677/-407 Luc, 5.97 ± 0.1). Taken together, we have identified a positive element between nucleotides -677 and -558. The element functioned only in the forward orientation (Fig. 2 WT-F and WT-R), indicating the nature of this DNA fragment as a promoter element. Within the promoter, five CAP sites (-635, -631, -623, -602, and -578) were found previously (17). NotI linker scanning mutation analysis of the promoter revealed that the sequences residing at -607/-568 were crucial for the basal promoter activity because mutations within this region completely abrogated promoter function (PM8-PM11; P < 0.01; Fig. 2). In addition, an AT-rich sequence (TAATAAA) residing at -636/-630 (PM5), 30 bp upstream of the core promoter (-607/-568), was identified. Within the PM5, two potential CAP sites (-631 and -635) were located, and mutation at this region led to a 49 ± 9% (P < 0.05) reduction in the promoter activity. This observation was consistent with the result of deletion mutation analysis. Deletion of the sequences -677/-617 also led to a similar (58%) reduction in the luciferase activity (Fig. 1). These results suggested that PM5 may play a role in modulating the core promoter function.
Two Inr elements mediate the function of the proximal promoter-, http://www.100md.com
Within the core promoter element (-607 to -568), there are no putative TATAA and CCAAT boxes. On the other hand, two pyrimidine-rich initiator elements (Inr, consensus YYANt/aYY) residing at -603/-598 (Inr I), overlapping the CAP site at -602, and -590/-584 (Inr II) at the close proximity to CAP site at -578, were identified (Fig. 3A). Functionally, as indicated by linker scanning and site-directed mutations (Fig. 2, CTAATT; PM8 gccgcT; TTAATTT; PM9: gcAATTT; PM10: TTAAgcg; and Fig. 3B, PMInr I: CTAATT to agccgg; PMInr II: TTAATTT to ggccggg), these two Inr motifs were shown to be essential for promoter activity. The site-directed mutation of either Inr motif resulted in a complete abolishment of the transcriptional activity. Interestingly, although the PM11 within the core promoter element does not overlap with the Inr motifs, mutation of this region also led to the abolishment of the promoter activity (Fig. 2).
As several important regions were identified, we next sought to characterize the protein(s) binding to these Inr motifs, PM11 and PM5 regions. We found that, using {alpha} T3–1 nuclear extract, three DNA-protein complexes were formed when the core promoter region (PP8–11) was used as a probe (Fig. 3C). The binding of these proteins with the DNA element is specific because only the cold PP8–11 oligo, but not nonspecific (NSP) DNA, is effective in competitively removing these complexes (see Fig. 7B). To study the Inr-specific interaction, the Inr and Inr mutant oligos were used in competition assays. Inr II, but not the mutant (Inr II {Delta} ), is able to specifically remove complex B (Fig. 3C). As expected, Inr I also interacted with the same nuclear protein because the intensity of the same complex (complex B) was reduced in the presence of an increasing concentration of the Inr I competitor DNA. Interestingly, we found that Inr II has a higher affinity for the protein in complex B because the presence of 50-fold excess of the unlabeled competitor was already sufficient to completely abrogate Inr-specific complex B formation (Fig. 3C). Thus, it is likely that these two Inr motifs within the same core promoter element bind the same protein(s) with different affinities.
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Figure 7. The proximal and distal promoters of the hGnRHR gene share common protein factors. A, Putative Inr elements were found in both proximal and distal promoters. B, Competitive gel shift assays were performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as a probe (PP8–11, -608/-565). Formation of the retarded bands was completely abrogated when 200-fold of unlabeled competitor oligos containing either the proximal (PP8–11) or the distal (DP3/5) promoter element was included in the binding reaction. The 200-fold molar in excess of unlabeled competitor oligo containing only the Inr motif of the distal promoter (DP5) was able to abolish the formation of complexes A and B. In contrast, the NSP oligo failed to abolish the complex formation.7t, 百拇医药
Transcription factor class II (TFII) transcription regulator(s) binds to the Inr motifs to initiate the core promoter function7t, 百拇医药
To identify the protein factor(s) binding to the Inr elements of the core promoter, several consensus oligos of initiator-binding proteins [e.g. TFII-D, upstream stimulatory factor (USF), and Yin Yang-1 zinc finger transcription factor (YY1)] were used in the competition assay (Fig. 4A). Among these oligos, only TFII-D was able to competitively remove complexes A and B, suggesting the protein factors responsible for forming these complexes are TFII transcription regulators. We then tried to identify these proteins using Southwestern blot analysis. Several distinct bands of size ranging from 36–150 kDa were observed. The bands were specific because there was a dose-dependent increase in the band intensities and no signal was observed in the BSA control (Fig. 4B). Competitive Southwestern blot was also performed using PP8–11 as a probe and 200-fold excess of oligo Inr II (Fig. 4B, right) as a competitor. These data suggest that the Inr-specific proteins are 150 or 36 kDa in size.
fig.ommitteed)!, 百拇医药
Figure 4. The Inr-specific protein(s) is TFII transcription regulator. Multiple protein factors of sizes ranging from 36–150 kDa interact specifically with the core promoter PP8–11. A, Competition gel shift assay was performed with 10 µg T3–1 nuclear proteins using PP8–11 as probe. Only the consensus oligo of TFII-D can completely abrogate the formation of protein complexes A and B when 200-fold unlabeled competitor was included in the binding reaction. B, Fifteen or 30 µg of nuclear extract from T3–1 cell or BSA were separated in 10% SDS-PAGE, transblotted to nitrocellulose membrane, and subjected to Southwestern blot analysis using 32P-labeled PP8–11 oligo as probe (left). Thirty micrograms of nuclear extract from T3–1 cell or BSA were separated in 10% SDS-PAGE, transblotted to nitrocellulose membrane, and subjected to Southwestern blot analysis using 32P-labeled PP8–11 oligo as a probe and the unlabeled Inr II as a competitor (200-fold excess).)!, 百拇医药
To study the association between the protein factor(s) with the two Inr motifs, oligos PP8–11, PP8–11Mut-InrI, and PP8–11Mut-InrII (mutation at either Inr I or Inr II) were used as probes for the gel shift assays (Fig. 5A). Three DNA-protein complexes (A–C) were found using the PP8–11 and PP8–11Mut-Inr I as probes, whereas only complex C was observed when the PP8–11Mut-InrII probe was used. Interestingly, the band intensity of the complex C was increased when using these two mutated oligos as probes. It is possible that the mutated sequences aid the binding of protein(s) to form complex C. Further experimentation is needed to verify this suggestion. In the competitive EMSA with an increasing concentration of the PP8–11(Mut-Inr I/II) with mutations of both Inr I and Inr II motifs, we observed a dose-dependent reduction only in complex C (Fig. 5B). Taken together, these results suggested that although both Inr I and Inr II interact with an Inr-specific protein in complex B, this protein has a higher affinity for the Inr II motif, and the initial interaction of the protein with Inr II may be necessary to recruit additional protein(s) for the formation of complex A.
fig.ommitteedm, 百拇医药
Figure 5. Protein factor(s) in complex B has a higher affinity toward Inr II, and the interaction of protein factor(s) in complex B with Inr II may be able to facilitate the recruitment of protein factor(s) for the formation of complex A. A, Gel shift assays were performed with 10 µg T3–1 nuclear proteins using PP8–11, PP8–11(Mut-Inr I), and PP8–11(Mut-InrII) as probes. Three DNA-protein complexes (A–C) were observed when using PP8–11 and PP8–11(Mut-Inr I) as the probes. However, the formation of complexes A and B was abolished when the probe PP8–11(Mut-Inr II) was used. B, Competition gel shift assay was performed with 10 µg T3–1 nuclear proteins using PP8–11 as probe. Only the formation of complex C, but not complexes A and B, was abrogated when 50- to 200-fold unlabeled PP8–11(Mut-Inr I/II) oligo was included in the binding reaction.m, 百拇医药
EMSA studies of PM5 and PM11m, 百拇医药
In addition to the Inr motifs, DNA sequences within the PM11 region of PP8–11 were also indicated to be transcriptionally active (Fig. 2). To investigate the potential protein factors interacting with PM11, competitive EMSA was performed using the oligo PM11(-579/-566) and PP8–11(Mut-PM11) as the competitors (Fig. 6, A and B). The short competitor oligo with only the PM11(-579/-566) DNA sequences had no effect on the formation of DNA protein complexes A–C (Fig. 6A). On the other hand, the competitor oligo containing the core promoter PP8–11 with mutation of PM11, at 50-fold excess, could effectively compete out all of the complexes (Fig. 6B). Although we were unable to detect protein factor(s) that interacts with this motif, it is possible that the protein(s) binding to PM11(-579/-566) is required only transiently to assist the formation of the preinitiation complex at the Inr motifs. Similarly, no protein was observed to interact with the DNA sequences of PM5 in an EMSA study (data not shown); it is possible that mutation of PM5 disrupts the two potential CAP sites (-635, -631) for transcription initiation, resulting in the partial loss of function. Further experimentation is needed to clarify the functions of PM5 and PM11 and the protein factors interacting with these motifs.
fig.ommitteeda4#%i^, 百拇医药
Figure 6. Protein factor(s) is transiently interacting with region PM11. A, Competition gel shift assay was performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as probe. None of the protein complexes can be removed by the short probe containing only the region PM11, even when 200-fold in excess unlabeled probe was included in the binding reaction. B, Competition gel shift assay was performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as probe. The formation of all the protein complexes was abrogated when 50-fold of the unlabeled PP8–11(Mut-PM11) oligo was included in the binding reaction.a4#%i^, 百拇医药
The proximal and distal promoters of the hGnRHR gene share common nuclear proteinsa4#%i^, 百拇医药
The proximal promoter residing at -677/-558 exhibits similar properties when compared with the distal promoter at -1705/-1674 (9). By DNA sequence analysis, we found that both promoters contain the consensus Inr motifs (Fig. 7A; proximal promoter, -590/-584, TTAATTT, and -603/-597, CTAATTT; distal promoter, -1682/-1676, TCATTTC). On the basis of this observation, we postulated that these promoters interact with similar transcription factors. By competitive gel shift assay, the presence of 200-fold excess of the unlabeled distal promoter (Fig. 7B, DP3–5) could remove all of the proteins binding onto the proximal promoter (PP8–11). The Inr element of the distal promoter (Fig. 7B, DP5) was able to compete out complexes A and B but not complex C. It is not surprising that this distal Inr sequence was able to interact with two different proteins (A and B) because the consensus Inr sequence, YYANt/aYY, is known to bind a variety of transcription factors, e.g. TAFs [TATA-binding protein (TBP)-associated factors], TFII-I, USF-1, YY1, and RNA-polymerase II (18). There is also evidence that there is more than one TAF (TAFII150-TAFII250 heterodimers) interacting with the same Inr (19). In addition, TFII-I has also been suggested to be able to recognize several independent DNA elements because it has six helix-loop-helix domains (18). As a control, 200-fold excess of the NSP had no effect on these complexes (Fig. 7B, NSP). Our data suggested that the distal and proximal promoters of the hGnRHR gene use similar nuclear factors and the Inr-specific protein(s) is able to interact with the Inr motifs in these promoters.
Discussionmwwu!2, http://www.100md.com
In human, identical GnRHR cDNAs were isolated from pituitary, ovary, and placenta. This observation suggests that the differential responsiveness of these tissues to GnRH (3, 4, 5) is the result of the difference in receptor number and/or post-translational modification of the receptors expressed on the cell surface. It is highly possible that multiple functional promoters are involved in the regulation of the human GnRHR gene in various tissues. Differential usage of CAP sites was evident in human brain and pituitary by primer extension and 5' RACE studies. Five and 18 CAP sites were found in the brain (16) and the pituitary (17), respectively. By Northern blot analysis, it was revealed that the human pituitary contains three different GnRHR transcripts, i.e. 5.0, 2.5, and 1.5 kb (17), and only two of them (2.5 and 1.5 kb) were detected in the human placenta (2). PCR studies confirmed that these GnRHR transcripts are fully spliced, and hence they are not alternatively spliced products. It is therefore likely that several functional promoters are present to regulate the hGnRHR gene expression in different cell types within the hypothalamic-pituitary-gonadal axis.
Because there are 11 CAP sites locating at the region -818 to -578, this observation prompted us to search for the promoter element responsible for these CAP sites. By deletion mutation analysis and transient transfection studies in mouse gonadotrope-derived {alpha} T3–1 cell, a promoter element was identified at -677/-558, relative to ATG codon, which is functionally active in pituitary cell (Fig. 1) and extrapituitary cells (data not shown). The core promoter element was found in -607/-568 (PM8-PM11) by block replacement mutation studies. In common eukaryotic genes, transcription is dependent on both or one of the two core promoter elements, the TATA box and its functional analog, the initiator (Inr) (20). DNA sequence analysis revealed that no TATA or CCAAT box was identified, whereas two Inr motifs (I and II) are present within PP8–11 and these motifs are functional, as indicated by site-directed mutations. These data suggest that the proximal promoter is a TATA-Inr+ promoter. Inr is a pyrimidine-rich element that overlaps with the transcription start site, and it is capable of directing transcription initiation in a gene that lacks a TATA box (21, 22, 23, 24). The Inr element has been demonstrated to be able to select the CAP sites for gene transcription, and hence mutation of Inr element has a direct effect on the position of CAP sites (25). In mouse galectin-1 gene promoter, the Inr element overlapping with the TATA box was found to be responsible for directing transcription initiation from an alternative CAP site (26). Thus, it is possible that these two Inr motifs in the human GnRHR proximal promoter are responsible for the transcription initiation of the CAP sites in the close proximity. In addition, the different binding affinities of the Inr-specific protein factors to these two Inr motifs may be responsible for selecting the appropriate CAP sites for this promoter element.
In common eukaryotic genes, transcription is mediated by ordered assembly of a set of transcription factors at the core promoter element to form the transcription initiation complex. Several models have been suggested to explain the mechanism of transcription initiation through initiator element. In the first model, the TAFs are able to interact with Inr elements to direct transcription. The second model suggests that RNA-polymerase II is able to recognize the Inr element directly. In the third model, the initiator-specific proteins, such as TFII-I, YY1, or USF, bind to the Inr motif to recruit TBP and other transcription factors to form the preinitiation complex (18). Although we were unable to clarify the identity of the Inr-specific protein by super-shift assays using TFII-I and TFII-D antibodies (data not shown) by competitive gel shift assays and Southwestern blot analysis, four protein factors of sizes 36, 40, 105, and 150 kDa were found to interact specifically with the core promoter. Several protein factors of similar sizes that are involved in transcription initiation could potentially interact with the proximal promoter element or the Inr motifs within this region. TFII-D (36 kDa) is required in transcription initiation in both TATA+Inr- and TATA-Inr+ promoters. In TATA+Inr- promoter, TFII-D is the first protein binding to the TATA element to initiate the assembly of the other factors into a preinitiation complex (18, 20). On the other hand, some studies suggested that TFII-D alone is not sufficient to direct Inr-mediated transcription; instead, several cofactors such as cofactor of Inr function (CIF)150 and TAFs are required to stabilize its binding to the core promoter (27, 28). CIF150 (150 kDa), a human homolog of TAFII150, is an essential cofactor for TFII-D-dependent initiator function because it can interact with the hTAFII135 to stabilize the binding of TFII-D to the core promoter element. In addition, Sp1 (95 or 105 kDa) can also interact with hTAFII135 to promote the recruitment of CIF150 to the core promoter (27, 28). TFII-I (120 kDa) is needed for the transcription initiation of TATA-Inr+ promoter by interacting directly with the Inr element within the core promoter to recruit TFII-D to form the preinitiation complex (18, 20, 29). IBP39 (39 kDa) is a novel initiator binding protein isolated from a primitive eukaryote Trichomonas vaginalis by DNA affinity chromatography. It was suggested that the mammalian homologs of IBP39 are: 1) components of the CIF, or 2) DNA binding domains of TAFII150 and TAFII250 (30). Further experimentation, such as protein purification by DNA affinity chromatography followed by peptide sequencing or MALDI-TOF mass spectrum analysis, is required to further identify these protein factors (31, 32).
Because both distal and proximal promoters of the hGnRHR gene are active in pituitary and extrapituitary cells (data not shown) and they share common protein factors, it is interesting to understand how these promoters can differentially regulate the human GnRH receptor gene transcription in a temporal and spatial manner. The presence of multiple silencer and activator/enhancer elements may act as molecular switches to turn on and off these promoters in time- and cell-specific conditions. For example, extracellular signals such as {gamma} -aminobutyric acid have been shown to be able to induce cell-specific down-regulation of the human type A {gamma} -aminobutyric acid receptor ß1 subunit gene by mediating the binding of different factors to the Inr element (33). In addition, cAMP and PMA were demonstrated to modulate the MCAM gene transcription by altering the amount or affinity of the protein factors binding to the Inr element (34). Recently, we have shown that pituitary adenylate cyclase-activating polypeptide regulates the hGnRHR gene expression in gonadotropes by controlling the activities of a silencer element in the vicinity of the distal promoter (35). In fact, TAFs distinguish different promoters by recognizing sequence variations of the Inr elements (36). Thus, under the influence of external modulators, these Inr elements in the hGnRHR gene may exhibit different affinities toward different TAFs, and the binding of TAFs in the absence of TBP may determine the responsiveness of the core promoter to various activators as indicated previously (19). In summary, the Inr motifs present in the distal and proximal promoters of the hGnRH receptor gene are essential for the function of these promoters, and several protein factors (36–150 kDa) were identified to interact specifically with the core promoter. The hypothesis that these Inr motifs are involved in the differential regulation of these promoters remains to be investigated in the future.
Acknowledgments]l\l, 百拇医药
We thank Mr. K. H. Hui for his technical support and Mr. C. K. Cheng for his comments.]l\l, 百拇医药
Received June 5, 2002.]l\l, 百拇医药
Accepted for publication October 28, 2002.]l\l, 百拇医药
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References]l\l, 百拇医药
Peng C, Fan NC, Ligier M, Vaananen J, Leung PCK 1994 Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135:1740–1746]l\l, 百拇医药
Cheng KW, Nathwani PS, Leung PC 2000 Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. Endocrinology 141:2340–2349]l\l, 百拇医药
Kakar SS, Grantham K, Musgrove LC, Devor D, Sellers JC, Neill JD 1994 Rat gonadotropin-releasing hormone (GnRH) receptor: tissue expression and hormonal regulation of its mRNA. Mol Cell Endocrinol 101:151–157]l\l, 百拇医药
Currie AJ, Fraser HM, Sharpe RM 1981 Human placental receptors for luteinizing hormone releasing hormone. Biochem Biophys Res Commun 99:332–338
Iwashita M, Evans MI, Catt KJ 1986 Characterization of a gonadotropin-releasing hormone receptor site in term placenta and chorionic villi. J Clin Endocrinol Metab 62:127–133s.!|'\, http://www.100md.com
Ngan ESW, Cheng PKW, Leung PCK, Chow BKC 1999 Steroidogenic factor-1 intercts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression. Endocrinology 140:2452–2462s.!|'\, http://www.100md.com
Cheng KW, Ngan ESW, Kang SK, Chow BKC, Leung PCK 2000 Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1. Endocrinology 141:3611–3622s.!|'\, http://www.100md.com
Kang SK, Cheng KW, Ngan ESW, Chow BKC, Choi KC, Leung PCK 2000 Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. Mol Cell Endocrinol 162:157–166s.!|'\, http://www.100md.com
Ngan ESW, Leung PCK, Chow BKC 2000 Identification of an upstream promoter in the human gonadotropin-releasing hormone receptor gene. Biochem Biophys Res Comm 270:766–772
Maniatis T, Fritsch EF, Sambrook J 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory/:2to, 百拇医药
Chow BKC, Ting V, Tufaro F, MacGillivray RTA 1991 Characterization of a novel liver-specific enhancer in the human prothrombin gene. J Biol Chem 266:18927–18933/:2to, 百拇医药
Lee KAW, Bindereif A, Green MR 1988 A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech 5:22–31/:2to, 百拇医药
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhi K 1997 Mobility shift DNA binding assay using gel eletrophoresis. Current protocols in molecular biology. New York: J. Wiley and Sons; 12.2.1–12.2.7/:2to, 百拇医药
Wieczorek E, Lin Z, Perkins EB, Law DJ, Merchant JL, Zehner ZE 2000 The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1. J Biol Chem 275:12879–12888/:2to, 百拇医药
Motulsky HJ, Searle P 1995 GraphPad Prism user’s guide, version 2. San Diego, Graph-Pad Software
Fan NC, Peng C, Krisinger J, Leung PCK 1995 The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals. Mol Cell Endocrinol 107:R1–R8(6g3%, 百拇医药
Kakar SS 1997 Molecular structure of the human gonadotropin-releasing hormone receptor gene. Euro J Endocrinol 137:183–192(6g3%, 百拇医药
Morikawa N, Clarke TR, Novina CD, Watanabe K, Haqq C, Weiss M, Roy AL, Donahoe PK 2000 Human Mullerian-inhibiting substance promoter contains a functional TFII-I-binding initiator. Biol Reprod 63:1075–1083(6g3%, 百拇医药
Chalkley GE, Verrijzer CP 1999 DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator. EMBO J 17:4835–4845(6g3%, 百拇医药
Manzano-Winkler B, Novina CD, Roy AL 1996 TFII is required for transcription of the naturally TATA-less but initiator-containing Vß promoter. J Biol Chem 271:12076–12081(6g3%, 百拇医药
Hariharan N, Perry RP 1990 Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region. Proc Natl Acad Sci USA 87:1526–1530
Javahery R, Khachi K, Lo K, Zenzie-Gregory B, Smale ST 1994 DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol Cell Biol 14:116–127$mu#, 百拇医药
Lo K, Smale ST 1996 Generality of a functional initiator consensus sequence. Gene 182:13–22$mu#, 百拇医药
Smale ST, Baltimore D 1989 The "initiator" as a transcription control element. Cell 57:103–113$mu#, 百拇医药
Liston DR, Johnson PJ 1999 Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 19:2380–2388$mu#, 百拇医药
Gregorio ED, Chiariotti L, Nocera PPD 2001 The overlap of Inr and TATA elements sets the use of alternative transcriptional start sites in the mouse galectin-1 gene promoter. Gene 268:215–223$mu#, 百拇医药
Kaufmann J, Verrijzer CP, Shao J, Smale ST 1996 CIF, an essential cofactor for TFIID-dependent initiator function. Genes Dev 10:873–886$mu#, 百拇医药
Kaufmann J, Ahrens K, Koop R, Smale ST, Muller R 1998 CIF150, a human cofactor for transcription factor IID-dependent initiator function. Mol Cell Biol 18:233–239
Johansson E, Skogman E, Thelander L 1995 The TATA-less promoter of mouse ribonucleotide reductase R1 gene contains a TFII-I binding initiator element essential for cell cycle-regulated transcription. J Biol Chem 270:30162–30167-y@s, 百拇医药
Liston DR, Lau AO, Ortiz D, Smale ST, Johnson PJ 2001 Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis. Mol Cell Biol 21:7872–7882-y@s, 百拇医药
Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C 1993 Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90:5011–5015-y@s, 百拇医药
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ 2000 A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672-y@s, 百拇医药
Russek SJ, Bandopadhyay S, Farb DH 2000 An initiator element mediates autologous downregulation of the human type A {gamma} -aminobutyric acid receptor ß 1 subunit gene. Proc Natl Acad Sci USA 97:8600–8605-y@s, 百拇医药
Karlen S, Braathen LR 2000 Role of the initiator element in the regulation of the melanoma cell adhesion molecule gene. J Invest Dermatol 115:668–673-y@s, 百拇医药
Ngan ESW, Leung PCK, Chow BKC 2001 Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene. Mol Cell Endocrinol 176:135–144-y@s, 百拇医药
Verrijzer CP, Chen JL, Yokomori K, Tjian R 1995 Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II. Cell 81:1115–1125(Ruby L. C. Hoo Elly S. W. Ngan Peter C. K. Leung and Billy K. C. Chow)
Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3' CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5' and 3' deletion mutant analysis. The promoter element drove a 13.1 ± 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived {alpha} T3–1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36–150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.
Introduction5i[}*r, 百拇医药
GnRH IS A DECAPEPTIDE released from the hypothalamus that plays a pivotal role in regulating human reproductive functions. In addition to the pituitary gonadotropes, GnRH receptor (GnRHR) mRNA transcripts have been detected in extrapituitary tissues including the ovary and placenta (1, 2). In human, identical GnRHR cDNAs were isolated from pituitary, ovary, and placenta. This observation suggests that the differential responsiveness of these tissues to GnRH (3, 4, 5) is the result of the difference in receptor number and/or post-translational modification of the receptors expressed on the cell surface. For these reasons, cell-specific transcriptional regulation of the human GnRHR gene has recently been a focus of interest.5i[}*r, 百拇医药
A variety of regulatory factors have been identified to be responsible for the proper expression of human GnRHR (hGnRHR) in various tissues. Previously, we have shown that a gonadotrope-specific element at -134 within the first exon of the gene mediates the gonadotrope-specific expression of the human GnRHR gene (6). More recently, a putative activator protein-1-binding motif located at -1000 of the human GnRHR 5'-flanking region has been functionally identified to be involved in the GnRH-mediated down-regulation in gonadotropes (7). In contrast to pituitary gonadotropes, down-regulation of GnRHR mRNA levels was not observed in placental cells after GnRH agonist treatment. Instead, an increase in GnRHR mRNA levels was observed in JEG-3 and IEVT cells, and this elevated expression was found to be coupled to both protein kinase C and A pathways (2). In ovarian cells (OVCAR-3), as indicated by gel shift assays, a combination of various cell-specific regulatory factors conferred different levels of hGnRHR gene expression in the ovary and pituitary (8).
Recently, we have identified an upstream promoter (-1705/-1674, relative to ATG) in the vicinity of a CAP site at -1673 found in the human pituitary (9). In this report, a proximal promoter element, containing two functional Inr motifs was located at -677/-558, which is in close proximity to a cluster of CAP sites from -635 to -578 in the pituitary. Several protein factors (36–150 kDa) were identified to interact with the core promoter to mediate its function. Interestingly, the distal and proximal promoters were found to interact with a common group of protein factors. The present report thus provides an insight for a better understanding of the transcriptional regulation of the hGnRHR gene using multiple promoter elements.'y.#&, http://www.100md.com
Materials and Methods'y.#&, http://www.100md.com
Cell lines'y.#&, http://www.100md.com
T3–1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD). All cells were incubated at 37 C with 5% CO2 in medium supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin (Life Technologies, Inc.).
Plasmids and DNA manipulations|96x}qx, 百拇医药
Escherichia coli strains JM109 and DH5{alpha} were used as the host strains for subcloning and sequencing. DNA manipulations were performed essentially as previously described (10). Deletion mutants, p1022 Luc and p2197/-771 Luc, were constructed from the clone p2200 Luc (6) by digestion with PstI, BglII, and SpeI (Life Technologies, Inc.). Deletion mutants, p677/-1 Luc, p677/-207 Luc, and p677/-407 Luc, were constructed from p2200 Luc using sequence specific primers (Table 1). Other 3' and 5' deletion mutants were obtained by exonuclease III/S1 nuclease digestion (Pharmacia LKB Biotechnology, Piscataway, NJ) of the plasmids p2200 Luc or p1022/-558 Luc. Twelve NotI block replacement mutants and two Inr site-directed mutants of p677/-558 Luc were generated by a three-step PCR mutagenesis method (11) using the mutagenic primers, PM 1–12, pInr I, or pInr II, and universal primers MP, MP-B, reverse, T7, and 5F-5I (Table 1). Mutant plasmids were identified by restriction mapping and DNA sequence analysis. Plasmid DNA used for transfection experiments was prepared by the QIAGEN Midi Preps kit (QIAGEN, Valencia, CA). Enzymes and oligoprimers were purchased from Life Technologies, Inc.
fig.ommitteed2st%:), http://www.100md.com
Table 1. Oligonucleotides used in the current study2st%:), http://www.100md.com
Transient transfection assay2st%:), http://www.100md.com
One day before transfection, cells were seeded onto a 35-mm well (six-well plate, Costar, San Diego, CA). The seeding density used for {alpha} T3–1 was 5 x 105. A mixture containing 5 µg promoter-luciferase plasmid DNA, 2.5 µg pSV-ß-gal, 20 µg lipofectamine (Life Technologies, Inc.), and 200 µl serum-free medium (DMEM) was prepared, and transfection was performed following the manufacturer’s protocol. After overnight incubation, 1 ml of the medium supplemented with 20% fetal bovine serum was added. Cell lysate were prepared after 48 h by washing the cells with ice-cold PBS twice, followed by the addition of 200 µl reporter lysis buffer (Promega Corp., Madison, WI). The cell lysate was centrifuged at 12,000 x g for 2 min.2st%:), http://www.100md.com
Luciferase and ß-galactosidase measurements
To assay for the promoter activity, 100 µl luciferase substrate solution (Promega Corp.) was automatically injected into 20 µl cell lysate, and luciferase activity was measured as light emission using a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). Galactosidase activity was determined by incubating the cell lysate (100 µl) in 100 mM sodium phosphate buffer (pH 7.3), 1 mM MgCl2, 50 mM ß-mercaptoethanol, and 0.7 mg/ml O-nitrophenyl ß-D-galactopyranoside for 15 min at 37 C, and absorbance at 420 nm was measured using a spectrophotometer (UV160A, Shimadzu, Columbia, MD). For each transfection study, luciferase activity was determined and normalized on the basis of the ß-galactosidase activity.n(#q2de, http://www.100md.com
Gel mobility shift assaysn(#q2de, http://www.100md.com
Double-stranded oligonucleotides were end-labeled using the Ready-To-Go T4 polynucleotide kinase labeling kit (Amersham Pharmacia Biotech, Arlington Heights, IL) and (-32P)ATP (5000 Ci/nmol; Amersham Pharmacia Biotech). Nuclear extract was prepared from {alpha} T3–1 cells as previously described (12). Mobility shift assays were performed essentially as described earlier (12, 13) with 1 µg polydeoxyinosinic deoxycytidylic acid as a nonspecific competitor.
Southwestern blot analysis;0z\/7c, http://www.100md.com
Southwestern blot analysis was performed as previously described (14). Fifteen or 30 µg of nuclear extract from {alpha} T3–1 cells or BSA was separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. Proteins on the membrane were allowed to renature in the TNED buffer [50 mM NaCl, 10 mM Tris (pH 7.5), 0.1 mM EDTA (pH 7.5), and 1 mM diothiothreitol] containing 5% nonfat milk overnight at room temperature. The membrane was then washed three times with TNED buffer and hybridized in the same buffer containing 32P-labeled oligo PP8–11 (50 pmol) as a probe and oligo Inr II (Table 1) as a competitor (200-fold excess).;0z\/7c, http://www.100md.com
Statistical analysis;0z\/7c, http://www.100md.com
The promoter-luciferase construct was tested by three independent transfection experiments within each study, and the study was repeated three times (n = 9). When appropriate and unless otherwise stated, the transfection data were analyzed by either one-way or two-way ANOVA followed by Tukey’s test (Fig. 1) or Dunnett’s test (Figs. 2 and 3B) with the negative control (pGL2-basic in Fig. 1) or the wild-type (WT-F in Fig. 2 and p677/-558 Luc in Fig. 3B) as the independent variable (15).
fig.ommitteed;*p46, 百拇医药
Figure 1. Localization of a putative proximal promoter (-677/-558) responsible to a cluster of the 3' CAP sites from -635 to -578 (relative to ATG). Five micrograms of various 5' or 3' deletion construct was cotransfected with 2.5 µg pSV-ß-gal into {alpha} T3–1 cells using the lipofectamine reagent. At 48 h post transfection, cell lysate was prepared and used for luciferase and ß-galactosidase assays. Luciferase values are normalized by ß-galactosidase expression and are shown as the fold increases in relative promoter activities compared with that in the control (pGL2-Basic). Values reported in the figure represent the mean ± SEM of at least three independent studies, each in triplicate. Bars bearing different letters (a, b, c, d) are statistically different (by ANOVA, P < 0.05).;*p46, 百拇医药
fig.ommitteed;*p46, 百拇医药
Figure 2. The sequence residing at -607 to -568 is essential for the promoter function. A family of block replacement NotI scanning mutants spanning the region -677 to -558 was constructed and used for transient transfection studies. Values are shown as the percentage changes in relative promoter activities compared with that of the positive control, p677/-558 Luc (WT). The bottom panel is the DNA sequence of the promoter, and the boldface and shaded sequences are the sites of NotI scanning mutations PM1–12. Arrows represent the previously identified CAP sites in the human pituitary. Functionally important sequences are underlined. All luciferase activities are normalized by ß-galactosidase expression, and values reported represent the mean ± SEM of three independent studies, each in triplicate. Bars bearing asterisks are statistically different (by ANOVA, **, P < 0.01; *, P < 0.05) from the wild-type constructs.
fig.ommitteedp@, http://www.100md.com
Figure 3. Two Inr motifs are essential for the proximal promoter function. A, The sequence of the synthetic oligonucleotide (sense strand). The oligo contains the functional region of the proximal promoter (PP8–11, -608/-565). Arrows represent the putative CAP sites identified in the human pituitary. B, Functional assays for the Inr (by ANOVA; **, P < 0.01) from the positive control (p677/-558 Luc). C, Competitive gel shift assays were performed with 10 µg T3–1 nuclear proteins using PP8–11 (-608/-565) as a probe and with increasing concentrations of competitor oligos containing Inr I, Inr II (50-, 100-, and 200-fold), or their mutants InrI and InrII (100- and 200-fold) as indicated. In the presence of unlabeled Inr oligos, the intensities of complex B are significantly reduced. However, an equal amount of Inr oligos had no observable effects.p@, http://www.100md.com
Resultsp@, http://www.100md.com
Identification of a proximal promoter elementp@, http://www.100md.com
The 5' flanking region (2.3 kb) of the hGnRHR gene was previously isolated (16). Eighteen transcription initiation start sites were identified in the human pituitary, and 11 of them were found to be clustered in the region -818 to -578 (-818, -808, -730, -721, -699, -683, -635, -631, -623, -602, and -578), relative to the ATG (17). To localize a putative promoter element for these CAP sites, progressive 5' and 3' deletion mutations followed by transient transfections using the gonadotrope-derived {alpha} T3–1 cells were conducted (Fig. 1). The 5' deletion mutant p1022 Luc, containing a 1022-bp DNA fragment (-1022/+1) with multiple CAP sites, was found to be transcriptionally active, which gave rise to a 3.8 ± 0.2-fold increase in the promoter activity similar to that of the 2.2-kb construct (p2200 Luc; 3.0 ± 0.1-fold; P > 0.05). Because the longest hGnRHR transcript contains a 5' untranslated region that is more than 500 bases, two 3' deletion mutants (p2197/-558 Luc, p2197/-771 Luc) were constructed to search for the 3' boundary of the promoter element. The mutant containing the region -2197/-558 retained a similar promoter activity (p2197/-558 Luc; 4.8 ± 0.3-fold; P < 0.05) as the full-length construct (p2200 Luc), whereas deletion of the region from -578 to -771 resulted in a complete loss of the promoter function (p2197/-771Luc; 0.3 ± 0.04-fold; P > 0.05). These data strongly suggested that the region from -1022 to -558 contains a functional element, and this idea is confirmed by the construct p1022/-558 Luc (Fig. 1). To define the 5' boundary of this regulatory element, a nested family of 5' deletion mutants was prepared. Deletion from -1022 (p1022/-558 Luc) to -927 (p927/-558 Luc) resulted in no significant change in promoter activity. However, further deletion from -927 to either -837 or -771 led to a marked increase in promoter activity, suggesting that a negative regulatory element is present in this region. Compared with the -771 deletion mutant, additional deletion to -677 had only a minor effect, whereas deletion to -617 caused a substantial drop in promoter activity (p617/-558 Luc; 5.4 ± 0.4-fold; P < 0.05). Several 3' deletion mutants were constructed to further confirm the 3' boundary, and these constructs had promoter activities much lower than that of the p677/-558 Luc (p677/-1 Luc, 4.45 ± 0.1-fold; p677/-207 Luc, 2.61 ± 0.1-fold; and p-677/-407 Luc, 5.97 ± 0.1). Taken together, we have identified a positive element between nucleotides -677 and -558. The element functioned only in the forward orientation (Fig. 2 WT-F and WT-R), indicating the nature of this DNA fragment as a promoter element. Within the promoter, five CAP sites (-635, -631, -623, -602, and -578) were found previously (17). NotI linker scanning mutation analysis of the promoter revealed that the sequences residing at -607/-568 were crucial for the basal promoter activity because mutations within this region completely abrogated promoter function (PM8-PM11; P < 0.01; Fig. 2). In addition, an AT-rich sequence (TAATAAA) residing at -636/-630 (PM5), 30 bp upstream of the core promoter (-607/-568), was identified. Within the PM5, two potential CAP sites (-631 and -635) were located, and mutation at this region led to a 49 ± 9% (P < 0.05) reduction in the promoter activity. This observation was consistent with the result of deletion mutation analysis. Deletion of the sequences -677/-617 also led to a similar (58%) reduction in the luciferase activity (Fig. 1). These results suggested that PM5 may play a role in modulating the core promoter function.
Two Inr elements mediate the function of the proximal promoter-, http://www.100md.com
Within the core promoter element (-607 to -568), there are no putative TATAA and CCAAT boxes. On the other hand, two pyrimidine-rich initiator elements (Inr, consensus YYANt/aYY) residing at -603/-598 (Inr I), overlapping the CAP site at -602, and -590/-584 (Inr II) at the close proximity to CAP site at -578, were identified (Fig. 3A). Functionally, as indicated by linker scanning and site-directed mutations (Fig. 2, CTAATT; PM8 gccgcT; TTAATTT; PM9: gcAATTT; PM10: TTAAgcg; and Fig. 3B, PMInr I: CTAATT to agccgg; PMInr II: TTAATTT to ggccggg), these two Inr motifs were shown to be essential for promoter activity. The site-directed mutation of either Inr motif resulted in a complete abolishment of the transcriptional activity. Interestingly, although the PM11 within the core promoter element does not overlap with the Inr motifs, mutation of this region also led to the abolishment of the promoter activity (Fig. 2).
As several important regions were identified, we next sought to characterize the protein(s) binding to these Inr motifs, PM11 and PM5 regions. We found that, using {alpha} T3–1 nuclear extract, three DNA-protein complexes were formed when the core promoter region (PP8–11) was used as a probe (Fig. 3C). The binding of these proteins with the DNA element is specific because only the cold PP8–11 oligo, but not nonspecific (NSP) DNA, is effective in competitively removing these complexes (see Fig. 7B). To study the Inr-specific interaction, the Inr and Inr mutant oligos were used in competition assays. Inr II, but not the mutant (Inr II {Delta} ), is able to specifically remove complex B (Fig. 3C). As expected, Inr I also interacted with the same nuclear protein because the intensity of the same complex (complex B) was reduced in the presence of an increasing concentration of the Inr I competitor DNA. Interestingly, we found that Inr II has a higher affinity for the protein in complex B because the presence of 50-fold excess of the unlabeled competitor was already sufficient to completely abrogate Inr-specific complex B formation (Fig. 3C). Thus, it is likely that these two Inr motifs within the same core promoter element bind the same protein(s) with different affinities.
fig.ommitteed7t, 百拇医药
Figure 7. The proximal and distal promoters of the hGnRHR gene share common protein factors. A, Putative Inr elements were found in both proximal and distal promoters. B, Competitive gel shift assays were performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as a probe (PP8–11, -608/-565). Formation of the retarded bands was completely abrogated when 200-fold of unlabeled competitor oligos containing either the proximal (PP8–11) or the distal (DP3/5) promoter element was included in the binding reaction. The 200-fold molar in excess of unlabeled competitor oligo containing only the Inr motif of the distal promoter (DP5) was able to abolish the formation of complexes A and B. In contrast, the NSP oligo failed to abolish the complex formation.7t, 百拇医药
Transcription factor class II (TFII) transcription regulator(s) binds to the Inr motifs to initiate the core promoter function7t, 百拇医药
To identify the protein factor(s) binding to the Inr elements of the core promoter, several consensus oligos of initiator-binding proteins [e.g. TFII-D, upstream stimulatory factor (USF), and Yin Yang-1 zinc finger transcription factor (YY1)] were used in the competition assay (Fig. 4A). Among these oligos, only TFII-D was able to competitively remove complexes A and B, suggesting the protein factors responsible for forming these complexes are TFII transcription regulators. We then tried to identify these proteins using Southwestern blot analysis. Several distinct bands of size ranging from 36–150 kDa were observed. The bands were specific because there was a dose-dependent increase in the band intensities and no signal was observed in the BSA control (Fig. 4B). Competitive Southwestern blot was also performed using PP8–11 as a probe and 200-fold excess of oligo Inr II (Fig. 4B, right) as a competitor. These data suggest that the Inr-specific proteins are 150 or 36 kDa in size.
fig.ommitteed)!, 百拇医药
Figure 4. The Inr-specific protein(s) is TFII transcription regulator. Multiple protein factors of sizes ranging from 36–150 kDa interact specifically with the core promoter PP8–11. A, Competition gel shift assay was performed with 10 µg T3–1 nuclear proteins using PP8–11 as probe. Only the consensus oligo of TFII-D can completely abrogate the formation of protein complexes A and B when 200-fold unlabeled competitor was included in the binding reaction. B, Fifteen or 30 µg of nuclear extract from T3–1 cell or BSA were separated in 10% SDS-PAGE, transblotted to nitrocellulose membrane, and subjected to Southwestern blot analysis using 32P-labeled PP8–11 oligo as probe (left). Thirty micrograms of nuclear extract from T3–1 cell or BSA were separated in 10% SDS-PAGE, transblotted to nitrocellulose membrane, and subjected to Southwestern blot analysis using 32P-labeled PP8–11 oligo as a probe and the unlabeled Inr II as a competitor (200-fold excess).)!, 百拇医药
To study the association between the protein factor(s) with the two Inr motifs, oligos PP8–11, PP8–11Mut-InrI, and PP8–11Mut-InrII (mutation at either Inr I or Inr II) were used as probes for the gel shift assays (Fig. 5A). Three DNA-protein complexes (A–C) were found using the PP8–11 and PP8–11Mut-Inr I as probes, whereas only complex C was observed when the PP8–11Mut-InrII probe was used. Interestingly, the band intensity of the complex C was increased when using these two mutated oligos as probes. It is possible that the mutated sequences aid the binding of protein(s) to form complex C. Further experimentation is needed to verify this suggestion. In the competitive EMSA with an increasing concentration of the PP8–11(Mut-Inr I/II) with mutations of both Inr I and Inr II motifs, we observed a dose-dependent reduction only in complex C (Fig. 5B). Taken together, these results suggested that although both Inr I and Inr II interact with an Inr-specific protein in complex B, this protein has a higher affinity for the Inr II motif, and the initial interaction of the protein with Inr II may be necessary to recruit additional protein(s) for the formation of complex A.
fig.ommitteedm, 百拇医药
Figure 5. Protein factor(s) in complex B has a higher affinity toward Inr II, and the interaction of protein factor(s) in complex B with Inr II may be able to facilitate the recruitment of protein factor(s) for the formation of complex A. A, Gel shift assays were performed with 10 µg T3–1 nuclear proteins using PP8–11, PP8–11(Mut-Inr I), and PP8–11(Mut-InrII) as probes. Three DNA-protein complexes (A–C) were observed when using PP8–11 and PP8–11(Mut-Inr I) as the probes. However, the formation of complexes A and B was abolished when the probe PP8–11(Mut-Inr II) was used. B, Competition gel shift assay was performed with 10 µg T3–1 nuclear proteins using PP8–11 as probe. Only the formation of complex C, but not complexes A and B, was abrogated when 50- to 200-fold unlabeled PP8–11(Mut-Inr I/II) oligo was included in the binding reaction.m, 百拇医药
EMSA studies of PM5 and PM11m, 百拇医药
In addition to the Inr motifs, DNA sequences within the PM11 region of PP8–11 were also indicated to be transcriptionally active (Fig. 2). To investigate the potential protein factors interacting with PM11, competitive EMSA was performed using the oligo PM11(-579/-566) and PP8–11(Mut-PM11) as the competitors (Fig. 6, A and B). The short competitor oligo with only the PM11(-579/-566) DNA sequences had no effect on the formation of DNA protein complexes A–C (Fig. 6A). On the other hand, the competitor oligo containing the core promoter PP8–11 with mutation of PM11, at 50-fold excess, could effectively compete out all of the complexes (Fig. 6B). Although we were unable to detect protein factor(s) that interacts with this motif, it is possible that the protein(s) binding to PM11(-579/-566) is required only transiently to assist the formation of the preinitiation complex at the Inr motifs. Similarly, no protein was observed to interact with the DNA sequences of PM5 in an EMSA study (data not shown); it is possible that mutation of PM5 disrupts the two potential CAP sites (-635, -631) for transcription initiation, resulting in the partial loss of function. Further experimentation is needed to clarify the functions of PM5 and PM11 and the protein factors interacting with these motifs.
fig.ommitteeda4#%i^, 百拇医药
Figure 6. Protein factor(s) is transiently interacting with region PM11. A, Competition gel shift assay was performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as probe. None of the protein complexes can be removed by the short probe containing only the region PM11, even when 200-fold in excess unlabeled probe was included in the binding reaction. B, Competition gel shift assay was performed with 10 µg {alpha} T3–1 nuclear proteins using PP8–11 as probe. The formation of all the protein complexes was abrogated when 50-fold of the unlabeled PP8–11(Mut-PM11) oligo was included in the binding reaction.a4#%i^, 百拇医药
The proximal and distal promoters of the hGnRHR gene share common nuclear proteinsa4#%i^, 百拇医药
The proximal promoter residing at -677/-558 exhibits similar properties when compared with the distal promoter at -1705/-1674 (9). By DNA sequence analysis, we found that both promoters contain the consensus Inr motifs (Fig. 7A; proximal promoter, -590/-584, TTAATTT, and -603/-597, CTAATTT; distal promoter, -1682/-1676, TCATTTC). On the basis of this observation, we postulated that these promoters interact with similar transcription factors. By competitive gel shift assay, the presence of 200-fold excess of the unlabeled distal promoter (Fig. 7B, DP3–5) could remove all of the proteins binding onto the proximal promoter (PP8–11). The Inr element of the distal promoter (Fig. 7B, DP5) was able to compete out complexes A and B but not complex C. It is not surprising that this distal Inr sequence was able to interact with two different proteins (A and B) because the consensus Inr sequence, YYANt/aYY, is known to bind a variety of transcription factors, e.g. TAFs [TATA-binding protein (TBP)-associated factors], TFII-I, USF-1, YY1, and RNA-polymerase II (18). There is also evidence that there is more than one TAF (TAFII150-TAFII250 heterodimers) interacting with the same Inr (19). In addition, TFII-I has also been suggested to be able to recognize several independent DNA elements because it has six helix-loop-helix domains (18). As a control, 200-fold excess of the NSP had no effect on these complexes (Fig. 7B, NSP). Our data suggested that the distal and proximal promoters of the hGnRHR gene use similar nuclear factors and the Inr-specific protein(s) is able to interact with the Inr motifs in these promoters.
Discussionmwwu!2, http://www.100md.com
In human, identical GnRHR cDNAs were isolated from pituitary, ovary, and placenta. This observation suggests that the differential responsiveness of these tissues to GnRH (3, 4, 5) is the result of the difference in receptor number and/or post-translational modification of the receptors expressed on the cell surface. It is highly possible that multiple functional promoters are involved in the regulation of the human GnRHR gene in various tissues. Differential usage of CAP sites was evident in human brain and pituitary by primer extension and 5' RACE studies. Five and 18 CAP sites were found in the brain (16) and the pituitary (17), respectively. By Northern blot analysis, it was revealed that the human pituitary contains three different GnRHR transcripts, i.e. 5.0, 2.5, and 1.5 kb (17), and only two of them (2.5 and 1.5 kb) were detected in the human placenta (2). PCR studies confirmed that these GnRHR transcripts are fully spliced, and hence they are not alternatively spliced products. It is therefore likely that several functional promoters are present to regulate the hGnRHR gene expression in different cell types within the hypothalamic-pituitary-gonadal axis.
Because there are 11 CAP sites locating at the region -818 to -578, this observation prompted us to search for the promoter element responsible for these CAP sites. By deletion mutation analysis and transient transfection studies in mouse gonadotrope-derived {alpha} T3–1 cell, a promoter element was identified at -677/-558, relative to ATG codon, which is functionally active in pituitary cell (Fig. 1) and extrapituitary cells (data not shown). The core promoter element was found in -607/-568 (PM8-PM11) by block replacement mutation studies. In common eukaryotic genes, transcription is dependent on both or one of the two core promoter elements, the TATA box and its functional analog, the initiator (Inr) (20). DNA sequence analysis revealed that no TATA or CCAAT box was identified, whereas two Inr motifs (I and II) are present within PP8–11 and these motifs are functional, as indicated by site-directed mutations. These data suggest that the proximal promoter is a TATA-Inr+ promoter. Inr is a pyrimidine-rich element that overlaps with the transcription start site, and it is capable of directing transcription initiation in a gene that lacks a TATA box (21, 22, 23, 24). The Inr element has been demonstrated to be able to select the CAP sites for gene transcription, and hence mutation of Inr element has a direct effect on the position of CAP sites (25). In mouse galectin-1 gene promoter, the Inr element overlapping with the TATA box was found to be responsible for directing transcription initiation from an alternative CAP site (26). Thus, it is possible that these two Inr motifs in the human GnRHR proximal promoter are responsible for the transcription initiation of the CAP sites in the close proximity. In addition, the different binding affinities of the Inr-specific protein factors to these two Inr motifs may be responsible for selecting the appropriate CAP sites for this promoter element.
In common eukaryotic genes, transcription is mediated by ordered assembly of a set of transcription factors at the core promoter element to form the transcription initiation complex. Several models have been suggested to explain the mechanism of transcription initiation through initiator element. In the first model, the TAFs are able to interact with Inr elements to direct transcription. The second model suggests that RNA-polymerase II is able to recognize the Inr element directly. In the third model, the initiator-specific proteins, such as TFII-I, YY1, or USF, bind to the Inr motif to recruit TBP and other transcription factors to form the preinitiation complex (18). Although we were unable to clarify the identity of the Inr-specific protein by super-shift assays using TFII-I and TFII-D antibodies (data not shown) by competitive gel shift assays and Southwestern blot analysis, four protein factors of sizes 36, 40, 105, and 150 kDa were found to interact specifically with the core promoter. Several protein factors of similar sizes that are involved in transcription initiation could potentially interact with the proximal promoter element or the Inr motifs within this region. TFII-D (36 kDa) is required in transcription initiation in both TATA+Inr- and TATA-Inr+ promoters. In TATA+Inr- promoter, TFII-D is the first protein binding to the TATA element to initiate the assembly of the other factors into a preinitiation complex (18, 20). On the other hand, some studies suggested that TFII-D alone is not sufficient to direct Inr-mediated transcription; instead, several cofactors such as cofactor of Inr function (CIF)150 and TAFs are required to stabilize its binding to the core promoter (27, 28). CIF150 (150 kDa), a human homolog of TAFII150, is an essential cofactor for TFII-D-dependent initiator function because it can interact with the hTAFII135 to stabilize the binding of TFII-D to the core promoter element. In addition, Sp1 (95 or 105 kDa) can also interact with hTAFII135 to promote the recruitment of CIF150 to the core promoter (27, 28). TFII-I (120 kDa) is needed for the transcription initiation of TATA-Inr+ promoter by interacting directly with the Inr element within the core promoter to recruit TFII-D to form the preinitiation complex (18, 20, 29). IBP39 (39 kDa) is a novel initiator binding protein isolated from a primitive eukaryote Trichomonas vaginalis by DNA affinity chromatography. It was suggested that the mammalian homologs of IBP39 are: 1) components of the CIF, or 2) DNA binding domains of TAFII150 and TAFII250 (30). Further experimentation, such as protein purification by DNA affinity chromatography followed by peptide sequencing or MALDI-TOF mass spectrum analysis, is required to further identify these protein factors (31, 32).
Because both distal and proximal promoters of the hGnRHR gene are active in pituitary and extrapituitary cells (data not shown) and they share common protein factors, it is interesting to understand how these promoters can differentially regulate the human GnRH receptor gene transcription in a temporal and spatial manner. The presence of multiple silencer and activator/enhancer elements may act as molecular switches to turn on and off these promoters in time- and cell-specific conditions. For example, extracellular signals such as {gamma} -aminobutyric acid have been shown to be able to induce cell-specific down-regulation of the human type A {gamma} -aminobutyric acid receptor ß1 subunit gene by mediating the binding of different factors to the Inr element (33). In addition, cAMP and PMA were demonstrated to modulate the MCAM gene transcription by altering the amount or affinity of the protein factors binding to the Inr element (34). Recently, we have shown that pituitary adenylate cyclase-activating polypeptide regulates the hGnRHR gene expression in gonadotropes by controlling the activities of a silencer element in the vicinity of the distal promoter (35). In fact, TAFs distinguish different promoters by recognizing sequence variations of the Inr elements (36). Thus, under the influence of external modulators, these Inr elements in the hGnRHR gene may exhibit different affinities toward different TAFs, and the binding of TAFs in the absence of TBP may determine the responsiveness of the core promoter to various activators as indicated previously (19). In summary, the Inr motifs present in the distal and proximal promoters of the hGnRH receptor gene are essential for the function of these promoters, and several protein factors (36–150 kDa) were identified to interact specifically with the core promoter. The hypothesis that these Inr motifs are involved in the differential regulation of these promoters remains to be investigated in the future.
Acknowledgments]l\l, 百拇医药
We thank Mr. K. H. Hui for his technical support and Mr. C. K. Cheng for his comments.]l\l, 百拇医药
Received June 5, 2002.]l\l, 百拇医药
Accepted for publication October 28, 2002.]l\l, 百拇医药
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References]l\l, 百拇医药
Peng C, Fan NC, Ligier M, Vaananen J, Leung PCK 1994 Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135:1740–1746]l\l, 百拇医药
Cheng KW, Nathwani PS, Leung PC 2000 Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. Endocrinology 141:2340–2349]l\l, 百拇医药
Kakar SS, Grantham K, Musgrove LC, Devor D, Sellers JC, Neill JD 1994 Rat gonadotropin-releasing hormone (GnRH) receptor: tissue expression and hormonal regulation of its mRNA. Mol Cell Endocrinol 101:151–157]l\l, 百拇医药
Currie AJ, Fraser HM, Sharpe RM 1981 Human placental receptors for luteinizing hormone releasing hormone. Biochem Biophys Res Commun 99:332–338
Iwashita M, Evans MI, Catt KJ 1986 Characterization of a gonadotropin-releasing hormone receptor site in term placenta and chorionic villi. J Clin Endocrinol Metab 62:127–133s.!|'\, http://www.100md.com
Ngan ESW, Cheng PKW, Leung PCK, Chow BKC 1999 Steroidogenic factor-1 intercts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression. Endocrinology 140:2452–2462s.!|'\, http://www.100md.com
Cheng KW, Ngan ESW, Kang SK, Chow BKC, Leung PCK 2000 Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1. Endocrinology 141:3611–3622s.!|'\, http://www.100md.com
Kang SK, Cheng KW, Ngan ESW, Chow BKC, Choi KC, Leung PCK 2000 Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. Mol Cell Endocrinol 162:157–166s.!|'\, http://www.100md.com
Ngan ESW, Leung PCK, Chow BKC 2000 Identification of an upstream promoter in the human gonadotropin-releasing hormone receptor gene. Biochem Biophys Res Comm 270:766–772
Maniatis T, Fritsch EF, Sambrook J 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory/:2to, 百拇医药
Chow BKC, Ting V, Tufaro F, MacGillivray RTA 1991 Characterization of a novel liver-specific enhancer in the human prothrombin gene. J Biol Chem 266:18927–18933/:2to, 百拇医药
Lee KAW, Bindereif A, Green MR 1988 A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech 5:22–31/:2to, 百拇医药
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhi K 1997 Mobility shift DNA binding assay using gel eletrophoresis. Current protocols in molecular biology. New York: J. Wiley and Sons; 12.2.1–12.2.7/:2to, 百拇医药
Wieczorek E, Lin Z, Perkins EB, Law DJ, Merchant JL, Zehner ZE 2000 The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1. J Biol Chem 275:12879–12888/:2to, 百拇医药
Motulsky HJ, Searle P 1995 GraphPad Prism user’s guide, version 2. San Diego, Graph-Pad Software
Fan NC, Peng C, Krisinger J, Leung PCK 1995 The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals. Mol Cell Endocrinol 107:R1–R8(6g3%, 百拇医药
Kakar SS 1997 Molecular structure of the human gonadotropin-releasing hormone receptor gene. Euro J Endocrinol 137:183–192(6g3%, 百拇医药
Morikawa N, Clarke TR, Novina CD, Watanabe K, Haqq C, Weiss M, Roy AL, Donahoe PK 2000 Human Mullerian-inhibiting substance promoter contains a functional TFII-I-binding initiator. Biol Reprod 63:1075–1083(6g3%, 百拇医药
Chalkley GE, Verrijzer CP 1999 DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator. EMBO J 17:4835–4845(6g3%, 百拇医药
Manzano-Winkler B, Novina CD, Roy AL 1996 TFII is required for transcription of the naturally TATA-less but initiator-containing Vß promoter. J Biol Chem 271:12076–12081(6g3%, 百拇医药
Hariharan N, Perry RP 1990 Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region. Proc Natl Acad Sci USA 87:1526–1530
Javahery R, Khachi K, Lo K, Zenzie-Gregory B, Smale ST 1994 DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol Cell Biol 14:116–127$mu#, 百拇医药
Lo K, Smale ST 1996 Generality of a functional initiator consensus sequence. Gene 182:13–22$mu#, 百拇医药
Smale ST, Baltimore D 1989 The "initiator" as a transcription control element. Cell 57:103–113$mu#, 百拇医药
Liston DR, Johnson PJ 1999 Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 19:2380–2388$mu#, 百拇医药
Gregorio ED, Chiariotti L, Nocera PPD 2001 The overlap of Inr and TATA elements sets the use of alternative transcriptional start sites in the mouse galectin-1 gene promoter. Gene 268:215–223$mu#, 百拇医药
Kaufmann J, Verrijzer CP, Shao J, Smale ST 1996 CIF, an essential cofactor for TFIID-dependent initiator function. Genes Dev 10:873–886$mu#, 百拇医药
Kaufmann J, Ahrens K, Koop R, Smale ST, Muller R 1998 CIF150, a human cofactor for transcription factor IID-dependent initiator function. Mol Cell Biol 18:233–239
Johansson E, Skogman E, Thelander L 1995 The TATA-less promoter of mouse ribonucleotide reductase R1 gene contains a TFII-I binding initiator element essential for cell cycle-regulated transcription. J Biol Chem 270:30162–30167-y@s, 百拇医药
Liston DR, Lau AO, Ortiz D, Smale ST, Johnson PJ 2001 Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis. Mol Cell Biol 21:7872–7882-y@s, 百拇医药
Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C 1993 Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90:5011–5015-y@s, 百拇医药
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ 2000 A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672-y@s, 百拇医药
Russek SJ, Bandopadhyay S, Farb DH 2000 An initiator element mediates autologous downregulation of the human type A {gamma} -aminobutyric acid receptor ß 1 subunit gene. Proc Natl Acad Sci USA 97:8600–8605-y@s, 百拇医药
Karlen S, Braathen LR 2000 Role of the initiator element in the regulation of the melanoma cell adhesion molecule gene. J Invest Dermatol 115:668–673-y@s, 百拇医药
Ngan ESW, Leung PCK, Chow BKC 2001 Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene. Mol Cell Endocrinol 176:135–144-y@s, 百拇医药
Verrijzer CP, Chen JL, Yokomori K, Tjian R 1995 Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II. Cell 81:1115–1125(Ruby L. C. Hoo Elly S. W. Ngan Peter C. K. Leung and Billy K. C. Chow)