Transactivation of the Parathyroid Hormone Promoter by Specificity Proteins and the Nuclear Factor Y Complex
http://www.100md.com
内分泌学杂志 2005年第8期
Division of Nephrology, Bone and Mineral Metabolism (A.P.A., H.H.M., N.J.K.) and Departments of Physiology (O.-K.P.-S.) and Molecular and Cellular Biochemistry (K.D.S.), University of Kentucky Medical Center, Lexington, Kentucky 40536
Address all correspondence and requests for reprints to: N. J. Koszewski, University of Kentucky Medical Center, Division of Nephrology, Bone and Mineral Metabolism, Room MN562, 800 Rose Street, Lexington, Kentucky 40536-0298. E-mail: njhosz0@uky.edu.
Abstract
We previously identified a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters that acted as an enhancer of gene transcription and bound Sp1 and Sp3 proteins present in parathyroid gland nuclear extracts. More recently, a nuclear factor (NF)-Y element (NF-Yprox) was also described by our group, which was located approximately 30 bp downstream from the Sp1 site in the human PTH (hPTH) promoter and by itself acted as a weak enhancer of gene transcription. We now report that Sp proteins and NF-Y can synergistically enhance transcription of a minimal hPTH promoter construct. Positioning of the Sp1 DNA element appears to be critical for this synergism because deviations of one half of a helical turn caused an approximate 60% decrease in transactivation. Finally, examination of the bovine PTH (bPTH) promoter also revealed Sp1/NF-Y synergism, in conjunction with the identification of an analogous NF-Y binding site similarly positioned downstream from the bPTH Sp1 element. In summary, synergistic transactivation of the hPTH and bPTH promoters is observed by Sp proteins and the NF-Y complex. The conservation of this transactivation in the human and bovine promoters suggests that this may be a principle means of enhancing PTH gene transcription.
Introduction
SPECIFICITY PROTEINS and the nuclear factor-Y (NF-Y) complex are thought to be ubiquitously expressed transcription factors associated with basal expression of a host of gene products (for review see Refs. 1 and 2). Others have shown that these factors are also likely involved in tissue-specific gene expression (3, 4, 5, 6). Several reports in the literature have indicated that specificity protein (Sp) family members and NF-Y can cooperatively enhance transcription of a target gene (7, 8, 9). It has also been reported that Sp proteins can interact directly with the A-subunit of the NF-Y complex (7, 10). Similarly, the human cystathionine-?-synthase-1b promoter exhibited synergistic activation by Sp1 and NF-Y, which could be attenuated by alternatively spliced isoforms of the NF-Y A-subunit (3).
Our group recently identified and characterized a highly conserved Sp1 DNA element present in mammalian PTH promoters (11). High levels of expression of both Sp3 and Sp1 were noted in parathyroid glands, and addition of phosphatase to parathyroid gland nuclear extracts stabilized their interactions with this DNA element. In addition, two NF-Y binding sites were discovered in the human PTH (hPTH) promoter, and abundant expression of NF-Y was also detected in parathyroid glands (12, 13). The distal NF-Y element partially overlapped the aforementioned Sp1 site, whereas a proximal element located approximately 30 bp downstream partially overlapped the previously characterized vitamin D response element (14). These two NF-Y DNA elements were shown to work in concert to synergistically enhance transcription of the hPTH promoter.
Reports in the literature indicate the importance of spacing between response elements for achieving full transcriptional potential. For example, three NF-Y elements are present in the cyclin B2 cell-cycle promoter, and correct spacing between them is essential for interactions with the p300 coactivator (15). In addition, the spacing of the two NF-Y elements in the hPTH promoter was highly similar to the arrangement of NF-Y sites in the human -globin and topoisomerase II? promoters, both of which are dependent on these sites for full promoter activity (16, 17). In addition, an Sp1 DNA element was also located approximately 30 bp upstream from one of the NF-Y elements in the topoisomerase II? promoter, and Sp protein interactions with this site made a significant contribution to synergistic transactivation of the gene (17). Based on these similarities in the arrangement of DNA response elements, we initiated a study of the PTH promoter to determine whether synergism between Sp proteins and NF-Y played a role in the transcription of this gene. We now report that coexpression of Sp proteins and NF-Y complex also leads to synergistic transactivation of the hPTH promoter, with alignment of the Sp1 DNA element essential for full activation. Furthermore, we identified a similar arrangement of DNA response elements and synergism in the bovine PTH (bPTH) promoter, suggesting that this may be a conserved mechanism to strongly enhance transcription of the PTH gene.
Materials and Methods
General
All enzymes were purchased from New England BioLabs (Beverly, MA) unless otherwise specified. Protease inhibitor cocktail (Complete, Mini) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The rabbit anti-NF-Y B-subunit and anti-estrogen receptor (ER) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The opossum kidney (OK) and Drosophila SL2 cell lines were purchased from American Type Culture Collection (Manassas, VA).
PCR and preparation of PTH promoter constructs
The hPTH wild-type (hPTHp/WT; –177 to +21) and mutant NF-Y response element promoter constructs linked to a luciferase reporter gene were described in detail previously (12, 13). Mutation of the Sp1 response element in the hPTH promoter was accomplished by two-step PCR as previously described (12). Oligonucleotides used for PCR amplification are listed in Table 1. Briefly, two separate PCR were performed to generate mutant promoter fragments with overlapping ends. The first reaction involved the following primer pairs that selectively inactivated the Sp1 binding site: hPTHPromF and HP-R-Sp1mut. The second amplification used hPTHPromR and HP-F-Sp1mut. PCR was performed by initial denaturation at 94 C for 3 min and then for 30 cycles as follows: 94 C for 30 sec, 56 C for 15 sec, and 72 C for 45 sec. After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the hPTHPromF and hPTHPromR primers and conditions as described above to generate the mutant Sp1 hPTH promoter fragment. The mutant promoter was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
TABLE 1. Oligonucleotides used in PCR amplification
Construction of the hPTHp/–5 spacing mutant in the hPTH promoter was accomplished by two-step PCR as described above using the following primer pairs to generate the overlapping mutant promoter fragments, with the removed 5-bp sequence highlighted as being between the underlined nucleotide pair (Table 1): hPTHPromF and HP-R-(–5); hPTHPromR and HP-F-(–5). After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the hPTHPromF and hPTHPromR primers and conditions as described above to generate the mutant hPTHp/–5 promoter fragment. The mutant promoter was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter. Similarly, construction of the hPTHp/+5 spacing mutant was also accomplished by two-step PCR as described above using the following primer pairs, with the inserted sequence underlined (Table 1): hPTHPromF and HP-R-(+5); hPTHPromR and HP-F-(+5).
The bPTH promoter (–170 to +24) luciferase reporter plasmid (bPTHp/luc) was prepared analogous to hPTHp/luc using bPTHPromF and bPTHPromR and bovine genomic DNA as described previously (13). The promoter product was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
Mutation of the NF-Y binding site in the bPTH promoter was also performed by two-step PCR as described above. Generation of the two overlapping mutant bovine promoter fragments was accomplished using the following combinations of paired PCR primers: bPTHPromF and BP-R-NF-Ymut; bPTHPromR and BP-F-NF-Ymut, where mutant positions are underlined, that selectively inactivated the binding site for NF-Y. PCR was performed by initial denaturation at 94 C for 3 min and then for 30 cycles as follows: 94 C for 30 sec, 56 C for 15 sec, and 72 C for 45 sec. After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the bPTHPromF and bPTHPromR primers and conditions as described above to generate the mutant NF-Y bPTH promoter fragment. The mutant promoter product was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
All reporter constructs were subjected to manual sequencing analysis to verify sequence identity.
Transient transfection
Drosophila SL2 cells were maintained in Drosophila SL2 media supplemented with 10% fetal bovine serum at 27 C. Cells were distributed in 24-well plates the day before transfection and transfected in triplicate wells with the indicated PTH promoter luciferase reporter construct (100 ng), p97b-?-galactosidase expression vector (50 ng), indicated pPac expression vectors, and carrier plasmid DNA made up to 500 ng total DNA per well. SL2 cells were transfected using Cellfectin (4 μl/well; Invitrogen Corp., Carlsbad, CA) for 3 h in media lacking serum, followed by supplementation to 7% serum. After 42 h, lysates were prepared by washing the cells with PBS (2x), followed by overlaying with lysis buffer and two rounds of freeze-thawing. Luciferase activities from individual wells were determined and normalized with respect to values for ?-galactosidase enzymatic activity, and average values were calculated ± SE from triplicate samples. Results are reflective of at least two independent experiments.
Preparation of nuclear extracts
Cultured OK cells were maintained in DMEM/F-12 (1:1) with 10% charcoal-stripped fetal bovine serum containing penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 C. The cells were detached with trypsin, washed with approximately 4 C PBS, incubated on ice in 3 vol of a similarly cold low-salt buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM EDTA, 2.0 mM dithiothreitol, 10% glycerol, and 1x protease inhibitor cocktail] for 20 min followed by cell disruption with a Teflon Dounce homogenizer. After a 30-min spin at 100,000 x g, the supernatants were removed and the nuclear pellets resuspended in 1 vol cold high-salt buffer (same as above with 400 mM KCl) and incubated on ice for 30 min with occasional gentle mixing. Samples were then spun at 100,000 x g for 30 min. The supernatant fractions were collected, aliquoted into individual tubes, snap-frozen, and stored at –70 C before use.
EMSA
Oligonucleotides (top strands indicated) used in the EMSA are listed in Table 2. The double-stranded oligonucleotide probe for the bPTH NF-Y was radiolabeled using Taq DNA polymerase (Invitrogen) and [32P-]dATP (3000 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), and the radiolabeled DNA fragment was gel purified before use in binding reactions. OK cell nuclear extracts were added to a binding solution [20 μl final volume; buffer components were 120 mM KCl, 20 mM Tris (pH 7.5), 1.5 mM EDTA, 2 mM dithiothreitol, 5% glycerol, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 10 mM NaF, 100 μM Na3VO4, 1.0 μg poly(deoxyinosine-deoxycytidine), and 100 μM leupeptin] for 30 min at 4 C. Where indicated, samples were incubated with antiserum for 30 min before addition of the radiolabeled DNA probe. For DNA competition experiments, the excess (250-fold) unlabeled oligonucleotide was also allowed to incubate with the sample for 30 min before addition of the radiolabeled DNA probe. After a 30-min incubation at 4 C with the radiolabeled DNA probe, samples were loaded onto prerun 5% polyacrylamide gels (29:1) and electrophoresis performed at approximately 14 V/cm for approximately 2 h 20 min with buffer cooling. Gels were transferred and dried, and autoradiography was performed.
TABLE 2. Oligonucleotides used in EMSA
Results
The location of a proximal NF-Y response element in the hPTH promoter approximately 30 bp downstream from an Sp1 DNA binding site suggested that the two factors could potentially interact with each other to synergistically activate transcription (3, 7, 10, 17). However, previous data from our laboratory indicated that Sp proteins may also be competing with the NF-Y complex for binding to a distal NF-Y DNA element that partially overlaps with the aforementioned Sp1 binding site (13). To address this topic, expression vectors for Sp1, the NF-Y complex, or both were cotransfected into Drosophila SL2 cells together with mutant hPTH promoter constructs that selectively inactivated the NF-Y elements (Fig. 1A), and we compared these with the wild-type promoter. As can be seen in Fig. 1B, cotransfection of Sp1 or NF-Y with the wild-type promoter resulted in the expected strong enhancement of reporter gene activity, whereas simultaneous expression of both factors in the SL2 cells produced a seemingly additive response, as noted previously (13). We next investigated the mutant hPTH promoter construct that inactivated the distal NF-Y binding site (NF-Ydist) thus alleviating competition between the two factors binding to the overlapping DNA elements. In this scenario, expression of Sp1 still resulted in strong enhancement of gene activity, whereas NF-Y alone had a much more muted capacity to stimulate the promoter, in keeping with previous results (12). However, coexpression of both factors resulted in strong synergistic transactivation of the mutant promoter that far exceeded the results for either factor alone. When both distal and proximal NF-Y binding sites were mutated, the strong synergistic response with Sp1 was largely lost, suggesting that proximal element was indeed critical for conveying this activity.
FIG. 1. Sp1 and NF-Y interactions with the wild-type (WT) and mutant (Mut) hPTH promoter constructs. A, Schematic of the hPTH promoter constructs, which were linked to a luciferase reporter gene. B, Drosophila SL2 cells were transfected in triplicate with the indicated promoter constructs (100 ng) and expression vectors (5 ng Sp1; 5 ng/subunit NF-Y). Cells were harvested after 42 h and assessed for luciferase activity. Values were normalized to the activity of a ?-galactosidase expression vector, and averages and SE calculated from the triplicate samples. Values above bars indicate fold increase relative to construct in the absence of expressed transcription factors.
To confirm that an intact Sp1 binding site was required for this activity, the Sp1 DNA element in the hPTH promoter was mutated and this construct examined for its transcriptional response to the various factors. Coexpression of Sp1 now produced only a minor enhancement of transcription, whereas inclusion of the NF-Y complex produced the characteristic robust increase (44-fold) in reporter activity from the mutant Sp1 hPTH promoter (Fig. 2A). However, coexpression of both Sp1 and NF-Y now failed to stimulate transcription above that seen with NF-Y alone, indicating that the Sp1 binding site was required to elicit a synergistic response in these cells.
FIG. 2. A, Dependence on an intact Sp1 binding site for the observed Sp1/NF-Y synergism with the hPTH promoter. A mutant hPTH promoter construct was generated that prevented Sp1 from binding to the hPTH promoter. Transfection was carried out in Drosophila SL2 cells with the indicated expression vectors (5 ng Sp1; 5 ng/subunit NF-Y), and luciferase values were normalized to the activity of ?-galactosidase. Values above bars indicate fold increase relative to construct in absence of expressed transcription factors. B, An intact NF-Y complex is required for synergistic transactivation with Sp1. Transfection analysis was done in Drosophila SL2 cells using the mutant NF-Ydist hPTH promoter construct. Cells were transfected with a constant amount of Sp1 expression vector (5 ng) and the indicated individual components of the NF-Y heterotrimeric complex (5 ng/subunit). Cells were harvested and assessed for luciferase activity as above. Values above bars indicate fold increase relative to construct in the presence of Sp1 alone.
We next investigated whether an intact NF-Y heterotrimeric complex was required for the synergistic response with Sp1. Accordingly, the NF-Ydist mutant hPTH promoter construct was cotransfected with an expression vector for Sp1 and different combinations of expression plasmids for the three individual subunits that comprise the NF-Y complex (Fig. 2B). A modest amount of enhancement was observed when the A- and B-subunits were expressed with Sp1 in the transfection experiment; however, the greatest induction (22-fold) was seen when all three subunits were coexpressed with Sp1 in the Drosophila SL2 cells, thus confirming that an intact complex was required for the observed synergism.
In an earlier report we noted a high level of expression of both Sp1 and Sp3 in the parathyroid gland (11); thus, the next set of experiments sought to ascertain whether the observed synergism was restricted to Sp1 or whether Sp3 could produce a similar response. The NF-Ydist mutant hPTH promoter construct was cotransfected with different combinations of Sp1 or Sp3 together with NF-Y (Fig. 3). As seen previously (Fig. 1), there was a strong synergistic enhancement of reporter activity when both Sp1 and the NF-Y complex were coexpressed in the SL2 cells (Fig. 3A). Similarly, coexpression of Sp3 and the NF-Y complex also produced a synergistic response from this promoter (Fig. 3B). Thus, the NF-Y complex appears to be capable of interacting with either of these Sp proteins to strongly enhance hPTH promoter activity.
FIG. 3. Comparison of different Sp family members’ ability to synergize with NF-Y on the hPTH promoter. The mutant NF-Ydist hPTH promoter construct (Fig. 1, Dist Mut) was used and NF-Y transcriptional activity assessed in conjunction with coexpression of Sp1 (left) or Sp3 (right), both of which are present in normal parathyroid glands (11 ). Transfection was carried out in Drosophila SL2 cells in the presence or absence of the indicated expression vectors (5 ng Sp1 or Sp3; 5 ng/subunit NF-Y) and assessed for luciferase activity as described previously. Values above bars indicate fold increase relative to construct in the absence of expressed transcription factors.
Attention then returned to the wild-type hPTH promoter to assess the effects of titrating the NF-Y complex in the absence or presence of a constant amount of low-level expression of Sp1 protein. As can be seen in Fig. 4, under these conditions, Sp1 alone (2 ng expression vector) produced a 6-fold enhancement of reporter gene activity, whereas transfection of half as much NF-Y complex (1 ng expression vector per subunit) produced a 12-fold increase in luciferase activity. The combination of these amounts of transfected Sp1 and NF-Y vectors, however, yielded a robust 69-fold increase in reporter readout from the wild-type hPTH promoter. A doubling of the transfected NF-Y complex alone (2 ng) essentially doubled luciferase activity (to 27-fold), whereas this combination of equivalent amounts of Sp1 and NF-Y still produced a strong, synergistic increase (to 110-fold) in promoter output. Finally, although strong activation (82-fold) of the promoter was evident with the maximum amount of transfected NF-Y (10 ng), the synergism with Sp1 was less evident. Coexpression of both factors now produced a 117-fold increase above control levels, but this represented only a 43% increase relative to NF-Y alone and was essentially unchanged from the previous 1:1 combination of Sp1 and NF-Y expression.
FIG. 4. Titration of NF-Y on Sp1 activity using the wild-type hPTH promoter construct. Transfection was carried out in Drosophila SL2 cells with the indicated expression vectors. Numbers in the table refer to amounts (in nanograms) of transfected expression vectors. Numbers above the bars refer to the fold induction above the baseline value in the absence of expressed transcription factors.
Reports of the importance in spacing between enhancer elements for cooperative or synergistic transcriptional responses prompted an examination of this issue regarding the distance between the Sp1 and the proximal NF-Y binding sites (NF-Yprox) (4, 18, 19). Two different mutant hPTH promoter constructs were made, which involved either decreasing the distance between these sites by approximately half a helical turn (hPTHp/–5) or increasing the distance between them by a similar amount (hPTHp/+5). As can be seen in Fig. 5A, decreasing the distance between the two NF-Y DNA elements caused an increase in NF-Y-stimulated activity and fold inducibility, whereas increasing the distance resulted in an overall decrease in reporter activity, although the fold induction was unchanged compared with the wild-type promoter. In contrast, cotransfection of Sp1 alone exhibited a marked, comparable (hPTHp/–5, –68%; hPTHp/+5, –60%) reduction of luciferase activity for both of the mutant promoter constructs compared with the wild-type sequence (Fig. 5B). The combination of transfecting equivalent amounts of Sp1 and NF-Y expression vectors was also evaluated and revealed a pattern that was analogous to that of Sp1 alone (Fig. 5C). To minimize the contribution of the NF-Y/NF-Y interaction in this latter case, the transfection was repeated by comparing the individual factors with their joint coexpression. In addition, the amount of added NF-Y complex expression vectors was also reduced to 20% of Sp1 expression vector to limit the amount of NF-Y/NF-Y synergistic transactivation. As can be seen in Fig. 5D, under these conditions, the combination of both factors still managed to synergize and enhance wild-type reporter activity much greater than either factor on its own. Meanwhile, as noted above, there was a marked decrease in Sp1 activity from the mutant hPTHp/–5 promoter, whereas the amount of reporter expression associated with NF-Y was slightly elevated compared with the wild-type sequence. However, the combination of the two factors, while still yielding enhanced activity, was decreased by approximately 60% relative to the wild-type promoter. Thus, it appears that the positioning of the Sp1 DNA element is critical for achieving the maximal enhanced transcriptional response, either from Sp1 alone or in concert with the NF-Y complex.
FIG. 5. Effect of altering the distance between Sp1 and NF-Y DNA elements on transactivation of the hPTH promoter. A, Drosophila SL2 cells were transfected in the absence (black bars) or presence (white bars) of expression vectors for the NF-Y complex (5 ng/subunit). B, Analogous transfection in the absence (black bars) or presence (white bars) of expression vector for Sp1 (5 ng). C, Analogous transfection in the absence (black bars) or presence (white bars) of expression vectors for both the NF-Y complex and Sp1 (5 ng each as in A and B). D, Transfection with the indicated reporter constructs and expression vectors for Sp1 (5 ng) or the NF-Y complex (1 ng/subunit) as indicated. Numbers refer to the fold induction above the baseline value in the absence of expressed transcription factors.
Previous data had shown that the liganded vitamin D receptor (VDR) heterodimer complex could repress NF-Y-enhanced transcription from the hPTH promoter (12). Accordingly, SL2 cells were transfected with the combination of Sp1 and NF-Y expression vectors to produce strong synergistic transcription of the luciferase reporter gene and then titrated with increasing amounts of the VDR heterodimer complex to assess the effects of hormone treatment on this synergism. As seen in Fig. 6, treatment with vitamin D caused a 78% decline in transcriptional activity at the lower VDR ratio, which increased to 88% suppression at the higher ratio. Thus, similar to the earlier results with NF-Y alone, the ligand-bound VDR heterodimer complex was also able to strongly suppress Sp1/NF-Y synergistic transactivation of the hPTH promoter.
FIG. 6. Suppression of Sp1/NF-Y synergistic transactivation of the hPTH promoter by vitamin D. Drosophila SL2 cells were transfected with the combination of Sp1 (5 ng) and NF-Y vectors (1 ng/subunit) as indicated. pPac expression vectors for human VDR and retinoid X receptor (RXR) were cotransfected in the indicated amounts relative to the Sp1 vector. Cells were treated with or without 100 nM 1,25-dihydroxyvitamin D3 (1,25D3) for 42 h and analyzed. Numbers above the bars refer to the fold induction compared with absence of expressed transcription factors. Numbers in parentheses indicate percentage decrease in the presence vs. the absence of hormone.
Previous work demonstrated that the Sp1 DNA element was conserved in other mammalian PTH promoters, including the bovine gene (11, 13) (data not shown). The next set of experiments was designed to evaluate whether Sp1/NF-Y transcriptional synergism observed with the hPTH promoter was also evident for the bPTH gene. The analogous bPTH promoter construct was transfected into Drosophila SL2 cells and coexpressed with Sp1, NF-Y, or the combination of the two transcription factors. As seen in Fig. 7A, inclusion of Sp1 in the transfection experiment resulted in modest enhancement of promoter activity, whereas NF-Y alone had no effect. However, expression of the combination of the two factors resulted in a dramatic increase in luciferase activity. These data were comparable to the results obtained with the mutant NF-Ydist hPTH promoter construct (Fig. 1), suggesting that a functional NF-Y element is present in the bovine promoter.
FIG. 7. Analysis of the bPTH promoter. A, The bPTHp/luc reporter construct was transfected into Drosophila SL2 cells together with the indicated expression vectors (5 ng Sp1; 5 ng/subunit NF-Y). B, Sequences surrounding the known NF-Yprox site (underlined) in the hPTH promoter in comparison with the corresponding region from the bPTH promoter (underlined). C, Mobility shift analysis using OK cell nuclear extracts and bPTH NF-Yprox oligonucleotide. Lane 1, Control binding reaction; lane 2, competition with excess unlabeled bPTH NF-Yprox oligonucleotide; lane 3, competition with Sp1 consensus element; lane 4, competition with hPTH NF-Yprox oligonucleotide; lane 5, competition with bPTH cAMP response element binding protein oligonucleotide; lane 6, competition with consensus NF-Y element; lane 7, addition of anti-NF-Y B-subunit antibody; lane 8, addition of anti-ER antibody. Arrowhead indicates position of the bound NF-Y complex. D, Transfection analysis of bPTH wild-type (bPTHwt) (white bars) and NF-Yprox mutant (Prox mut) (gray bars) promoters in Drosophila SL2 cells with the indicated expression vectors (5 ng Sp1; 1 ng/subunit NF-Y).
A sequence comparison between the human and bovine genes (Fig. 7B) suggested that an imperfect NF-Y binding site may also exist in the bovine promoter at a comparable position relative to the NF-Yprox site in the human gene. To evaluate this possibility, mobility shift experiments were pursued with a radiolabeled oligonucleotide from this region of the bPTH promoter, in combination with OK cell nuclear extracts previously used to demonstrate specific binding by the NF-Y complex to the NF-Yprox element in the hPTH promoter (12). As can be seen in Fig. 7C, a complex was readily evident under these conditions that could be competed with an excess of the corresponding unlabeled bPTH oligonucleotide but not by an excess of a consensus Sp1 DNA element (lanes 1–3). Furthermore, the corresponding region from the hPTH promoter and a consensus NF-Y DNA element could also compete for this complex (Fig. 7, lanes 4 and 6), whereas the cAMP response element binding protein DNA element from the bPTH promoter (20) failed to disrupt binding of the bound band (Fig. 7, lane 5). A rabbit anti-NF-Y B-subunit antibody was able to block formation of the complex, which was not recognized by a rabbit anti-ER antibody (Fig. 7, lanes 7 and 8). Thus, analogous to the situation with the hPTH promoter (12), specific binding by the NF-Y complex was evident with the corresponding region from the bovine promoter.
To confirm that this NF-Y binding site was responsible for the observed synergism, a mutant bPTH promoter construct was generated that selectively prevented binding by NF-Y to this site in mobility shift assays (data not shown). Both wild-type and mutant bPTH promoters were transfected into Drosophila SL2 cells and coexpressed with either Sp1, NF-Y, or a combination of the two factors to assess reporter gene activity. As can be seen in Fig. 7D (white bars), the wild-type bPTH promoter produced the expected strong synergism when both Sp1 and NF-Y were expressed in the cells, whereas there was only a modest increase in transcriptional activity in the presence of both factors when the mutant promoter was evaluated (gray bars). Thus, the identified NF-Y binding site in the bPTH promoter appears to be the principal transducer of the functional synergism observed between Sp1 and NF-Y in the SL2 cells.
Discussion
The current study extends our previous work examining factors that control transcription of the PTH promoter. Synergism was noted in a previous study of the NF-Y complex and its distal and proximal binding sites in the hPTH promoter (12). In the present findings, strong synergistic transactivation was also observed with the hPTH promoter when the combination of Sp proteins and NF-Y complex were coexpressed with the hPTH and bPTH promoter constructs. The titration data using the wild-type hPTH promoter (Fig. 4) indicated that at low relative concentrations of expression of each of the factors, the Sp1/NF-Y synergism far exceeded the synergistic NF-Y/NF-Y response. In combination with data from the bPTH promoter demonstrating similar Sp1/NF-Y synergism, the ability of NF-Y to bind to the proximal element and transcriptionally synergize with Sp proteins bound upstream suggests this may be a principle means of strongly enhancing PTH gene expression.
Previous work by Mantovani and colleagues (16) indicated a strict spacing requirement between two NF-Y DNA binding sites. Positioning of the Sp1 element relative to the NF-Yprox site suggests that this is also the case in PTH promoters. Physical interaction between Sp1 and the NF-Y complex has been reported (10, 21); therefore, it seems plausible that disruption of the spacing between their respective elements in the PTH promoter would have a negative impact on their protein-protein contacts and, hence, synergistic transactivation. Alternatively, Sp1 has been shown to interact directly with components of transcription factor IID (22, 23), and insertion or deletion of 5 bp caused a significant decrease in Sp1-mediated transcriptional activity from the PTH promoter, as well as synergistic transactivation by the combination of Sp1 and NF-Y (Fig. 5). Thus, we would speculate that proper alignment of the Sp1 element to position Sp1 in such a way as to facilitate interactions with components of the transcriptional machinery may be viewed as a critical feature for full activation of the PTH promoter, either by Sp1 alone or in conjunction with the NF-Y complex.
The aforementioned similarities between the hPTH and bPTH promoters in Sp1/NF-Y synergism suggests a common mechanism for enhancing PTH gene transcription; however, there are still notable differences in transcription factor binding sites between the two species. The NF-Ydist DNA element is unique to the human gene (13), as is the strong NF-Y/NF-Y synergistic enhancement of hPTH promoter activity (compare Figs. 1 and 6, and see Ref. 12). In addition, the NF-Yprox element in the human gene adjoins a repressor binding site for the VDR (12), but we have been unable to demonstrate directly or indirectly an analogous receptor binding site in the bPTH promoter (data not shown). Thus, expression of the human gene appears to contain additional layers of control not evident in its bovine counterpart and will require additional studies to uncover their overall significance and what, if any, selective advantage this affords.
Analogous to previous experiments (12), the liganded VDR heterodimer complex is able to suppress Sp1/NF-Y synergistic transactivation of the hPTH promoter (Fig. 6). Therefore, the repressive response exerted by vitamin D on hPTH promoter activity can now also be extended to take into account the capacity of NF-Y to interact with its proximal element and synergize with either Sp proteins or NF-Y binding at their distal positions. This suggests that control of this proximal position, either by NF-Y or the VDR heterodimer, may represent a key determinant of hPTH promoter activity in terms of either gene enhancement or repression. Although the VDR responds to circulating hormone concentrations, it remains to be seen whether modulation of NF-Y activity, through processes such as phosphorylation (24, 25), may also be a possible target of external stimuli that could increase or decrease its ability to interact with particularly the proximal element to alter PTH gene expression.
Although noted for their ubiquitous expression and involvement in basal transcriptional activity of numerous genes (1, 2, 26), several studies have determined that the expression of Sp proteins and the NF-Y complex are likely involved in tissue-specific gene expression as well (4, 5, 6, 27). Key questions remain, therefore, in delineating whether one or both of these proteins are involved in tissue-specific expression of PTH in the parathyroid gland or whether they simply represent factors relegated to basal transcriptional expression. Likewise, it will be of interest to determine whether either of these factors plays some role in various hypo- or hyperparathyroid disease states.
Acknowledgments
We thank H. Gravatte for excellent technical assistance. In addition, we acknowledge the generous contributions by Dr. T. Osborne (Irvine, CA) for providing the individual NF-Y A, B, and C subunit Drosophila pPac expression vectors and Dr. G. Suske (Marburg, Germany) for providing the pPac Sp1 and Sp3 expression vectors.
References
Kaczynski J, Cook T, Urrutia R 2003 Sp1- and Kruppel-like transcription factors. Genome Biol 4:206
Mantovani R 1999 The molecular biology of the CCAAT-binding factor NF-Y. Gene 239:15–27
Ge Y, Jensen TL, Matherly LH, Taub JW 2002 Synergistic regulation of human cystathionine-?-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. Biochim Biophys Acta 1579:73–80
Rodenburg RJ, Holthuizen PE, Sussenbach JS 1997 A functional Sp1 binding site is essential for the activity of the adult liver-specific human insulin-like growth factor II promoter. Mol Endocrinol 11:237–250
Valor LM, Campos-Caro A, Carrasco-Serrano C, Ortiz JA, Ballesta JJ, Criado M 2002 Transcription factors NF-Y and Sp1 are important determinants of the promoter activity of the bovine and human neuronal nicotinic receptor ?4 subunit genes. J Biol Chem 277:8866–8876
Zhang LP, Stroud J, Eddy CA, Walter CA, McCarrey JR 1999 Multiple elements influence transcriptional regulation from the human testis-specific PGK2 promoter in transgenic mice. Biol Reprod 60:1329–1337
Yamada K, Tanaka T, Miyamoto K, Noguchi T 2000 Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions. J Biol Chem 275:18129–18137
Iwano S, Saito T, Takahashi Y, Fujita K, Kamataki T 2001 Cooperative regulation of CYP3A5 gene transcription by NF-Y and Sp family members. Biochem Biophys Res Commun 286:55–60
Murakami Y, Ikeda U, Shimada K, Kawakami K 1997 Promoter of the Na,K-ATPase 3 subunit gene is composed of cis elements to which NF-Y and Sp1/Sp3 bind in rat cardiocytes. Biochim Biophys Acta 1352:311–324
Roder K, Wolf SS, Larkin KJ, Schweizer M 1999 Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1. Gene 234:61–69
Alimov AP, Langub MC, Malluche HH, Koszewski NJ 2003 Sp3/Sp1 in the parathyroid gland: identification of an Sp1 deoxyribonucleic acid element in the parathyroid hormone promoter. Endocrinology 144:3138–3147
Koszewski NJ, Alimov AP, Park-Sarge OK, Malluche HH 2004 Suppression of the human parathyroid hormone promoter by vitamin D involves displacement of NF-Y binding to the vitamin D response element. J Biol Chem 279:42431–42437
Alimov AP, Langub MC, Malluche HH, Park-Sarge OK, Koszewski NJ 2004 Contrasting mammalian parathyroid hormone (PTH) promoters: nuclear factor-Y binds to a deoxyribonucleic acid element unique to the human PTH promoter and acts as a transcriptional enhancer. Endocrinology 145:2713–2720
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 89:8097–8101
Salsi V, Caretti G, Wasner M, Reinhard W, Haugwitz U, Engeland K, Mantovani R 2003 Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function. J Biol Chem 278:6642–6650
Liberati C, di Silvio A, Ottolenghi S, Mantovani R 1999 NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices. J Mol Biol 285:1441–1455
Lok CN, Lang AJ, Mirski SE, Cole SP 2002 Characterization of the human topoisomerase II? (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites. Biochem J 368:741–751
Inoue J, Sato R, Maeda M 1998 Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. J Biochem (Tokyo) 123:1191–1198
Danilition SL, Frederickson RM, Taylor CY, Miyamoto NG 1991 Transcription factor binding and spacing constraints in the human ?-actin proximal promoter. Nucleic Acids Res 19:6913–6922
Deutsch PJ, Hoeffler JP, Jameson JL, Lin JC, Habener JF 1988 Structural determinants for transcriptional activation by cAMP- responsive DNA elements. J Biol Chem 263:18466–18472
Liang F, Schaufele F, Gardner DG 2001 Functional interaction of NF-Y and Sp1 is required for type a natriuretic peptide receptor gene transcription. J Biol Chem 276:1516–1522
Gill G, Pascal E, Tseng ZH, Tjian R 1994 A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci USA 91:192–196
Hoey T, Weinzierl RO, Gill G, Chen JL, Dynlacht BD, Tjian R 1993 Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell 72:247–260
Yun J, Chae HD, Choi TS, Kim EH, Bang YJ, Chung J, Choi KS, Mantovani R, Shin DY 2003 Cdk2-dependent phosphorylation of the NF-Y transcription factor and its involvement in the p53–p21 signaling pathway. J Biol Chem 278:36966–36972
Chae HD, Yun J, Bang YJ, Shin DY 2004 Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions. Oncogene 23:4084–4088
Li L, He S, Sun JM, Davie JR 2004 Gene regulation by Sp1 and Sp3. Biochem Cell Biol 82:460–471
Ge Y, Konrad MA, Matherly LH, Taub JW 2001 Transcriptional regulation of the human cystathionine ?-synthase-1b basal promoter: synergistic transactivation by transcription factors NF-Y and Sp1/Sp3. Biochem J 357:97–105(Alexander P. Alimov, Ok-K)
Address all correspondence and requests for reprints to: N. J. Koszewski, University of Kentucky Medical Center, Division of Nephrology, Bone and Mineral Metabolism, Room MN562, 800 Rose Street, Lexington, Kentucky 40536-0298. E-mail: njhosz0@uky.edu.
Abstract
We previously identified a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters that acted as an enhancer of gene transcription and bound Sp1 and Sp3 proteins present in parathyroid gland nuclear extracts. More recently, a nuclear factor (NF)-Y element (NF-Yprox) was also described by our group, which was located approximately 30 bp downstream from the Sp1 site in the human PTH (hPTH) promoter and by itself acted as a weak enhancer of gene transcription. We now report that Sp proteins and NF-Y can synergistically enhance transcription of a minimal hPTH promoter construct. Positioning of the Sp1 DNA element appears to be critical for this synergism because deviations of one half of a helical turn caused an approximate 60% decrease in transactivation. Finally, examination of the bovine PTH (bPTH) promoter also revealed Sp1/NF-Y synergism, in conjunction with the identification of an analogous NF-Y binding site similarly positioned downstream from the bPTH Sp1 element. In summary, synergistic transactivation of the hPTH and bPTH promoters is observed by Sp proteins and the NF-Y complex. The conservation of this transactivation in the human and bovine promoters suggests that this may be a principle means of enhancing PTH gene transcription.
Introduction
SPECIFICITY PROTEINS and the nuclear factor-Y (NF-Y) complex are thought to be ubiquitously expressed transcription factors associated with basal expression of a host of gene products (for review see Refs. 1 and 2). Others have shown that these factors are also likely involved in tissue-specific gene expression (3, 4, 5, 6). Several reports in the literature have indicated that specificity protein (Sp) family members and NF-Y can cooperatively enhance transcription of a target gene (7, 8, 9). It has also been reported that Sp proteins can interact directly with the A-subunit of the NF-Y complex (7, 10). Similarly, the human cystathionine-?-synthase-1b promoter exhibited synergistic activation by Sp1 and NF-Y, which could be attenuated by alternatively spliced isoforms of the NF-Y A-subunit (3).
Our group recently identified and characterized a highly conserved Sp1 DNA element present in mammalian PTH promoters (11). High levels of expression of both Sp3 and Sp1 were noted in parathyroid glands, and addition of phosphatase to parathyroid gland nuclear extracts stabilized their interactions with this DNA element. In addition, two NF-Y binding sites were discovered in the human PTH (hPTH) promoter, and abundant expression of NF-Y was also detected in parathyroid glands (12, 13). The distal NF-Y element partially overlapped the aforementioned Sp1 site, whereas a proximal element located approximately 30 bp downstream partially overlapped the previously characterized vitamin D response element (14). These two NF-Y DNA elements were shown to work in concert to synergistically enhance transcription of the hPTH promoter.
Reports in the literature indicate the importance of spacing between response elements for achieving full transcriptional potential. For example, three NF-Y elements are present in the cyclin B2 cell-cycle promoter, and correct spacing between them is essential for interactions with the p300 coactivator (15). In addition, the spacing of the two NF-Y elements in the hPTH promoter was highly similar to the arrangement of NF-Y sites in the human -globin and topoisomerase II? promoters, both of which are dependent on these sites for full promoter activity (16, 17). In addition, an Sp1 DNA element was also located approximately 30 bp upstream from one of the NF-Y elements in the topoisomerase II? promoter, and Sp protein interactions with this site made a significant contribution to synergistic transactivation of the gene (17). Based on these similarities in the arrangement of DNA response elements, we initiated a study of the PTH promoter to determine whether synergism between Sp proteins and NF-Y played a role in the transcription of this gene. We now report that coexpression of Sp proteins and NF-Y complex also leads to synergistic transactivation of the hPTH promoter, with alignment of the Sp1 DNA element essential for full activation. Furthermore, we identified a similar arrangement of DNA response elements and synergism in the bovine PTH (bPTH) promoter, suggesting that this may be a conserved mechanism to strongly enhance transcription of the PTH gene.
Materials and Methods
General
All enzymes were purchased from New England BioLabs (Beverly, MA) unless otherwise specified. Protease inhibitor cocktail (Complete, Mini) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The rabbit anti-NF-Y B-subunit and anti-estrogen receptor (ER) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The opossum kidney (OK) and Drosophila SL2 cell lines were purchased from American Type Culture Collection (Manassas, VA).
PCR and preparation of PTH promoter constructs
The hPTH wild-type (hPTHp/WT; –177 to +21) and mutant NF-Y response element promoter constructs linked to a luciferase reporter gene were described in detail previously (12, 13). Mutation of the Sp1 response element in the hPTH promoter was accomplished by two-step PCR as previously described (12). Oligonucleotides used for PCR amplification are listed in Table 1. Briefly, two separate PCR were performed to generate mutant promoter fragments with overlapping ends. The first reaction involved the following primer pairs that selectively inactivated the Sp1 binding site: hPTHPromF and HP-R-Sp1mut. The second amplification used hPTHPromR and HP-F-Sp1mut. PCR was performed by initial denaturation at 94 C for 3 min and then for 30 cycles as follows: 94 C for 30 sec, 56 C for 15 sec, and 72 C for 45 sec. After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the hPTHPromF and hPTHPromR primers and conditions as described above to generate the mutant Sp1 hPTH promoter fragment. The mutant promoter was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
TABLE 1. Oligonucleotides used in PCR amplification
Construction of the hPTHp/–5 spacing mutant in the hPTH promoter was accomplished by two-step PCR as described above using the following primer pairs to generate the overlapping mutant promoter fragments, with the removed 5-bp sequence highlighted as being between the underlined nucleotide pair (Table 1): hPTHPromF and HP-R-(–5); hPTHPromR and HP-F-(–5). After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the hPTHPromF and hPTHPromR primers and conditions as described above to generate the mutant hPTHp/–5 promoter fragment. The mutant promoter was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter. Similarly, construction of the hPTHp/+5 spacing mutant was also accomplished by two-step PCR as described above using the following primer pairs, with the inserted sequence underlined (Table 1): hPTHPromF and HP-R-(+5); hPTHPromR and HP-F-(+5).
The bPTH promoter (–170 to +24) luciferase reporter plasmid (bPTHp/luc) was prepared analogous to hPTHp/luc using bPTHPromF and bPTHPromR and bovine genomic DNA as described previously (13). The promoter product was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
Mutation of the NF-Y binding site in the bPTH promoter was also performed by two-step PCR as described above. Generation of the two overlapping mutant bovine promoter fragments was accomplished using the following combinations of paired PCR primers: bPTHPromF and BP-R-NF-Ymut; bPTHPromR and BP-F-NF-Ymut, where mutant positions are underlined, that selectively inactivated the binding site for NF-Y. PCR was performed by initial denaturation at 94 C for 3 min and then for 30 cycles as follows: 94 C for 30 sec, 56 C for 15 sec, and 72 C for 45 sec. After isolation of the two separate PCR products, aliquots of each were mixed and PCR performed again using the bPTHPromF and bPTHPromR primers and conditions as described above to generate the mutant NF-Y bPTH promoter fragment. The mutant promoter product was isolated, digested with BamHI/XhoI, and ligated into the same sites of the luciferase reporter.
All reporter constructs were subjected to manual sequencing analysis to verify sequence identity.
Transient transfection
Drosophila SL2 cells were maintained in Drosophila SL2 media supplemented with 10% fetal bovine serum at 27 C. Cells were distributed in 24-well plates the day before transfection and transfected in triplicate wells with the indicated PTH promoter luciferase reporter construct (100 ng), p97b-?-galactosidase expression vector (50 ng), indicated pPac expression vectors, and carrier plasmid DNA made up to 500 ng total DNA per well. SL2 cells were transfected using Cellfectin (4 μl/well; Invitrogen Corp., Carlsbad, CA) for 3 h in media lacking serum, followed by supplementation to 7% serum. After 42 h, lysates were prepared by washing the cells with PBS (2x), followed by overlaying with lysis buffer and two rounds of freeze-thawing. Luciferase activities from individual wells were determined and normalized with respect to values for ?-galactosidase enzymatic activity, and average values were calculated ± SE from triplicate samples. Results are reflective of at least two independent experiments.
Preparation of nuclear extracts
Cultured OK cells were maintained in DMEM/F-12 (1:1) with 10% charcoal-stripped fetal bovine serum containing penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 C. The cells were detached with trypsin, washed with approximately 4 C PBS, incubated on ice in 3 vol of a similarly cold low-salt buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM EDTA, 2.0 mM dithiothreitol, 10% glycerol, and 1x protease inhibitor cocktail] for 20 min followed by cell disruption with a Teflon Dounce homogenizer. After a 30-min spin at 100,000 x g, the supernatants were removed and the nuclear pellets resuspended in 1 vol cold high-salt buffer (same as above with 400 mM KCl) and incubated on ice for 30 min with occasional gentle mixing. Samples were then spun at 100,000 x g for 30 min. The supernatant fractions were collected, aliquoted into individual tubes, snap-frozen, and stored at –70 C before use.
EMSA
Oligonucleotides (top strands indicated) used in the EMSA are listed in Table 2. The double-stranded oligonucleotide probe for the bPTH NF-Y was radiolabeled using Taq DNA polymerase (Invitrogen) and [32P-]dATP (3000 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), and the radiolabeled DNA fragment was gel purified before use in binding reactions. OK cell nuclear extracts were added to a binding solution [20 μl final volume; buffer components were 120 mM KCl, 20 mM Tris (pH 7.5), 1.5 mM EDTA, 2 mM dithiothreitol, 5% glycerol, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 10 mM NaF, 100 μM Na3VO4, 1.0 μg poly(deoxyinosine-deoxycytidine), and 100 μM leupeptin] for 30 min at 4 C. Where indicated, samples were incubated with antiserum for 30 min before addition of the radiolabeled DNA probe. For DNA competition experiments, the excess (250-fold) unlabeled oligonucleotide was also allowed to incubate with the sample for 30 min before addition of the radiolabeled DNA probe. After a 30-min incubation at 4 C with the radiolabeled DNA probe, samples were loaded onto prerun 5% polyacrylamide gels (29:1) and electrophoresis performed at approximately 14 V/cm for approximately 2 h 20 min with buffer cooling. Gels were transferred and dried, and autoradiography was performed.
TABLE 2. Oligonucleotides used in EMSA
Results
The location of a proximal NF-Y response element in the hPTH promoter approximately 30 bp downstream from an Sp1 DNA binding site suggested that the two factors could potentially interact with each other to synergistically activate transcription (3, 7, 10, 17). However, previous data from our laboratory indicated that Sp proteins may also be competing with the NF-Y complex for binding to a distal NF-Y DNA element that partially overlaps with the aforementioned Sp1 binding site (13). To address this topic, expression vectors for Sp1, the NF-Y complex, or both were cotransfected into Drosophila SL2 cells together with mutant hPTH promoter constructs that selectively inactivated the NF-Y elements (Fig. 1A), and we compared these with the wild-type promoter. As can be seen in Fig. 1B, cotransfection of Sp1 or NF-Y with the wild-type promoter resulted in the expected strong enhancement of reporter gene activity, whereas simultaneous expression of both factors in the SL2 cells produced a seemingly additive response, as noted previously (13). We next investigated the mutant hPTH promoter construct that inactivated the distal NF-Y binding site (NF-Ydist) thus alleviating competition between the two factors binding to the overlapping DNA elements. In this scenario, expression of Sp1 still resulted in strong enhancement of gene activity, whereas NF-Y alone had a much more muted capacity to stimulate the promoter, in keeping with previous results (12). However, coexpression of both factors resulted in strong synergistic transactivation of the mutant promoter that far exceeded the results for either factor alone. When both distal and proximal NF-Y binding sites were mutated, the strong synergistic response with Sp1 was largely lost, suggesting that proximal element was indeed critical for conveying this activity.
FIG. 1. Sp1 and NF-Y interactions with the wild-type (WT) and mutant (Mut) hPTH promoter constructs. A, Schematic of the hPTH promoter constructs, which were linked to a luciferase reporter gene. B, Drosophila SL2 cells were transfected in triplicate with the indicated promoter constructs (100 ng) and expression vectors (5 ng Sp1; 5 ng/subunit NF-Y). Cells were harvested after 42 h and assessed for luciferase activity. Values were normalized to the activity of a ?-galactosidase expression vector, and averages and SE calculated from the triplicate samples. Values above bars indicate fold increase relative to construct in the absence of expressed transcription factors.
To confirm that an intact Sp1 binding site was required for this activity, the Sp1 DNA element in the hPTH promoter was mutated and this construct examined for its transcriptional response to the various factors. Coexpression of Sp1 now produced only a minor enhancement of transcription, whereas inclusion of the NF-Y complex produced the characteristic robust increase (44-fold) in reporter activity from the mutant Sp1 hPTH promoter (Fig. 2A). However, coexpression of both Sp1 and NF-Y now failed to stimulate transcription above that seen with NF-Y alone, indicating that the Sp1 binding site was required to elicit a synergistic response in these cells.
FIG. 2. A, Dependence on an intact Sp1 binding site for the observed Sp1/NF-Y synergism with the hPTH promoter. A mutant hPTH promoter construct was generated that prevented Sp1 from binding to the hPTH promoter. Transfection was carried out in Drosophila SL2 cells with the indicated expression vectors (5 ng Sp1; 5 ng/subunit NF-Y), and luciferase values were normalized to the activity of ?-galactosidase. Values above bars indicate fold increase relative to construct in absence of expressed transcription factors. B, An intact NF-Y complex is required for synergistic transactivation with Sp1. Transfection analysis was done in Drosophila SL2 cells using the mutant NF-Ydist hPTH promoter construct. Cells were transfected with a constant amount of Sp1 expression vector (5 ng) and the indicated individual components of the NF-Y heterotrimeric complex (5 ng/subunit). Cells were harvested and assessed for luciferase activity as above. Values above bars indicate fold increase relative to construct in the presence of Sp1 alone.
We next investigated whether an intact NF-Y heterotrimeric complex was required for the synergistic response with Sp1. Accordingly, the NF-Ydist mutant hPTH promoter construct was cotransfected with an expression vector for Sp1 and different combinations of expression plasmids for the three individual subunits that comprise the NF-Y complex (Fig. 2B). A modest amount of enhancement was observed when the A- and B-subunits were expressed with Sp1 in the transfection experiment; however, the greatest induction (22-fold) was seen when all three subunits were coexpressed with Sp1 in the Drosophila SL2 cells, thus confirming that an intact complex was required for the observed synergism.
In an earlier report we noted a high level of expression of both Sp1 and Sp3 in the parathyroid gland (11); thus, the next set of experiments sought to ascertain whether the observed synergism was restricted to Sp1 or whether Sp3 could produce a similar response. The NF-Ydist mutant hPTH promoter construct was cotransfected with different combinations of Sp1 or Sp3 together with NF-Y (Fig. 3). As seen previously (Fig. 1), there was a strong synergistic enhancement of reporter activity when both Sp1 and the NF-Y complex were coexpressed in the SL2 cells (Fig. 3A). Similarly, coexpression of Sp3 and the NF-Y complex also produced a synergistic response from this promoter (Fig. 3B). Thus, the NF-Y complex appears to be capable of interacting with either of these Sp proteins to strongly enhance hPTH promoter activity.
FIG. 3. Comparison of different Sp family members’ ability to synergize with NF-Y on the hPTH promoter. The mutant NF-Ydist hPTH promoter construct (Fig. 1, Dist Mut) was used and NF-Y transcriptional activity assessed in conjunction with coexpression of Sp1 (left) or Sp3 (right), both of which are present in normal parathyroid glands (11 ). Transfection was carried out in Drosophila SL2 cells in the presence or absence of the indicated expression vectors (5 ng Sp1 or Sp3; 5 ng/subunit NF-Y) and assessed for luciferase activity as described previously. Values above bars indicate fold increase relative to construct in the absence of expressed transcription factors.
Attention then returned to the wild-type hPTH promoter to assess the effects of titrating the NF-Y complex in the absence or presence of a constant amount of low-level expression of Sp1 protein. As can be seen in Fig. 4, under these conditions, Sp1 alone (2 ng expression vector) produced a 6-fold enhancement of reporter gene activity, whereas transfection of half as much NF-Y complex (1 ng expression vector per subunit) produced a 12-fold increase in luciferase activity. The combination of these amounts of transfected Sp1 and NF-Y vectors, however, yielded a robust 69-fold increase in reporter readout from the wild-type hPTH promoter. A doubling of the transfected NF-Y complex alone (2 ng) essentially doubled luciferase activity (to 27-fold), whereas this combination of equivalent amounts of Sp1 and NF-Y still produced a strong, synergistic increase (to 110-fold) in promoter output. Finally, although strong activation (82-fold) of the promoter was evident with the maximum amount of transfected NF-Y (10 ng), the synergism with Sp1 was less evident. Coexpression of both factors now produced a 117-fold increase above control levels, but this represented only a 43% increase relative to NF-Y alone and was essentially unchanged from the previous 1:1 combination of Sp1 and NF-Y expression.
FIG. 4. Titration of NF-Y on Sp1 activity using the wild-type hPTH promoter construct. Transfection was carried out in Drosophila SL2 cells with the indicated expression vectors. Numbers in the table refer to amounts (in nanograms) of transfected expression vectors. Numbers above the bars refer to the fold induction above the baseline value in the absence of expressed transcription factors.
Reports of the importance in spacing between enhancer elements for cooperative or synergistic transcriptional responses prompted an examination of this issue regarding the distance between the Sp1 and the proximal NF-Y binding sites (NF-Yprox) (4, 18, 19). Two different mutant hPTH promoter constructs were made, which involved either decreasing the distance between these sites by approximately half a helical turn (hPTHp/–5) or increasing the distance between them by a similar amount (hPTHp/+5). As can be seen in Fig. 5A, decreasing the distance between the two NF-Y DNA elements caused an increase in NF-Y-stimulated activity and fold inducibility, whereas increasing the distance resulted in an overall decrease in reporter activity, although the fold induction was unchanged compared with the wild-type promoter. In contrast, cotransfection of Sp1 alone exhibited a marked, comparable (hPTHp/–5, –68%; hPTHp/+5, –60%) reduction of luciferase activity for both of the mutant promoter constructs compared with the wild-type sequence (Fig. 5B). The combination of transfecting equivalent amounts of Sp1 and NF-Y expression vectors was also evaluated and revealed a pattern that was analogous to that of Sp1 alone (Fig. 5C). To minimize the contribution of the NF-Y/NF-Y interaction in this latter case, the transfection was repeated by comparing the individual factors with their joint coexpression. In addition, the amount of added NF-Y complex expression vectors was also reduced to 20% of Sp1 expression vector to limit the amount of NF-Y/NF-Y synergistic transactivation. As can be seen in Fig. 5D, under these conditions, the combination of both factors still managed to synergize and enhance wild-type reporter activity much greater than either factor on its own. Meanwhile, as noted above, there was a marked decrease in Sp1 activity from the mutant hPTHp/–5 promoter, whereas the amount of reporter expression associated with NF-Y was slightly elevated compared with the wild-type sequence. However, the combination of the two factors, while still yielding enhanced activity, was decreased by approximately 60% relative to the wild-type promoter. Thus, it appears that the positioning of the Sp1 DNA element is critical for achieving the maximal enhanced transcriptional response, either from Sp1 alone or in concert with the NF-Y complex.
FIG. 5. Effect of altering the distance between Sp1 and NF-Y DNA elements on transactivation of the hPTH promoter. A, Drosophila SL2 cells were transfected in the absence (black bars) or presence (white bars) of expression vectors for the NF-Y complex (5 ng/subunit). B, Analogous transfection in the absence (black bars) or presence (white bars) of expression vector for Sp1 (5 ng). C, Analogous transfection in the absence (black bars) or presence (white bars) of expression vectors for both the NF-Y complex and Sp1 (5 ng each as in A and B). D, Transfection with the indicated reporter constructs and expression vectors for Sp1 (5 ng) or the NF-Y complex (1 ng/subunit) as indicated. Numbers refer to the fold induction above the baseline value in the absence of expressed transcription factors.
Previous data had shown that the liganded vitamin D receptor (VDR) heterodimer complex could repress NF-Y-enhanced transcription from the hPTH promoter (12). Accordingly, SL2 cells were transfected with the combination of Sp1 and NF-Y expression vectors to produce strong synergistic transcription of the luciferase reporter gene and then titrated with increasing amounts of the VDR heterodimer complex to assess the effects of hormone treatment on this synergism. As seen in Fig. 6, treatment with vitamin D caused a 78% decline in transcriptional activity at the lower VDR ratio, which increased to 88% suppression at the higher ratio. Thus, similar to the earlier results with NF-Y alone, the ligand-bound VDR heterodimer complex was also able to strongly suppress Sp1/NF-Y synergistic transactivation of the hPTH promoter.
FIG. 6. Suppression of Sp1/NF-Y synergistic transactivation of the hPTH promoter by vitamin D. Drosophila SL2 cells were transfected with the combination of Sp1 (5 ng) and NF-Y vectors (1 ng/subunit) as indicated. pPac expression vectors for human VDR and retinoid X receptor (RXR) were cotransfected in the indicated amounts relative to the Sp1 vector. Cells were treated with or without 100 nM 1,25-dihydroxyvitamin D3 (1,25D3) for 42 h and analyzed. Numbers above the bars refer to the fold induction compared with absence of expressed transcription factors. Numbers in parentheses indicate percentage decrease in the presence vs. the absence of hormone.
Previous work demonstrated that the Sp1 DNA element was conserved in other mammalian PTH promoters, including the bovine gene (11, 13) (data not shown). The next set of experiments was designed to evaluate whether Sp1/NF-Y transcriptional synergism observed with the hPTH promoter was also evident for the bPTH gene. The analogous bPTH promoter construct was transfected into Drosophila SL2 cells and coexpressed with Sp1, NF-Y, or the combination of the two transcription factors. As seen in Fig. 7A, inclusion of Sp1 in the transfection experiment resulted in modest enhancement of promoter activity, whereas NF-Y alone had no effect. However, expression of the combination of the two factors resulted in a dramatic increase in luciferase activity. These data were comparable to the results obtained with the mutant NF-Ydist hPTH promoter construct (Fig. 1), suggesting that a functional NF-Y element is present in the bovine promoter.
FIG. 7. Analysis of the bPTH promoter. A, The bPTHp/luc reporter construct was transfected into Drosophila SL2 cells together with the indicated expression vectors (5 ng Sp1; 5 ng/subunit NF-Y). B, Sequences surrounding the known NF-Yprox site (underlined) in the hPTH promoter in comparison with the corresponding region from the bPTH promoter (underlined). C, Mobility shift analysis using OK cell nuclear extracts and bPTH NF-Yprox oligonucleotide. Lane 1, Control binding reaction; lane 2, competition with excess unlabeled bPTH NF-Yprox oligonucleotide; lane 3, competition with Sp1 consensus element; lane 4, competition with hPTH NF-Yprox oligonucleotide; lane 5, competition with bPTH cAMP response element binding protein oligonucleotide; lane 6, competition with consensus NF-Y element; lane 7, addition of anti-NF-Y B-subunit antibody; lane 8, addition of anti-ER antibody. Arrowhead indicates position of the bound NF-Y complex. D, Transfection analysis of bPTH wild-type (bPTHwt) (white bars) and NF-Yprox mutant (Prox mut) (gray bars) promoters in Drosophila SL2 cells with the indicated expression vectors (5 ng Sp1; 1 ng/subunit NF-Y).
A sequence comparison between the human and bovine genes (Fig. 7B) suggested that an imperfect NF-Y binding site may also exist in the bovine promoter at a comparable position relative to the NF-Yprox site in the human gene. To evaluate this possibility, mobility shift experiments were pursued with a radiolabeled oligonucleotide from this region of the bPTH promoter, in combination with OK cell nuclear extracts previously used to demonstrate specific binding by the NF-Y complex to the NF-Yprox element in the hPTH promoter (12). As can be seen in Fig. 7C, a complex was readily evident under these conditions that could be competed with an excess of the corresponding unlabeled bPTH oligonucleotide but not by an excess of a consensus Sp1 DNA element (lanes 1–3). Furthermore, the corresponding region from the hPTH promoter and a consensus NF-Y DNA element could also compete for this complex (Fig. 7, lanes 4 and 6), whereas the cAMP response element binding protein DNA element from the bPTH promoter (20) failed to disrupt binding of the bound band (Fig. 7, lane 5). A rabbit anti-NF-Y B-subunit antibody was able to block formation of the complex, which was not recognized by a rabbit anti-ER antibody (Fig. 7, lanes 7 and 8). Thus, analogous to the situation with the hPTH promoter (12), specific binding by the NF-Y complex was evident with the corresponding region from the bovine promoter.
To confirm that this NF-Y binding site was responsible for the observed synergism, a mutant bPTH promoter construct was generated that selectively prevented binding by NF-Y to this site in mobility shift assays (data not shown). Both wild-type and mutant bPTH promoters were transfected into Drosophila SL2 cells and coexpressed with either Sp1, NF-Y, or a combination of the two factors to assess reporter gene activity. As can be seen in Fig. 7D (white bars), the wild-type bPTH promoter produced the expected strong synergism when both Sp1 and NF-Y were expressed in the cells, whereas there was only a modest increase in transcriptional activity in the presence of both factors when the mutant promoter was evaluated (gray bars). Thus, the identified NF-Y binding site in the bPTH promoter appears to be the principal transducer of the functional synergism observed between Sp1 and NF-Y in the SL2 cells.
Discussion
The current study extends our previous work examining factors that control transcription of the PTH promoter. Synergism was noted in a previous study of the NF-Y complex and its distal and proximal binding sites in the hPTH promoter (12). In the present findings, strong synergistic transactivation was also observed with the hPTH promoter when the combination of Sp proteins and NF-Y complex were coexpressed with the hPTH and bPTH promoter constructs. The titration data using the wild-type hPTH promoter (Fig. 4) indicated that at low relative concentrations of expression of each of the factors, the Sp1/NF-Y synergism far exceeded the synergistic NF-Y/NF-Y response. In combination with data from the bPTH promoter demonstrating similar Sp1/NF-Y synergism, the ability of NF-Y to bind to the proximal element and transcriptionally synergize with Sp proteins bound upstream suggests this may be a principle means of strongly enhancing PTH gene expression.
Previous work by Mantovani and colleagues (16) indicated a strict spacing requirement between two NF-Y DNA binding sites. Positioning of the Sp1 element relative to the NF-Yprox site suggests that this is also the case in PTH promoters. Physical interaction between Sp1 and the NF-Y complex has been reported (10, 21); therefore, it seems plausible that disruption of the spacing between their respective elements in the PTH promoter would have a negative impact on their protein-protein contacts and, hence, synergistic transactivation. Alternatively, Sp1 has been shown to interact directly with components of transcription factor IID (22, 23), and insertion or deletion of 5 bp caused a significant decrease in Sp1-mediated transcriptional activity from the PTH promoter, as well as synergistic transactivation by the combination of Sp1 and NF-Y (Fig. 5). Thus, we would speculate that proper alignment of the Sp1 element to position Sp1 in such a way as to facilitate interactions with components of the transcriptional machinery may be viewed as a critical feature for full activation of the PTH promoter, either by Sp1 alone or in conjunction with the NF-Y complex.
The aforementioned similarities between the hPTH and bPTH promoters in Sp1/NF-Y synergism suggests a common mechanism for enhancing PTH gene transcription; however, there are still notable differences in transcription factor binding sites between the two species. The NF-Ydist DNA element is unique to the human gene (13), as is the strong NF-Y/NF-Y synergistic enhancement of hPTH promoter activity (compare Figs. 1 and 6, and see Ref. 12). In addition, the NF-Yprox element in the human gene adjoins a repressor binding site for the VDR (12), but we have been unable to demonstrate directly or indirectly an analogous receptor binding site in the bPTH promoter (data not shown). Thus, expression of the human gene appears to contain additional layers of control not evident in its bovine counterpart and will require additional studies to uncover their overall significance and what, if any, selective advantage this affords.
Analogous to previous experiments (12), the liganded VDR heterodimer complex is able to suppress Sp1/NF-Y synergistic transactivation of the hPTH promoter (Fig. 6). Therefore, the repressive response exerted by vitamin D on hPTH promoter activity can now also be extended to take into account the capacity of NF-Y to interact with its proximal element and synergize with either Sp proteins or NF-Y binding at their distal positions. This suggests that control of this proximal position, either by NF-Y or the VDR heterodimer, may represent a key determinant of hPTH promoter activity in terms of either gene enhancement or repression. Although the VDR responds to circulating hormone concentrations, it remains to be seen whether modulation of NF-Y activity, through processes such as phosphorylation (24, 25), may also be a possible target of external stimuli that could increase or decrease its ability to interact with particularly the proximal element to alter PTH gene expression.
Although noted for their ubiquitous expression and involvement in basal transcriptional activity of numerous genes (1, 2, 26), several studies have determined that the expression of Sp proteins and the NF-Y complex are likely involved in tissue-specific gene expression as well (4, 5, 6, 27). Key questions remain, therefore, in delineating whether one or both of these proteins are involved in tissue-specific expression of PTH in the parathyroid gland or whether they simply represent factors relegated to basal transcriptional expression. Likewise, it will be of interest to determine whether either of these factors plays some role in various hypo- or hyperparathyroid disease states.
Acknowledgments
We thank H. Gravatte for excellent technical assistance. In addition, we acknowledge the generous contributions by Dr. T. Osborne (Irvine, CA) for providing the individual NF-Y A, B, and C subunit Drosophila pPac expression vectors and Dr. G. Suske (Marburg, Germany) for providing the pPac Sp1 and Sp3 expression vectors.
References
Kaczynski J, Cook T, Urrutia R 2003 Sp1- and Kruppel-like transcription factors. Genome Biol 4:206
Mantovani R 1999 The molecular biology of the CCAAT-binding factor NF-Y. Gene 239:15–27
Ge Y, Jensen TL, Matherly LH, Taub JW 2002 Synergistic regulation of human cystathionine-?-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. Biochim Biophys Acta 1579:73–80
Rodenburg RJ, Holthuizen PE, Sussenbach JS 1997 A functional Sp1 binding site is essential for the activity of the adult liver-specific human insulin-like growth factor II promoter. Mol Endocrinol 11:237–250
Valor LM, Campos-Caro A, Carrasco-Serrano C, Ortiz JA, Ballesta JJ, Criado M 2002 Transcription factors NF-Y and Sp1 are important determinants of the promoter activity of the bovine and human neuronal nicotinic receptor ?4 subunit genes. J Biol Chem 277:8866–8876
Zhang LP, Stroud J, Eddy CA, Walter CA, McCarrey JR 1999 Multiple elements influence transcriptional regulation from the human testis-specific PGK2 promoter in transgenic mice. Biol Reprod 60:1329–1337
Yamada K, Tanaka T, Miyamoto K, Noguchi T 2000 Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions. J Biol Chem 275:18129–18137
Iwano S, Saito T, Takahashi Y, Fujita K, Kamataki T 2001 Cooperative regulation of CYP3A5 gene transcription by NF-Y and Sp family members. Biochem Biophys Res Commun 286:55–60
Murakami Y, Ikeda U, Shimada K, Kawakami K 1997 Promoter of the Na,K-ATPase 3 subunit gene is composed of cis elements to which NF-Y and Sp1/Sp3 bind in rat cardiocytes. Biochim Biophys Acta 1352:311–324
Roder K, Wolf SS, Larkin KJ, Schweizer M 1999 Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1. Gene 234:61–69
Alimov AP, Langub MC, Malluche HH, Koszewski NJ 2003 Sp3/Sp1 in the parathyroid gland: identification of an Sp1 deoxyribonucleic acid element in the parathyroid hormone promoter. Endocrinology 144:3138–3147
Koszewski NJ, Alimov AP, Park-Sarge OK, Malluche HH 2004 Suppression of the human parathyroid hormone promoter by vitamin D involves displacement of NF-Y binding to the vitamin D response element. J Biol Chem 279:42431–42437
Alimov AP, Langub MC, Malluche HH, Park-Sarge OK, Koszewski NJ 2004 Contrasting mammalian parathyroid hormone (PTH) promoters: nuclear factor-Y binds to a deoxyribonucleic acid element unique to the human PTH promoter and acts as a transcriptional enhancer. Endocrinology 145:2713–2720
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 89:8097–8101
Salsi V, Caretti G, Wasner M, Reinhard W, Haugwitz U, Engeland K, Mantovani R 2003 Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function. J Biol Chem 278:6642–6650
Liberati C, di Silvio A, Ottolenghi S, Mantovani R 1999 NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices. J Mol Biol 285:1441–1455
Lok CN, Lang AJ, Mirski SE, Cole SP 2002 Characterization of the human topoisomerase II? (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites. Biochem J 368:741–751
Inoue J, Sato R, Maeda M 1998 Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. J Biochem (Tokyo) 123:1191–1198
Danilition SL, Frederickson RM, Taylor CY, Miyamoto NG 1991 Transcription factor binding and spacing constraints in the human ?-actin proximal promoter. Nucleic Acids Res 19:6913–6922
Deutsch PJ, Hoeffler JP, Jameson JL, Lin JC, Habener JF 1988 Structural determinants for transcriptional activation by cAMP- responsive DNA elements. J Biol Chem 263:18466–18472
Liang F, Schaufele F, Gardner DG 2001 Functional interaction of NF-Y and Sp1 is required for type a natriuretic peptide receptor gene transcription. J Biol Chem 276:1516–1522
Gill G, Pascal E, Tseng ZH, Tjian R 1994 A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci USA 91:192–196
Hoey T, Weinzierl RO, Gill G, Chen JL, Dynlacht BD, Tjian R 1993 Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell 72:247–260
Yun J, Chae HD, Choi TS, Kim EH, Bang YJ, Chung J, Choi KS, Mantovani R, Shin DY 2003 Cdk2-dependent phosphorylation of the NF-Y transcription factor and its involvement in the p53–p21 signaling pathway. J Biol Chem 278:36966–36972
Chae HD, Yun J, Bang YJ, Shin DY 2004 Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions. Oncogene 23:4084–4088
Li L, He S, Sun JM, Davie JR 2004 Gene regulation by Sp1 and Sp3. Biochem Cell Biol 82:460–471
Ge Y, Konrad MA, Matherly LH, Taub JW 2001 Transcriptional regulation of the human cystathionine ?-synthase-1b basal promoter: synergistic transactivation by transcription factors NF-Y and Sp1/Sp3. Biochem J 357:97–105(Alexander P. Alimov, Ok-K)