MBE Advance Access originally published online on January 6, 2007
Molecular Biology and Evolution 2007 24(3):814-826; doi:10.1093/molbev/msl210
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Research Articles |
Chimpanzee, Orangutan, Mouse, and Human Cell Cycle Promoters Exempt CCAAT Boxes and CHR Elements from Interspecies Differences
* Department of Internal Medicine II, Max Bürger Research Center, University of Leipzig, Leipzig, Germany
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
E-mail: engeland{at}medizin.uni-leipzig.de.
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Abstract |
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Mechanisms regulating the cell division cycle are well conserved among all eukaryotes. Consistently many proteins regulating the cell cycle are functionally interchangeable between many organisms. Cell division control is regulated on different levels of which the transcriptional level appears to be particularly important for controlling synthesis of many cell cycle proteins. We had earlier described transcription factor–binding sites essential for regulating genes important for the transition from the G2 phase to mitosis. A tandem repressor site named cell cycle–dependent element (CDE) and cell cycle genes homology region (CHR) are responsible for the correct expression during the cell cycle. Another feature of these G2/M-specific promoters is the activation through 2 or 3 CCAAT boxes binding the transcription factor nuclear factor-Y (NF-Y). These major activating sites have to be spaced 32 or 33 bp apart to be fully functional. We were interested in looking at the evolutionary changes in regulatory elements and overall promoter structure of 3 well-characterized cell cycle genes. Here, we compare the DNA sequences and functional features of the cdc25C, cyclin B1, and cyclin B2 promoters from humans, mouse, chimpanzee, and orangutan. We find numerous differences in the nucleotide sequence between mouse and primate promoters. However, CHR and CCAAT boxes stand out in that they are perfectly conserved in all promoters tested. The CDE site contains nucleotide exchanges between mouse and primate promoters. Comparing sequences and functions of chimpanzee, orangutan, and human promoters, we observe a complete conservation in nucleotide sequence of the regulatory elements. Functional assays of the cyclin B1, cyclin B2, and cdc25C promoters yield moderate variations in activity and thereby a good conservation of function. Although we find nucleotide differences in cell cycle promoters between orangutan and humans of about 5%, there are never changes in any of the CCAAT boxes or CDE/CHR sites in the cyclin B1, cyclin B2, and cdc25C promoters. Furthermore, we describe the influence of the tumor suppressor p53 and the transcriptional activator NF-Y on regulation of the newly cloned primate promoters.
Key Words: cell cycle • evolution of gene expression • primates • sequence alignment • functional promoter assays • transcription
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Introduction |
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Chimpanzees are man's most closely related relatives, but humans and chimpanzees vary in many morphological, behavioral, and cognitive aspects. One of these differences is the lower susceptibility to cancer of nonhuman primates as already described more than 30 years ago (McClure 1973
). Other reports, including more recent publications, support these observations (Seibold and Wolf 1973; Beniashvili 1989; Waters et al. 1998; Varki 2000). Various factors may cause these differences in cancer incidence, among them environmental influences, different life expectancies, and genetic differences.
Cancer is mostly a consequence of defects in cell cycle regulation. All steps during this complex process are strictly controlled to ensure proper DNA replication and correct division of 1 cell into 2 daughter cells. Central molecules, which regulate the transition from one cell cycle phase to the next, are cyclins and their corresponding cyclin-dependent kinases (cdk), which they regulate by complex formation. Cyclin B associates with the cdk1 (cdc2) protein to form the maturation-promoting factor (MPF), which is essential for the transition from the G2 phase to mitosis. An additional level of regulating MPF activity is exercised when cdk1 is dephosphorylated on threonine-14 and tyrosine-15 by the cell cycle phosphatase cdc25C (Strausfeld et al. 1991; Millar and Russell 1992). One general feature of cyclins is that their protein levels oscillate during the cell cycle (Evans et al. 1983). The B-type cyclins B1 and B2 appear in S phase and accumulate in G2 before disappearing at transition from metaphase to anaphase in mitosis (Brandeis and Hunt 1996). These oscillations are maintained by regulating synthesis and degradation. For these oscillators, transcriptional regulation determines the rate of protein synthesis (Lange-zu Dohna et al. 2000; Wasner, Tschöp, et al. 2003). Like cyclins B1 and B2, the mRNA level of cdc25C also changes periodically during the cell cycle (Sadhu et al. 1990). The fluctuations in synthesis of cyclins B1 and B2, and cdc25C are dependent on transcriptional control due to a tandem repressor element in the promoters that we named cell cycle–dependent element (CDE) and cell cycle genes homology region (CHR) (Zwicker, Lucibello, et al. 1995; Lange-zu Dohna et al. 2000; Wasner, Tschöp, et al. 2003). Repression dependent on both halves of the CDE/CHR element leads to transcriptional downregulation in G0 and G1. Relief of this repression in S und G2 phases allows cell cycle–dependent expression (Zwicker, Gross, et al. 1995; Lange-zu Dohna et al. 2000; Wasner, Tschöp, et al. 2003). Another common feature of these cell cycle promoters is their transcriptional activation through several CCAAT boxes. Trimeric nuclear factor-Y (NF-Y) binds to these elements and acts as the main activator (Zwicker, Gross, et al. 1995; Bolognese et al. 1999; Wasner, Haugwitz, et al. 2003; Wasner, Tschöp, et al. 2003).
Modulation of cdc25C, cyclin B1 and cyclin B2 transcription also plays an important role in causing cell cycle arrest and preparing for apoptosis. Transcription of the 3 genes is repressed by the tumor suppressor p53 (Krause et al. 2000, 2001; Manni et al. 2001). For p53, both functions as a transcriptional activator and as a repressor have been described. p53 can cause G1 and G2 growth arrest or apoptosis by repressing the transcription of genes whose protein products stimulate progression through the cell cycle and by activating inducers of cell cycle arrest and apoptosis (Ko and Prives 1996). Because cdc25C, cyclin B1, and cyclin B2 are proteins that play fundamental roles at the transition from the G2 phase to mitosis, downregulation by p53 leads to an arrest in G2 phase.
Differences between human and nonhuman primates in the amino acid sequence of these central cell cycle molecules could contribute to an altered susceptibility to cancer. However, humans and chimpanzees differ just in approximately 1.2% of the genomic DNA sequence. King and Wilson (1975) suggested that phenotypic differences between humans and chimpanzees are caused by changes in gene expression rather than by structural changes in the gene product. Recent studies come to similar conclusions (Enard et al. 2002; Caceres et al. 2003; Gu J and Gu X 2003; Karaman et al. 2003; Khaitovich et al. 2004; Uddin et al. 2004). They show that the expression level of about 10% of all genes expressed in brain differs between humans and chimpanzees. Although gene expression is a complex process, studies of allelic variation on the DNA sequence level within promoter regions of several genes show a profound influence of this variation on the gene expression level (Bray et al. 2003; Hoogendoorn et al. 2003; Trinklein et al. 2003).
From a human–chimpanzee promoter comparison, we know that as few as 3 changes in the promoter sequence can lead to big differences—in this case fivefold—in expression activity from the core promoter (Huby et al. 2001). Such variations in promoter activities could also lead to dramatic changes in other regulatory systems. Another example in which evolutionary changes in regulatory sites influence gene expression is the regulation of the PIG3 promoter through a microsatellite sequence by the tumor suppressor p53. It has been shown that a functional microsatellite length developed only with apes and humans (Contente et al. 2002).
We were interested in looking at the evolutionary changes in regulatory elements of 3 well-characterized cell cycle genes. In this report, we compare the DNA sequences of the cdc25C, cyclin B1, and cyclin B2 promoters from humans, mouse, chimpanzee, and orangutan. We describe the influence of p53 and NF-Y on activity of the newly cloned primate promoters as well as the extent by which these genes are regulated at 2 indicative points during the cell cycle.
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Materials and Methods |
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Cloning of the Chimpanzee and Orangutan Cell Cycle Promoters
The cyclin B1 promoters from chimpanzee and orangutan were amplified from genomic DNA with the primers B1-forward and B1-reverse already used for the human promoter (Wasner, Tschöp, et al. 2003
) to create DNA fragments flanked by KpnI and NcoI restriction sites. Similarly, the cyclin B2 promoters were also obtained by polymerase chain reaction (PCR) using the primers B2 Hu-5' and B2 Hu-3' (Wasner, Haugwitz, et al. 2003). This produced DNA fragments with restriction sites for BglII and NcoI. For the amplification of the cdc25C promoters from chimpanzee and orangutan, the primers 25c-5' and 25c-3' with restriction sites for BglII and NcoI were employed (Krause et al. 2001). The restriction sites were used to clone the fragments into the pGL3-basic firefly luciferase reporter plasmid (Promega, Mannheim, Germany) to create the constructs chimpB1-Luci, ouB1-Luci, chimpB2-Luci, ouB2-Luci, chimp-cdc25C-Luci, and ou-cdc25C-Luci.
The resulting promoter constructs used for the transfection experiments all extend at their 3' ends to the first methionine-coding 5'-ATG-3' of the gene. In the pGL3-basic constructs, these nucleotides are at the same time employed as the first codon for the luciferase gene. The 5' ends of the individual constructs, when counted from the first nucleotide upstream of the 5'-ATG-3' as the –1 nucleotide position, extend up to the following positions: chimpB1-Luci, nucleotide –1165; ouB1-Luci, nucleotide –1150; chimpB2-Luci, nucleotide –866; ouB2-Luci, nucleotide –857; chimp-cdc25C-Luci, nucleotide –1434; ou-cdc25C-Luci, nucleotide –1438; human B1h-wt, nucleotide –1165 (Wasner, Tschöp, et al. 2003); human hB2-Luci, nucleotide –857 (Wasner, Haugwitz, et al. 2003); and human cdc25C-wt-luci, nucleotide –1434 (Krause et al. 2001). Nucleotide sequences of the cloned fragments of the promoters used in the reporter constructs, including the sequences for the regulatory regions, are displayed in figures 1 and 3.
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Three independent clones of each promoter were sequenced (GATC Biotech AG, Konstanz, Germany). Each of the sequences was identical for one promoter and used for alignments with the OMIGA V 2.0 program (Oxford Molecular Ltd, Oxford, UK). The sequences were submitted to GenBank and accession numbers were assigned as follows: Pongo pygmaeus cyclin B1, AY788992 [GenBank] ; P. pygmaeus cyclin B2, AY788993 [GenBank] ; P. pygmaeus cdc25C, AY788994 [GenBank] ; Pan troglodytes cdc25C, AY788995 [GenBank] ; Pa. troglodytes cyclin B1, AY788996 [GenBank] ; and Pa. troglodytes cyclin B2, AY788997 [GenBank] . Reference numbers for other promoter sequences are Mus musculus cyclin B2, AY788998 [GenBank] ; Homo sapiens cdc25C, AY046902 [GenBank] ; M. musculus cdc25C, AF450244 [GenBank] ; M. musculus cyclin B1 AC112701 [GenBank] ; H. sapiens cyclin B1, NM_031966 [GenBank] ; and H. sapiens cyclin B2, AF545815 [GenBank] .
Targeted Mutation of Cell Cycle Promoters
To show the functional relevance of CDE, CHR, and CAAT-box elements, specific mutations were introduced to the promoter luciferase constructs. CDE and CHR point mutations were created with the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA) employing the following primers: 25C_h+c mCDE, 5'-GGA TAG GTT ACT GGG CTG TCA GAA GGT TTG AAT GG-3'; 25C_ou mCDE, 5'-GGA TAG GTT ACT GGG CGG TCA GAA GGT TTG AAT GG-3'; B1_ h+c+ou mCDE, 5'-GGC CAA TAA GGA GGG AGC ATT ACG GGG TTT A-3'; B2_ h+c+ou mCDE, 5'-CGC CCA ATG GGG CGC AAG CAT TAC GG TAT TTG-3'; 25C_ h+c mCHR, 5'-CTG GGC TGG CGG AAG GTG CAT ATG GTC AAC GCC-3'; 25C_ ou mCHR, 5'-CTG GGC GGG CGG AAG GTG CAT ATG GTC AAA CGC C-3'; B1_ h+c mCHR, 5'-GGA GCA GTG CGG GGT GCA TAT CTG AGG CTA GGC-3'; B1_ ou mCHR, 5'-GGA GCA GTG CGG GGT GCA TAC CTG AGG CTA GGC-3'; and B2_ h+c+ou mCHR, 5'-GGC GCA AGC GAC GCG GTA TGC ATA TCC TGG AAC-3'. Mutated nucleotides are indicated in bold. Reverse primers complementary to the forward primers were used. CCAAT-box deletions were introduced by overlapping PCR with these primers: 25C_ h+c+ou dCAAT, 5'-GAG CCC AAC GGT TAC TGG GC-3'; 25C_ h+c+ou dCAAT rev, 5'-CTC GGG TTG CCA ATG ACC CG-3'; B1_ h+c+ou dCAAT forw, 5'-CCG GCA GCC GAA GGA GGG AG-3'; B1_ h+c+ou dCAAT rev, 5'-GGC CGT CGG CTT CCT CCC TC-3'; B2_ h+c+ou dCAAT forw, 5'-GAA AAT TCA GGG GGC GCA AG-3'; and B2_ h+c+ou dCAAT rev, 5'-CTT TTA AGT CCC CCG CGT TC-3'.
Cell Culture and Transfection
NIH3T3 cells and SaOS-2 cells (DSMZ, Braunschweig, Germany) were cultured and transfected as described earlier (Dietz et al. 2002; Haugwitz et al. 2002). For cell cycle analysis, NIH3T3 cells in 12 well plates were transfected with 0.6 µg of the promoter luciferase constructs and 12 ng of the pRLnull control plasmid (Promega, Madison, WI). After transfection the cells were cultured for 48 h in Dulbecco's modified Eagle's medium without fetal calf serum (FCS, Biochrom, Berlin, Germany). After this time period, half of the cells were harvested for the 0-h time point. The other part was stimulated with 20% FCS to reenter the cell cycle and harvested 24 h after restimulation. Fluorescence activated cell sorting (FACS) analyses were performed as previously described (Haugwitz et al. 2002, 2004).
To analyze the influence of p53 on the promoters, SaOS-2 cells in 24-well plates were cotransfected with 150 ng of the luciferase reporter constructs, 25 ng of pRLnull plasmid, and 25 ng of a p53-expression plasmid or 25 ng of pUC19 as a control (Krause et al. 2001). After the transfection cells were cultured for 24 h in McCoy`s medium containing 15% FCS.
SaOS-2 cells were employed to examine the NF-Y–dependent activation of cell cycle promoters. The cells were cotransfected with the primate luciferase reporter constructs and a plasmid expressing a dominant-negative form of NF-YA (YA13m29, kindly provided by Roberto Mantovani). For these experiments, 150 ng of the luciferase reporter constructs, 25 ng of pRLnull, and 50 ng of YA13m29 or 25 ng pUC19 were used.
Luciferase Assays
Luciferase assays were performed with the Dual Luciferase Assay System (Promega) as suggested by the manufacturer. Three assays per data point were performed. Averages of the 3 measurements with standard deviations are given in the figures. The firefly luciferase activity was normalized to the activity of Renilla luciferase synthesized from the cotransfected pRLnull plasmid. Normalization was done by dividing firefly luciferase activity by Renilla activity for each measurement. In order to compensate for changes in Renilla activity by effectors like p53, we multiplied the 3 quotients of firefly activity divided by Renilla activity with the average of the Renilla activity from the set of measurements for 1 data point.
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Results and Discussion |
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The protein levels of cyclin A, cyclin B, and the phosphatase cdc25C fluctuate during the cell cycle. Changes in their synthesis are mainly transcriptionally regulated. This regulation is mediated by CCAAT boxes, CDE, and CHR elements in the promoters of the genes (Zwicker, Lucibello, et al. 1995
; Haugwitz et al. 2002; Wasner, Haugwitz, et al. 2003). Although binding of trimeric NF-Y to the CCAAT boxes activates the promoters, interactions of regulating proteins with the CDE/CHR tandem elements lead to transcriptional repression. We were interested how the regulatory elements of the cell cycle promoters differ among primates and what the changes are in comparison to mouse. To this end we cloned and sequenced cyclin B1, cyclin B2, and cdc25C promoters from chimpanzee and orangutan and compared their nucleotide sequences and basic functional properties in reporter assays with those of the promoters from mouse and humans.
Primate and Mouse cdc25C Promoters Are Conserved in the Nucleotide Sequence of CHR Sites and the Distance between 2 CCAAT Boxes
Comparisons of cdc25C promoters from primates and mouse showed that the mouse promoter lacks a CDE, whereas the genes from chimpanzee, orangutan, and humans display conserved CDE sites (fig. 1). Functional assays with the mouse cdc25C promoter had shown earlier that a CHR element without a CDE regulates the cell cycle–dependent transcription (Haugwitz et al. 2002). In addition, we find that 1 of the 3 NF-Y–binding CCAAT boxes in the primate promoters is missing in the mouse gene (fig. 1). Generally, we observe a strong conservation of the nucleotide sequences in promoters of chimpanzee and orangutan as the closest human relatives (fig. 1). In contrast to the mouse, all elements that are known to be relevant for the transcriptional regulation like CDE/CHR and CCAAT boxes are identical in their sequence. Furthermore, also distances between CDE and CHR elements as well as nucleotide distances between conserved CCAAT boxes are identical. Distances between regulating elements in cell cycle promoters have been shown to be essential for their ability to function (Zwicker, Lucibello, et al. 1995). Particularly the distance of 32 bp between CCAAT boxes has been found not only in the cdc25C promoter but also in a number of other G2/M-specific cell cycle promoters (Zwicker, Gross, et al. 1995; Salsi et al. 2003; Wasner, Haugwitz, et al. 2003). In the other parts of the promoters, single nucleotide exchanges appear. There is an identity of 99.0% in the nucleotide sequence of the human and chimpanzee cdc25C promoters. This numerical value is very close to the average identity of 99.4% in all noncoding genomic DNA regions between humans and chimpanzee (Wildman et al. 2003). The human and orangutan cdc25C promoter nucleotide sequences show 95.9% identity. Interestingly, we found one nucleotide exchange in this comparison directly next to the CDE (fig. 1). In order to examine if this specific or any of the other nucleotide differences may have an influence on the activity of the promoters, we tested the promoter activity with luciferase reporter assays.
Cell Cycle–Dependent Expression of cdc25C
The phosphatase cdc25C mRNA is expressed periodically during the cell cycle. In the G0 and G1 phases of the cell cycle, no reporter activity from the cdc25C promoter is observed (Zwicker, Lucibello, et al. 1995). In S phase, expression starts and reaches a maximum in G2 phase and mitosis (Haugwitz et al. 2002). Therefore, relative expression in G0 and G2/M is indicative for the cell cycle regulation of cdc25C promoters. We compared the activity of the human, chimpanzee, and orangutan promoters driving luciferase reporters in serum-starved and restimulated NIH3T3 cells 24 h after serum readdition. Furthermore, the newly identified primate promoters were mutated in the putative CDE and CHR elements identified by the sequence alignment. Mutants were functionally tested in reporter assays relative to human wild-type and mutant cdc25C promoters (fig. 2). FACS analyses indicate that the cells were mostly in G0 when serum starved and to a large part in G2/M when restimulated for 24 h (Haugwitz et al. 2004 and data not shown). Mutations in both the CDE and CHR elements generally lead to a diminished repression in G0 phase. Interestingly, mutants of CDE and CHR elements in cdc25C promoters of chimpanzee and orangutan vary in the extent of their deregulation in comparison to the human mutant promoters. Particularly the CHR in the orangutan promoter appears to be more relevant to cell cycle–dependent transcription than CHRs in the other promoters because its mutation leads nearly to complete loss of regulation (fig. 2). In summary, these observations show that transcription from the orangutan cdc25C promoter at these 2 cell cycle points is similar to that of the chimpanzee and human promoters, despite a number of nucleotide differences and in particular a T to G change upstream of the CDE in the orangutan gene (fig. 1). This is consistent with the fact that the mouse cdc25C promoter does not rely on a CDE upstream from the regulating CHR but nevertheless displays the same cell cycle regulation as the primate genes (Haugwitz et al. 2002). Interestingly, the cdc25C promoter from mouse also has a G in position –669, which together with 2 more changes in the CDE appears to be tolerable for cell cycle–dependent regulation of this promoter. It is tempting to conclude that a functional CDE has developed only from mouse to primates (figs. 1 and 2) (Haugwitz et al. 2002).
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Comparison of cyclin B1 and cyclin B2 Promoter Sequences
In earlier studies, the promoter sequences of human and mouse cyclins B1 and B2 have been compared. The overall nucleotide identity is quite low. However, CHR sites and functionally active CCAAT boxes stand out in that they are identical in both their sequences as well as their distances in all of the promoters (Wasner, Haugwitz, et al. 2003
; Wasner, Tschöp, et al. 2003) (fig. 3). Although the CHR is completely identical in the human and mouse genes, the CDEs show some nucleotide differences. However, the CHRs are different between cyclins B1 and B2 (fig. 3). Nevertheless, both CHR types are functional (Wasner, Haugwitz, et al. 2003; Wasner, Tschöp, et al. 2003). The comparison of the cyclin B1 and B2 promoters of human, chimpanzee, and orangutan shows a significant conservation in nucleotide sequences. Differences are restricted to regions outside the CDE/CHR sites and CCAAT boxes (fig. 3). The promoters of chimpanzee and humans show an identity of 98.2% (cyclin B1) and 97.5% (cyclin B2), respectively, whereas human and orangutan promoters are 94.4% (cyclin B1) and 96.3% (cyclin B2) identical, respectively. Recently, comparison of chimpanzee and human chromosome 5 yielded a conservation rate of 0.0067 (99.33%) changes per nucleotide in coding sequences and 0.0091 (99.09%) in noncoding regions when looking at chromosomal segments conserved in rodents (Schmutz et al. 2004). Therefore, nucleotide changes in cyclin B1 and B2 genes described here display a higher rate of changes (fig. 3). Taken together, these variations indicate that regulatory sites in the cyclin B promoters are under evolutionary constraint and are exempted from nucleotide changes.
Chimpanzee and Orangutan cyclin B Promoters Control Low Expression in G0 and High Expression in G2/M cells
Cyclins B1 and B2 are expressed periodically during the cell cycle. In human cells, the known regulatory mechanism is similar to that of the cdc25C phosphatase gene. Regulation comprises activation by the transcription factor NF-Y through CCAAT boxes and cell cycle–dependent repression through the CDE/CHR sites (Wasner, Haugwitz, et al. 2003; Wasner, Tschöp, et al. 2003). In order to compare the regulation during the cell cycle of the cyclin B1 and B2 promoters of chimpanzee and orangutan with that of humans, we tested 2 representative cell cycle states, G0 and G2/M. The luciferase reporter plasmids chimpB1-Luci, chimpB2-Luci, ouB1-Luci, and ouB2-Luci were transiently transfected into NIH3T3 cells. In order to test the function of putative CDE and CHR sites in the new chimpanzee and orangutan promoters, the elements were mutated and the respective promoters analyzed in the same assays as the wild-type promoters. Cells were serum starved to lead them into G0 phase of the cell cycle (0-h time point). Restimulation with serum for 24 h yields a cell population that is enriched in G2/M cells (24-h time point). The luciferase activity in G0 cells and G2 cells was measured and compared with the regulation by the human cyclin B1 and B2 promoters (fig. 4). Testing cell populations enriched in G0 or G2/M, we find that cyclin B1 and cyclin B2 promoters from chimpanzee and orangutan origin are transcriptionally regulated during the cell cycle the same way as the human promoters are. Looking at the chimpanzee and orangutan promoters mutated in the CDE and CHR sites and considering error margins in the experiments, we find that their regulation in the cell cycle is altered upon mutation of CDE/CHR elements in the same way as that of the respective human promoters (fig. 4A and B). From these results, we conclude in general that the CDE and CHR elements in the promoters of humans, chimpanzee, and orangutan are not only conserved in their sequence but have also a comparable function directing transcriptional repression in G0 cells and regulating relief of this repression in G2/M cells (Zwicker, Lucibello, et al. 1995).
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NF-Y Is the Main Activator
Although proteins interacting with the CDE and CHR of the cyclin B1, cyclin B2, and cdc25C promoters lead to a repression of the transcriptional activity, the NF-Y is the main activator of these promoters (Mantovani 1998
). Binding of heterotrimeric NF-Y at multiple CCAAT boxes (Mantovani 1999) leads to a strong increase in mRNA synthesis. For some years an elegant tool had been available to study the total contribution by NF-Y to the transcriptional activation of a particular promoter. Overexpression of dominant-negative form of the subunit A of NF-Y leads to inactivation of the trimeric NF-Y complex (Bolognese et al. 1999). Here, we employed this tool to test for the contribution of NF-Y in the activation of the chimpanzee and the orangutan promoters. Cotransfecting the promoter-luciferase constructs with the dominant-negative NF-YA mutant YA13m29 (NF-Y-mut) yielded a downregulation of each of the cdc25C, cyclin B1, and cyclin B2 promoters to a similar extent for humans, chimpanzee, and orangutan. In order to assess the contribution to promoter activation by the conserved CCAAT boxes, we deleted the CCAAT boxes and assayed the remaining contribution of NF-Y activation on the cloned promoter fragments by cotransfecting a plasmid expressing the dominant-negative NF-YA mutant (NF-Y-mut). NF-Y-mut inhibits the activation by endogenous wild-type NF-Y and thereby allows to estimate the remaining activation through NF-Y in the CCAAT-box deletion mutants (fig. 5). For the cdc25C promoter, NF-Y contributes about two-thirds to the total human, chimpanzee, and orangutan promoter activation. Most of this contribution is lost in all 3 promoters when the conserved CCAAT boxes are deleted (fig. 5A). In the cyclin B1 promoter this is different. NF-Y–dependent activation contributes from one-third to one-quarter to the total promoter activity. When the conserved CCAAT boxes in all 3 primate promoters are deleted, NF-Y-mut is still able to reduce activity of the mutant promoters to about the same level as the wild-type promoters. This indicates that NF-Y–dependent activation does not act through the conserved CCAAT boxes in those 3 promoters. Nevertheless, promoter function in the cyclin B1 promoters of the 3 organisms is conserved because they are regulated essentially in an identical way (fig. 5B). Overall, regulation of cyclin B2 is similar to that of cdc25C. About one-third in case of the chimpanzee and orangutan promoters to one-half when looking at the cyclin B2 promoter activation is dependent on NF-Y as seen by the lack of a further reduction by NF-Y-mut when assaying the CCAAT-box mutants of the promoters. Essentially all of this activation potential is lost in the cyclin B2 promoters from humans, chimpanzee, and orangutan when the CCAAT boxes are deleted (fig. 5C). This leads us to conclude that the conservation of the 3 CCAAT boxes in the cdc25C and cyclin B2 promoters (figs. 1 and 3) is responsible for the similar extent of NF-Y activation of the promoters in different primate species. Also the conservation of the 32-bp or 33-bp distance between CCAAT boxes appears to be important. It has been noticed earlier that the exact spacing is required to allow for binding of the histone acetyltransferase p300 in a complex with NF-Y to stimulate promoter activation (Salsi et al. 2003; Wasner, Tschöp, et al. 2003).
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CCAAT) leads to a reduced repression of some promoters by a dominant-negative NF-Y subunit A (NF-Y-mut). SaOS-2 cells were cotransfected with promoter luciferase constructs and 50 ng of a plasmid coding for a dominant-negative mutant of the NF-Y subunit A (NF-Y-mut). The expression level from the wild-type (wt) or the CCAAT box–deleted promoters (Influence of p53 on the Transcription of cdc25C, cyclin B1, and cyclin B2
We have previously shown that the tumor suppressor p53 can downregulate transcription of human cyclin B1, cyclin B2, and cdc25C promoters (Krause et al. 2000
, 2001; Manni et al. 2001). This regulation is not mediated by p53 consensus binding sites, but depends on larger parts of the promoters. Therefore nucleotide exchanges, insertions, and deletions—as they appear in the promoters of the chimpanzee and orangutan compared with that of humans—could influence the ability of p53 to downregulate the expression of cyclin B1, cyclin B2, or cdc25C.
What remains unresolved is the exact mechanism by which p53 downregulates promoters. For promoters activated by p53, the details are rather straightforward. p53 binds as a transcription factor to consensus binding sites and activates the target gene. For this mechanism, 2 kinds of activating DNA elements have been identified (Contente et al. 2002). Even though p53-dependent repression is independent of consensus sites in the target promoter, the DNA-binding domain of p53 is required also for repression (Krause et al. 2000, 2001). For the Tcf-4 target gene, chromatin immunoprecipitation experiments showed that not even a peripheral binding of p53 to the Tcf-4 promoter can be observed (Rother et al. 2004). Taken together, these observations may imply that p53 transcriptionally activates a gene, which then in turn interferes with transcription of p53 target genes.
A potential mediator for p53-dependent repression is NF-Y binding to CCAAT boxes. It appears as if p53 can compromise the activation by NF-Y in some promoters and thereby lead to downregulation of transcription as shown for the cdk1 (cdc2) and cdc25C promoters (Yun et al. 1999; Krause et al. 2001; Manni et al. 2001). Although we found that not all activation through the conserved CCAAT boxes is dependent on the transcription factor NF-Y (fig. 5), we were interested if the CCAAT boxes can serve as a mediator of p53-dependent repression. To this end, the conserved CCAAT boxes were deleted and tested to which extent p53-dependent repression is altered in the promoters from the 3 primate species (fig. 6). Wild-type and CCAAT-box deletion mutants of luciferase reporter constructs together with a plasmid expressing wild-type p53 were cotransfected into p53-negative SaOS-2 cells. The analyses showed that cdc25C, cyclin B1, and cyclin B2 promoters of different species origin are repressed by p53 (fig. 6).
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Generally, whatever activity changes from wild type to CCAAT box–deleted promoters occur in any of the 3 human promoters are found to a similar extend also in the respective chimpanzee or orangutan promoters. In all promoters tested, only a limited portion of the p53-dependent repression is lost when CCAAT boxes had been deleted (fig. 6). Interestingly, we had observed that in the case of cyclin B1 regulation most activation through NF-Y to be independent of the conserved CCAAT boxes (fig. 5B), but we find that nevertheless a small part of the p53-dependent regulation is lost upon mutation of the CCAAT boxes (fig. 6B). This observation may be explained by other factors than NF-Y being able to bind and act through these elements like the Y box–binding protein 1 YB-1 (Jurchott et al. 2003
). We conclude from the experiments that the partial mediation of p53-dependent repression through CCAAT boxes is conserved among humans, chimpanzee, and orangutan cyclin B1, cyclin B2, and cdc25C promoters. The remaining larger portion of the regulation has to be independent of the CCAAT boxes.
Interestingly, we find only small variations between the repression factors of wild-type human, chimpanzee, and orangutan promoters. Therefore, it appears unlikely that the observed changes in the nucleotide sequences between the promoters (figs. 1 and 3) have a major influence on p53-dependent downregulation. Nevertheless, the small differences observed may be due to the variations in the nucleotide sequences and may in particular be due to their impact on the basal transcriptional machinery. Importantly, our results suggest that p53-dependent repression of cyclin B1, cyclin B2, and cdc25C promoters is conserved from mouse to humans, showing a general conservation of this regulatory pathway. This is different from an observation indicating that PIG3 upregulation by p53 is an evolutionary young development (Contente et al. 2002).
In summary, we find numerous differences in the nucleotide sequence of cell cycle promoters between mouse and primate promoters. However, comparing sequences and function of chimpanzee, orangutan, and human promoters, we observe a complete conservation in nucleotide sequence of the regulatory elements. Functional assays of the cyclin B1, cyclin B2, and cdc25C promoters yield moderate variations in activity and thereby a good conservation of function. Although we find nucleotide differences in cell cycle promoters between orangutan and human of about 5%, there are never changes in any of the CCAAT boxes or CDE/CHR sites in the cyclin B1, cyclin B2, and cdc25C promoters.
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionAcknowledgementsReferences |
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We thank Svante Pääbo for support and comments on the manuscript. G.A.M. is the recipient for a graduate fellowship awarded by the Freistaat Sachsen. F.H. acknowledges funding by the Max-Planck-Gesellschaft. This work was supported by grants from the Bundesministerium für Bildung und Forschung through the Interdisciplinary Center for Clinical Research at the University of Leipzig and the Deutsche Forschungsgemeinschaft (to K.E.).
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Douglas Crawford, Associate Editor
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