Proliferating Cell Nuclear Antigen Gene Promoter Research Articles

Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

Down-Regulation of Proliferating Cell Nuclear Antigen Gene Expression Occurs during Cell Cycle Arrest Induced by Human Fecal Water in Colonic HT-29 Cells

Cancer is a disease in which the cell cycle is altered, and the elucidation of the mechanisms by which constituents of human fecal water influence the cell cycle can lead to noninvasive measurement of colon cancer risk. The purpose of the present study was to investigate the effect of human fecal water on HT-29 cell-cycle progression with sodium selenite as a reference for comparison. Both human fecal water (2.5-5.0%) and selenite (3-4 micro mol/L) significantly inhibited cell growth. Cell-cycle analysis revealed that human fecal water decreased the proportion of S + G2 phase cells and increased that of G1 phase cells. In contrast, selenite decreased G1 phase cells and increased proportions of S and G2 phase cells. Both 5% human fecal water and 4 micro mol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene p21(waf1). Interestingly, the mRNA levels of cyclin A and proliferating cell nuclear antigen (PCNA) were dramatically decreased by 69 and 62%, respectively, in HT-29 cells treated with fecal water but not selenite. In contrast, the mRNA level of DNA damage-inducible transcript 1, gadd45, was significantly increased by 2.28-fold in HT-29 cells treated with selenite but not fecal water. Furthermore, a PCNA gene promoter was cloned into a luciferase reporter construct and its activity was significantly reduced in a dose-dependent manner in cells treated with fecal water but not selenite. Collectively, these results suggest that human fecal water and selenite can differentially induce growth arrest genes, and that PCNA gene expression is uniquely and highly sensitive to human fecal water.

Serum responsiveness of the rat PCNA promoter involves the proximal ATF and AP-1 sites

We have previously shown that the rat PCNA (proliferating cell nuclear antigen) gene promoter is responsive to serum stimulation. In this study, the sequence of the promoter responsive to serum stimulation has been localized in the region between nucleotides −70 and +125 relative to the transcription initiation site. This region contains an ATF site (nucleotides −51 to −44) and an AP-1 site (nucleotides −64 to −58). Mutation at either the ATF or the AP-1 site reduced the serum responsiveness of the promoter. In gel mobility shift assays, nuclear extracts from serum stimulated cells, compared to those from quiescent cells, exhibit an increasing binding activity toward a promoter related oligonucleotide (−70 to −42) which includes the ATF site and the AP-1 site. Formation of the DNA:protein complexes requires the simultaneous involvement of ATF and AP-1 sites as either element can abrogate the complexes in the competition experiment. Both the distance and sequence are essential to complex formation. Moreover, ATF-1 but not ATF-2 (or CREB) has been identified as a major component of the complexes in the antibody supershift or interference experiment. The results of this study suggest that ATF-1 in association with other factors is involved in regulating the serum stimulation of the rat PCNA promoter activity via the proximal ATF and AP-1 sites.

Involvement of the DNA replication-related element (DRE) and DRE-binding factor (DREF) in transcriptional regulation of the Bombyx mori PCNA gene.

The promoter region of the Bombyx mori gene encoding the proliferating cell nuclear antigen (PCNA), and its activating factor(s) were analyzed to ascertain similarities with Drosophila regulatory elements. Full promoter activity was established to reside within the region from -466 to +347 base pairs with respect to the transcription initiation site. Within this region, we found a sequence similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is a promoter-activating sequence common to promoters of the Drosophila genes for DNA replication-related factors, including PCNA. A mutation in the DRE-like sequence of the B. mori PCNA gene promoter caused reduction of the promoter activity and also binding to the recombinant Drosophila DRE-binding factor (DREF). Furthermore, a factor(s) binding to the DRE sequence was detected in extracts of B. mori BmN4 cells. Monoclonal antibodies against Drosophila DREF inhibited the binding activity of the factor, as shown by gel mobility shift assays, and allowed specific detection of a 100 kDa protein on immuno Western blot analysis. These results suggest that the B. mori DREF homolog binds to DRE to regulate transcription of the PCNA gene.

Identification of CFDD (Common Regulatory Factor for DNA Replication and DREF Genes) and Role of Its Binding Site in Regulation of the Proliferating Cell Nuclear Antigen Gene Promoter

The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, we detected a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes.

Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development.

The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. We generated PCNA-LacZ fusion genes in which the 5'-flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulated the PCNA gene minimal promoter (-86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of beta-galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development.

Serum Responsiveness of the Rat PCNA Promoter

Expression of proliferating cell nuclear antigen (PCNA) gene is growth-regulated. The growth dependence of the rat PCNA gene promoter activity was investigated. Cultured cells were transfected with the promoter containing plasmid and recovered for 48 h in serum-free medium to become quiescent. Cells were then cultured in serum-containing medium and harvested at certain intervals after serum-stimulation, and the promoter-directed chloramphenicol acetyltransferase activities in cell extracts were measured. The promoter used in this study contained sequences between -693 and +125 in relation to the transcription initiation site. The promoter activity was found to be serumresponsive. However, the serum-responsiveness of the promoter became less obvious when the amount of the promoter increased; meanwhile, the promoter became active in the control unstimulated (or quiescent) cells. It was suspected that the dosage effect was due to the titration of the negative regulatory factor in quiescent cells. The titration experiment with a reporterless construct as competitor for regulatory factors showed that the excess of promoter molecules reduced the promoter activity in serum-stimulated cells, while causing a transiently increase of promoter activity in quiescent cells. Based on these results, it is postulated that the serum-responsiveness of the rat PCNA promoter is controlled by both negative and positive regulatory factors. Consistent with this proposition, promoter binding proteins of 105 and 114 kDa were identified only in serum-stimulated and quiescent cells, respectively, in addition to several other promoter binding proteins (ranging from 76 to 110 kDa) which were seen in both serum-stimulated and quiescent cells.

Distribution of PCNA during postblastoderm cell division cycles in the Drosophila melanogaster embryo: effect of a string- mutation.

We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string- mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to +137 with respect to the transcription initiation site) with string- mutant files, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.

Differential effect of p53 on the promoters of mouse DNA polymerase beta gene and proliferating-cell-nuclear-antigen gene.

A plasmid carrying the 5' flanking region of the mouse proliferating-cell-nuclear-antigen (PCNA) gene or DNA polymerase beta gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild-type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase beta gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase beta gene promoter, we identified the unique 10-bp palindromic sequence (-24 to -15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21-bp upstream from the transcription-initiation site.

Nucleotide sequence and promoter-specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene.

Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.

Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5′‐flanking region (– 1584 to + 47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5‐ to 9‐fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of El A resides in a common region(s) of 13S and 12S El A products. The major target region of El A was mapped within the 68 base‐pair region (‐21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2P, although the upstream region (–83 to – 21) including ATF(CREB)‐binding consensus had an additional effect in the transactivation.

Repression of the Drosophila Proliferating-Cell Nuclear Antigen Gene Promoter by zerknüllt Protein

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknüllt protein.

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants

The transgenic tobacco plants have been generated that express the E. coli beta-glucuronidase (GUS) gene under control of the promoter from the rice proliferating cell nuclear antigen (PCNA, DNA polymerase auxiliary protein) gene. GUS expression detected in situ by staining with the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), was restricted to meristems in the organs of the transgenic tobacco plants. This expression responded to the phytohormones which promote callus formation. Furthermore, in situ thymidine uptake showed that the GUS expression pattern corresponded well to the active sites of DNA synthesis. Deletion analysis of the 5' upstream sequence confined the GUS expression pattern to a fragment extending 263 bp upstream of the transcription start site of the rice PCNA gene. Thus, we have identified this fragment as a main regulatory element of the rice PCNA gene promoter.