CDC6 Promoter Research Articles

UHRF1 promotes androgen receptor-regulated CDC6 transcription and anti-androgen receptor drug resistance in prostate cancer through KDM4C-Mediated chromatin modifications.

The UHRF1 and CDC6, oncogenes play critical roles in therapeutic resistance. In the present study, we found that UHRF1 mediates androgen receptor (AR)-regulated CDC6 transcription in prostate cancer cells. In prostate cancer tissues and cell lines, levels of UHRF1 and CDC6 were simultaneously upregulated, and this was associated with worse survival. UHRF1 silencing significantly promoted the cytotoxicity and anti-prostate cancer efficacy of bicalutamide in mouse xenografts by inhibiting CDC6 gene expression. UHRF1 promoted AR-regulated CDC6 transcription by binding to the CCAAT motif near the androgen response element (ARE) in the CDC6 promoter. We further found that UHRF1 promoted androgen-dependent chromatin occupancy of AR protein by recruiting the H3K9me2/3-specific demethyltransferase KDM4C and modifying the intense heterochromatin status. Altogether, we found for the first time that UHRF1 promotes AR-regulated CDC6 transcription through a novel chromatin modification mechanism and contributes to anti-AR drug resistance in prostate cancer. Targeting AR and UHRF1 simultaneously may be a novel and promising therapeutic modality for prostate cancer.

Transcriptional repression of CDC6 and SLD2 during meiosis is associated with production of short heterogeneous RNA isoforms.

Execution of the meiotic and mitotic cell division programs requires distinct gene expression patterns. Unlike mitotic cells, meiotic cells reduce ploidy by following one round of DNA replication with two rounds of chromosome segregation (meiosis I and meiosis II). However, the mechanisms by which cells prevent DNA replication between meiosis I and meiosis II are not fully understood. Here, we show that transcriptional repression of two essential DNA replication genes, CDC6 and SLD2, is associated with production of shorter meiosis-specific RNAs containing the 3' end of both genes. Despite the short CDC6 RNA coding for a short protein (Cdc6short), this protein is not essential for meiosis and it does not have either a positive or negative impact on DNA replication. Production of CDC6short mRNA does not require the upstream CDC6 promoter (PCDC6) and is not a processed form of the full-length RNA. Instead, CDC6short depends on transcription initiation from within the ORF upon repression of PCDC6. Finally, using CDC6 genes from related yeast, we show that repression of full-length CDC6 mRNA is evolutionarily conserved and that this repression is consistently associated with production of unique short CDC6 RNAs. Together, these data demonstrate that meiotic cells transcriptionally repress full-length CDC6 and SLD2, and that inactivation of PCDC6 results in heterogeneous transcription initiation from within the CDC6 ORF.

The Circadian Clock Sets the Time of DNA Replication Licensing to Regulate Growth in Arabidopsis

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.

Combinatorial Transcription Regulation of Oncogene CDC6 in Androgen-Sensitive Prostate Cancer Cells

Previous studies showed that CDC6 gene transcription is mainly regulated by E2F transcription factors. We recently reported [ 1 ] a novel regulatory mechanism of aberrant CDC6 gene transcription in androgen-responsive prostate cancer (PCa) cells where FOXM1 transcription factor positively regulates CDC6 gene transcription and DNA replication. In addition to direct binding to the CDC6 promoter, FOXM1 regulates CDC6 gene transcription by elevating AR gene expression. Furthermore, FOXM1 and AR collaborate to regulate CDC6 gene transcription in a cell cycle-dependent manner. Supporting the significance of this mechanism, siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, inhibited PCa cell proliferation, and the effect of siomycin A was additive to that of bicalutamide, an antiandrogen commonly used to treat PCa patients.

FOXM1 and androgen receptor co-regulate CDC6 gene transcription and DNA replication in prostate cancer cells

CDC6 is a key component of the DNA replication initiation machinery, and its transcription is regulated by E2F or androgen receptor (AR) alone or in combination in prostate cancer (PCa) cells. Through both overexpression and knockdown approaches, we found that in addition to its effects on the E2F pathway, the cell proliferation specific transcription factor FOXM1 stimulated CDC6 transcription in cooperation with AR. We have identified a forkhead box motif in the CDC6 proximal promoter that is occupied by FOXM1 and is sufficient to drive FOXM1-regulated transcription. Indirectly, FOXM1 elevated AR protein levels and AR dependent transcription. Furthermore, FOXM1 and AR proteins physically interact. Using synchronized cultures, we observed that CDC6 expression is elevated near S phase of the cell cycle, at a time coinciding with elevated FOXM1 and AR expression and CDC6 promoter occupancy by both AR and FOXM1 proteins. Androgen increased the binding of AR protein to CDC6 promoter, and AR and FOXM1 knockdown decreased AR binding. These results provided new evidence for the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR, two highly expressed transcription factors in PCa cells. Functionally, the cooperation of FOXM1 and AR accelerated DNA synthesis and cell proliferation by affecting CDC6 gene expression. Furthermore, siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, inhibited PCa cell proliferation and its effect was additive to that of bicalutamide, an antiandrogen commonly used to treat PCa patients.

CDC6 expression is regulated by lineage-specific transcription factor GATA1

GATA1 is a hematopoietic transcription factor essential for expression of most genes encoding erythro-megakaryocytic proteins, i.e., globins and platelet glycoproteins. A role for GATA1 as a cell proliferation regulator has been proposed, as some of its bona fide targets comprise global regulators, such as c-KIT or c-MYC, or cell cycle factors, i.e., CYCLIN D or p21CIP1. In this study, we describe that GATA1 directly regulates the expression of replication licensing factor CDC6. Using reporter transactivation, electrophoretic mobility shift and chromatin immunoprecipitation assays, we show that GATA1 stimulates CDC6 transcription by binding to a canonical binding site located within a 166bp enhancer region upstream CDC6 promoter. This evolutionary conserved GATA binding site conforms to recently described chromatin occupancy rules, i.e., preferred bases within core WGATAR (TGATAA), 5' and 3' flanking bases (GGTGATAAGG) and distance to the transcription initiation site. We also found adjacent conserved binding sites for ubiquitously expressed transcription factor CP2, needed for GATA activity on CDC6 enhancer. Our results add to the growing evidence for GATA1 acting as a direct transcriptional regulator of the cell cycle machinery, thus linking cell proliferation control and specific gene expression programs during lineage differentiation.

Interaction between HMGA1 and Retinoblastoma Protein Is Required for Adipocyte Differentiation

It is generally accepted that the regulation of adipogenesis prevents obesity. However, the mechanisms controlling adipogenesis have not been completely defined. We have previously demonstrated that HMGA1 proteins play a critical role in adipogenesis. In fact, suppression of HMGA1 protein synthesis by antisense technology dramatically increased growth rate and impaired adipocyte differentiation in 3T3-L1 cells. Furthermore, we showed that HMGA1 strongly potentiates the capacity of the CCAAT/enhancer-binding protein beta (C/EBPbeta) transcriptional factor to transactivate the leptin promoter, an adipocytic-specific promoter. In this study we demonstrate that HMGA1 physically interacts with retinoblastoma protein (RB), which is also required in adipocyte differentiation. Moreover, we show that RB, C/EBPbeta, and HMGA1 proteins all cooperate in controlling both Id1 and leptin gene transcriptions, which are down- and up-regulated during adipocyte differentiation, respectively. We also demonstrate that HMGA1/RB interaction regulates CDC25A and CDC6 promoter activities, which are induced by E2F-1 protein during early adipocyte differentiation, by displacing HDAC1 from the RB-E2F1 complex. Furthermore, by using Hmga1(-/-) embryonic stem cells, which failed to undergo adipocyte differentiation, we show the crucial role of HMGA1 proteins in adipocyte differentiation due to its pivotal involvement in the formation of the RB-C/EBPbeta complex. Altogether these data demonstrate a key role of the interaction between HMGA1 and RB in adipocyte differentiation.

A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression

The androgen receptor (AR) plays a pivotal role in the onset and progression of prostate cancer by promoting cellular proliferation. Recent studies suggest AR is a master regulator of G1-S progression and possibly a licensing factor for DNA replication yet the mechanisms remain poorly defined. Here we report that AR targets the human Cdc6 gene for transcriptional regulation. Cdc6 is an essential regulator of DNA replication in eukaryotic cells and its mRNA expression is inversely modulated by androgen or antiandrogen treatment in androgen-sensitive prostate cancer cells. AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription. We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression. We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter. Significantly, AR silencing in prostate cancer cells markedly decreases Cdc6 expression and androgen-dependent cellular proliferation. Collectively, our results suggest that Cdc6 is a key regulatory target for AR and provide new insights into the mechanisms of prostate cancer cell proliferation.

A novel functional polymorphism in the Cdc6 promoter is associated with the risk for hepatocellular carcinoma

Cdc6 is essential for DNA replication and its deregulation is involved in carcinogenesis. To date, the biological significance of the polymorphism in Cdc6 promoter is still unknown. In this study, we aimed to evaluate the influence of the Cdc6 −515A >G polymorphism (rs4134994) on the individual's susceptibility to cancer and on the function of Cdc6. The Cdc6 −515A >G polymorphism was genotyped in 387 hepatocellular carcinoma (HCC) and 389 age- and sex-matched healthy subjects. The association between the genotypes and the risk for HCC was then estimated by unconditional logistic regression analysis with adjustment for age, sex and HBV status. Compared with the AA homozygotes, the homozygous GG genotype (adjusted OR = 0.36, 95% confidence interval (CI) = 0.18–0.72, P = 0.004) or the combined AG/GG genotypes (adjusted OR = 0.56, 95% CI = 0.36–0.86, P = 0.008) were statistically significantly associated with the reduced risk for HCC. Moreover, the analysis using luciferase reporter system showed that the G-allelic Cdc6 promoter displayed a decreased transcriptional activity compared with the A-allelic one. These results indicate that the individuals with G allele may have reduced Cdc6 expression and are therefore in reduced risk for HCC. Further investigation using electrophoretic mobility shift assay (EMSA) revealed that the G allele had a stronger binding strength to nuclear protein(s) which might function as negative regulator(s) for Cdc6 transcription. Our findings suggest that the −515A >G polymorphism may affect the Cdc6 promoter binding affinity with nuclear protein(s) and in turn the Cdc6 expression, which consequently modulates the individual's susceptibility to HCC.

Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells

Androgen receptor plays a critical role in the development and maintenance of cancers in the prostate. Earlier, we have shown that Cdc6, a regulatory protein for initiation of DNA replication, is down regulated in androgen-insensitive prostate cancer cells. In this report, we studied the involvement of androgen, mediated through androgen receptor (AR) in regulation of Cdc6 expression. Our results demonstrated that androgen treatment stimulated Cdc6 expression in xenograft tumors and androgen-sensitive prostate cancer cells. We also showed that androgen treatment stimulated Cdc6 transcription through possible interaction of AR with the ARE sequence in the Cdc6 promoter and that the stimulatory effect of androgen required intact E2F binding sites in the promoter. Androgen treatment differentially altered nuclear availability of E2F1 and E2F3, and increased the amount of hypophosphorylated retinoblastoma protein (pRb) in the nucleus in a time dependent fashion. We further showed that AR interacted with E2F transcription factors in a ligand-independent manner and that ligand-bound AR was less efficient in interacting with E2F proteins. DNA–protein interaction assays indicated that androgen treatment altered binding of E2F1 to the Cdc6 promoter in prostate cancer cells. We conclude that AR regulates Cdc6 transcription through interaction with the Cdc6 promoter, and complex formation with E2F1 and E2F3 in a differential manner.

Development and characterization of a synthetic promoter for selective expression in proliferating endothelial cells

Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues. Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter. The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues. These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors.

Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) are essential for normal ocular development and are expressed in numerous ocular cell types including lens epithelial cells, retinal pigment epithelial cells, Müller cells and endothelial cells. Endothelial cell proliferation is a common feature of proliferative retinopathies and involves abnormal growth of blood vessels within and on the surface of the retina. In an effort to inhibit the formation of these aberrant blood vessels, we cloned an IGF-IR ribozyme into an expression vector that limits expression of the ribozyme to proliferating endothelial cells. An endothelin enhancer and Cdc6 promoter chimera drives expression of the IGF-IR ribozyme. This promoter limited retinal expression of the reporter gene to proliferating endothelial cells in two mouse models of proliferative retinopathy. In addition, expression of the IGF-IR ribozyme by this promoter inhibited aberrant retinal angiogenesis in both models while preserving normal vessels. These results demonstrate the feasibility of IGF-IR ribozyme expression in a selective manner for safer treatment of abnormal angiogenesis associated with retinopathy.

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization.

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements.

Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function.

To explore mechanisms for specificity of function within the family of E2F transcription factors, we have identified proteins that interact with individual E2F proteins. A two-hybrid screen identified RYBP (Ring1- and YY1-binding protein) as a protein that interacts specifically with the E2F2 and E2F3 family members, dependent on the marked box domain in these proteins. The Cdc6 promoter contains adjacent E2F- and YY1-binding sites, and both are required for promoter activity. In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, can stimulate Cdc6 promoter activity synergistically, dependent on the marked box domain of E2F3. Using chromatin immunoprecipitation assays, we show that both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at G(1)/S of the cell cycle. In contrast, we detect no interaction of E2F1 with the Cdc6 promoter. We suggest that the ability of RYBP to mediate an interaction between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and provides a basis for specificity of E2F function.

Down-regulation of Cdc6, a Cell Cycle Regulatory Gene, in Prostate Cancer

CDC6 plays a critical role in regulation of the onset of DNA replication in eukaryotic cells. We have found that Cdc6 expression is down-regulated in prostate cancer as detected by semiquantitative reverse transcriptase-PCR of prostate cell lines and laser-captured microdissected prostate tissues. This result was substantiated by immunohistochemical analysis of paraffin-embedded tissue sections and immunoblot analysis of benign (BPH-1) and adenocarcinomatous prostatic cells. Furthermore, a 100-fold reduction in the transcription efficiency of the Cdc6 promoter-luciferase construct was noted in the metastatic PC3 cells compared with that in BPH-1 cells. Concentration of the E2F and Oct1 transcription factors that have putative binding sites in the Cdc6 promoter was substantially low in PC3 cells compared with BPH cells. Mutagenesis of the two E2F binding sites on the Cdc6 promoter resulted in increased promoter activity in PC3 cells owing to elimination of the negative regulation by pRb.E2F complex but not to the level of that obtained in BPH cells. We conclude that an altered interaction of transcription factors may be responsible for the down-regulation of Cdc6 transcription in PC3 cells. Our study suggests a potential use of the lack of CDC6 expression as an index of prostate cancer development.

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

The Saccharomyces cerevisiae CDC6 gene is transcribed at late mitosis and encodes a ATP/GTPase controlling S phase initiation.

We have compared the periodic fluctuation of mRNAs encoded by CDC6, a cell cycle gene controlling initiation of DNA replication, and CLN1, a G1 cyclin gene expressed at late G1. The maxima of CDC6 mRNA precede those of CLN1 mRNA by about 15 min in cells synchronized by release from pheromone arrest or from a cdc15 thermal arrest. CDC6 mRNA accumulates in cdc15-arrested telophase cells, decays around cell separation, and reappears during telophase and nuclear division of the next cycle. CDC6 transcription at late mitosis is not affected by the pheromone signaling pathway. The CDC6 mRNA fluctuation pattern is imposed to a CLN1-derived reporter gene if fused to the CDC6 promoter. The CDC6 gene was expressed in Escherichia coli as a glutathione-S-transferase fusion protein and purified by affinity chromatography. The Cdc6 protein binds rATP and rGTP upon UV cross-linking and catalyzes the DNA-independent hydrolysis of purine nucleoside triphosphates, but does not appear to interact directly with DNA. The Cdc6 protein may control the ATP-dependent initiation of replication by conferring ATPase activity to an origin-recognizing complex.

Mutation Analysis ofSaccharomyces cerevisiae CDC6Promoter: Defining Its UAS Domain and Cell Cycle Regulating Element

Using beta-galactosidase as the reporter gene, we carried out mutagenesis experiments to investigate the 5' promoter region of the CDC6 gene. Our results showed that the DNA element, between -262 and -170, is important for the upstream activating sequence (UAS) activities. On the basis of the DNA sequence, there is a Mlu I (-204) and a Mlu I-like (-216) element located within the middle of the UAS region. Insertion and deletion mutagenesis analysis of the Mlu I sequence has indicated that the internal CGCG sequence of the Mlu I site (ACGCGT) is important for gene expression. Furthermore, when DNA elements containing the Mlu I sites were subcloned into the tester plasmid, periodic expression of a reporter gene throughout the cell cycle was observed, as evidenced by the beta-galactosidase activities and lacZ mRNA. Because the possible transcriptional initiation sites of the CDC6 transcript have been previously defined (Zhou and Jong, 1990, J. Biol. Chem. 264, 9022-9029), we propose a model regarding the construct of the CDC6 promoter region. This 5' promoter construct contains a UAS region and a Mlu I element (MCB box) typical of a family of cell cycle-regulated genes involved in DNA metabolism. Previous genetic studies have not completely defined the CDC6 execution point in the functional yeast cell cycle map. Our results favor the possibility that the CDC6 gene is required, and directly involved, in the initiation of DNA replication.

CDC6 mRNA fluctuates periodically in the yeast cell cycle.

Using cultures synchronized by two independent procedures, alpha-factor arrest and centrifugal elutriation, we have investigated the expression of the Saccharomyces cerevisiae CDC6 gene through the cell cycle. Our results show that the CDC6 gene is periodically expressed in the yeast cell cycle. The level of CDC6 transcripts increases in late G1, reaching a peak (approximately 10-20-fold over the initial level) at about the G1/S phase boundary. The peak of CDC6 mRNA was observed to overlap or slightly precede that of the CDC8 message, and to obviously precede that of the histone H2A message by some 25 min. Unlike histone H2A mRNA, the CDC6 mRNA as well as CDC8 mRNA were not affected by hydroxyurea treatment. These results suggest that regulation of H2A mRNA is different from that of CDC6 or CDC8. We have studied the 5'-flanking regions of CDC6 and other cell cycle-regulated genes. DNA sequence analysis of the CDC6 promoter revealed two sequences, 5'-C/GACGCGNC/G-3' and 5'-PuGNAGAAA-3' (where Pu is a purine, and N is any nucleotide), which are repeated three times each. Similar sequence elements have also been found among several cell cycle-regulated genes, including the CDC8 gene, but are not found upstream of histone genes. The possible significance of these elements is discussed.