The promoter regions of the Myb-regulated Adora2B and Mcm4 genes co-localize with origins of DNA replication
© Gundelach et al; licensee BioMed Central Ltd. 2007
Received: 27 February 2007
Accepted: 06 September 2007
Published: 06 September 2007
The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. We have recently demonstrated that the chicken Gas41 gene, whose promoter co-localizes with an origin of DNA replication, is a bona fide Myb target gene. Because of this finding we have asked whether other Myb-regulated genes are also associated with DNA replication origins.
We show that the promoter region of the chicken adenosine receptor 2B gene (Adora2B), a known Myb-target gene, acts as a DNA replication origin. Furthermore, we have examined known replication origins for the presence of Myb binding sites. We found that the intergenic region between the genes for the minichromosome maintenance 4 protein (Mcm4) and the catalytic subunit of DNA-dependent protein kinase (Prkdc), whose human counterpart has been identified as a replication origin, contains a number of Myb binding sites. Our data show that this region also acts as an origin of replication in chicken cells. Interestingly, we found that the chicken Mcm4 gene is also Myb-regulated.
Our work identifies the chicken Mcm4 gene as a novel Myb target gene and presents evidence for the co-localization of two novel origins of DNA replication with Myb-regulated genes. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins.
The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor which is responsible for the ability of AMV to transform cells of the myelomonocytic lineage [1, 2]. v-myb is derived from the chicken c-myb gene, which plays a crucial role in the development of the hematopoietic system. c-myb is expressed in immature cells of all hematopoietic lineages and is silenced during the terminal differentiation of these cells. It is thought that c-myb is part of a genetic switch that allows hematopoietic progenitor cells to choose between alternative fates, such as proliferation, differentiation or apoptosis [2, 3].
The proteins encoded by v-myb and c-myb (v-Myb and c-Myb) bind to the sequence motif PyAAC(G/T)G  and control the expression of a number of genes, such as mim-1 , the lysozyme gene , bcl-2 [7, 8], tom-1 , c-kit , GBX2 , Pdcd4 , the genes for neutrophil elastase  and the A2B adenosine receptor  among others. Recent microarray analyses have suggested that Myb actually affects the expression of a much larger set of genes, either by direct binding or by indirect mechanisms .
Most of the work aimed at understanding how Myb activates the expression of its target genes has focused on the promoter regions of these genes and has provided a relatively simple picture of how Myb affects gene expression. In many cases, Myb binding sites have been identified in the promoter regions of these genes, suggesting that Myb acts primarily on the promoters of its targets. Recent in-depth analyses of some Myb target genes, however, have suggested a more complex picture in which the activation of certain target genes is mediated by cooperation of additional Myb-responsive enhancer elements with the target gene promoters. For example, the expression of the chicken mim-1 gene is controlled by interactions of Myb both with the promoter as well as with a powerful cell-specific enhancer located upstream of the gene . Similarly, in case of the chicken C/EBPβ gene Myb targets the promoter as well as an enhancer located downstream of the gene .
Recently, another regulatory DNA element has been mapped close to a Myb-regulated gene. We have shown that chicken Gas41, whose promoter region has been known for some time to act as an origin of DNA replication [18, 19], is a direct Myb target gene . Other work has shown that the Myb homolog of Drosophila melanogaster is involved in the control of the replication origin responsible for chorion gene amplification . These observations raised the question of whether DNA replication origins are also associated with other Myb target genes. Here, we demonstrate that the promoter region of the Myb-regulated chicken adenosine receptor 2B gene acts as an origin of DNA replication. Furthermore, we identify the chicken Mcm4 gene as a novel Myb target gene and demonstrate that the Mcm4 promoter is associated with a replication origin. Besides identifying two new DNA replication origins in chicken cells and a novel Myb-regulated gene our work suggests that a significant fraction of Myb-regulated genes might be associated with DNA replication origins.
The chicken Mcm4 gene is a Myb target gene
An origin of DNA replication is located upstream of the chicken Mcm4 gene
Identification of an origin of DNA-replication in the promoter region of the chicken adenosine receptor 2B gene
The identification of the chicken Gas41 gene, whose promoter region functions as a DNA replication origin, as a bona fide Myb target gene  has raised the question whether the co-localization of a Myb-regulated promoter with a replication origin is just a rare coincidence or a more common phenomenon? To address this question we used two different strategies: i.) We examined the promoter regions of known Myb-regulated genes for replication origin activity using the nascent strand abundance assay. ii.) We investigated the activity of v-Myb at the Mcm4-Prkdc intergenic region, which harbors several potential Myb binding sites and is associated with a replication origin in human cells .
Analysis of the adenosine receptor 2B gene, a known Myb target gene [14, 30], showed that the sequences around the 5' end of the gene were enriched in preparations of nascent DNA. This observation strongly suggested that the promoter of the A2B receptor co-localizes with a DNA replication origin. A further case of such co-localization was revealed by the analysis of the Mcm4-Prkdc intergenic region. Our data showed that v-Myb binds to this region both in vitro and in vivo and stimulates transcription of the chicken Mcm4 gene. Furthermore, we demonstrated that the Mcm4-Prkdc intergenic region is a replication origin also in chicken cells. Thus, chicken Mcm4 is a novel v-Myb target gene whose promoter region is also associated with a DNA replication origin. As these examples show, Myb-regulated genes and replication origins co-localize in some cases. However, it is also evident from the limited number of genes that have been analyzed so far that not every Myb target gene promoter is associated with a replication origin. For example, the promoter region of the mim-1 gene does not possess replication origin activity.
In the light of the co-localization of replication origins with several promoters containing clusters of Myb binding sites we wondered how frequently such clusters occur in the chicken genome. We screened approximately three million basepairs of randomly selected chicken genomic sequences for the presence of clusters of Myb binding motifs (AACG or AACTG) that are similar to those found at the MCM4, Adora2b and Gas41 replication origins. While on the average one such motif is present per 200 base pairs we found that clusters of at least six of such sites within stretches of 250 base pairs occur only once per approximately 25 kb. Although the number of such binding motif clusters is higher than the estimated number of replication origins in the chicken genome (one replication origin per approximately 100 kb, as extrapolated from mammalian cells [31, 32]), it appears unlikely that the concurrence of clusters of Myb binding motifs and DNA replication origins reported here is purely accidental. It will therefore be interesting to examine such clusters of Myb binding motifs for their potential role as replication origins.
The functional consequences, if any, of such co-localization are unclear at present. We have not observed that v-Myb or c-Myb influence the activity of the replication origins identified here. v-Myb or c-Myb, therefore, appear not to be involved in the control of origin activity. Recent work on the Myb homolog (Dm-Myb) of Drosophila melanogaster has provided evidence that Dm-Myb performs a non-transcriptional role in the control of the replication origin responsible for chorion gene amplification . Furthermore, it has been shown that some of the phenotypic consequences of mutation of Dm-Myb can be compensated by B-Myb, but not by c-Myb or A-Myb . It is therefore conceivable that B-Myb plays a role at the replication origins identified here. Another interesting possibility is that rather than being regulated by Myb the presence of a replication origin might affect the activation of the associated gene by Myb. For example, compacted chromatin structures will be opened during replication thereby providing transcription factors access to the DNA. It is therefore possible that the association with a replication origin imposes an additional level of control onto the adjacent gene, which would be expected to be cell cycle dependent. It is presently not known whether the Mcm4 and Adora2B genes are expressed in a cell cycle dependent manner. However, in this respect it is interesting to note that the phenotypic changes of myeloid cells transformed by a temperature-sensitive version of v-Myb that accompany activation of v-Myb by down-shifting the cells from the non-permissive to the permissive temperature were not observed when DNA replication was inhibited . This observation is consistent with the idea that the activation of at least some Myb-regulated genes depends on DNA replication. Clearly, further work is necessary until we can discern which of these possibilities, if any, is right.
Independent of a possible role of DNA replication origins in Myb-inducible gene expression our work is also of interest because we have identified two novel replication origins in chicken cells. So far, only very few replication origins are known in chicken cells, such as the origins located in the alpha and beta globin gene clusters [35, 36] and between the lysozyme and Gas41 genes [18, 19]. It is also interesting to note that the origin in the Mcm4-Prkdc intergenic region has been conserved between between chickens and humans.
Another interesting aspect of our work is the identification of the chicken Mcm4 gene as a Myb-regulated gene. MCM4, together with other MCM proteins, forms the hexameric MCM2-7 complex which functions as helicase during the S-phase of the cell cycle. The MCM2-7 complex is loaded onto the DNA prior to the initiation of DNA replication and constitutes an essential part of the pre-replication complex [for reviews see: [37–39]]. In addition to acting as a helicase in concert with the other MCM proteins forming the MCM2-7 complex there is evidence that MCM4 also performs a specific regulatory role. MCM4 is extensively phosphorylated when cells are exposed to inhibitors of DNA synthesis or to UV irradiation, leading to a block of DNA fork progression [40, 41], suggesting that MCM4 plays a role in the DNA replication block checkpoint system. In the normal course of the cell cycle MCM4 is phosphorylated by Cdk2, leading to the inhibition of the helicase function of the MCM complex [42, 43]. Blockage of this phosphorylation leads to reinitiation of replication and genomic instability. These observations suggest that MCM4 is a key component of the MCM complex whose manipulation can influence the control of DNA replication and genomic stability. Interestingly, elevated Mcm4 mRNA levels have been found in certain tumors, suggesting that increased Mcm4 expression can contribute to the malignant properties of the tumor cells [44, 45]. Recently, a hypomorph allele of mouse Mcm4 was identified and shown to cause chromosomal instability . Thus, Mcm4 is an interesting novel Myb target gene that provides a possible link between Myb, DNA-replication and checkpoint control.
We have identified the chicken Mcm4 gene as a novel Myb-regulated gene and shown that the Mcm4 promoter region is associated with an origin of DNA replication. Similarly, our work shows that the promoter of the Myb-regulated adenosine receptor 2B gene co-localizes with a replication origin. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins.
The BM2 and HD11 are lines of AMV-transformed chicken myeloblasts and MC29-transformed macrophages that were originally obtained from C. Moscovici and T. Graf, respectively. BM2 cells were grown in RPMI 1640 medium supplemented with 5 % fetal calf serum, 5 % chicken serum and 10 % tryptone phosphate broth (Gibco). HD11 cells were grown in basal Iscoves' medium supplemented with 8% fetal calf serum and 2% chicken serum. 10.4 is a derivative of the HD11 cell line expressing a v-Myb/ER fusion protein . 10.4 cells were grown in the same medium as HD11 cells. To activate the v-Myb/ER fusion protein the growth medium was supplemented with 2 μM β-estradiol for 24 hours.
Preparation of polyadenylated RNA and Northern blotting were performed as described . Mcm4 and Prkdc mRNAs were detected using cDNA clones of chicken Mcm4 (riken1_3305r1) and Prkdc (riken1_9d19r2), kindly provided by H. Arakawa and J.M. Buerstedde. As an internal control a specific probe for the ribosomal S17 gene was used.
Electrophoretic mobility shift assay
The following pairs of single-stranded oligonucleotides were annealed and used for electrophoretic mobility shift assays:
C/EBP_antisense: 5'- GGGATGTGTGGTTGTGAAAGAACCGAGCCAG-3'
After annealing, the oligonucleotides were radiolabeled by filling-in the ends using Klenow polymerase and α32P-dCTP. Preparation of bacterial full-length v-Myb protein and binding experiments were performed as described .
Approximately 108 BM2 cells were incubated in 1/10 cross-linking solution containing 0.1 M NaCl; 1 mM EDTA; 0.5 mM EGTA; 50 mM Tris-HCl, pH 8.0 and 11% formaldehyde for 10 min and quenched for 5 min in 125 mM glycine. After washing in ice-cold phosphate-buffered saline, cells were treated for 20 min each with washing solution A (0.25% Triton X100; 10 mM Tris-HCl, pH8.0, 10 mM EDTA; 0.5 mM EGTA) and B (200 mM NaCl; 10 mM Tris-HCl, pH8.0; 1 mM EDTA; 0.5 mM EGTA). Nuclei were sonicated on ice (4 times for 20 sec in 2 min intervals) in egg lysis buffer (ELB) (120 mM NaCl; 50 mM Tris-HCl, pH7.5; 20 mM NaF; 1 mM EDTA; 6 mM EGTA; 15 mM sodiumpyrophosphate; 1 mM phenylmethylsulfonyl fluoride and 0.1% Nonidet P-40). DNA-protein complexes were incubated with two different Myb-specific antibodies raised against the DNA-binding domain of v-Myb , normal rabbit serum or anti-Flag antibody (Sigma). After precipitation with protein-A-sepharose the samples were washed several times in ELB buffer. Following elution with 0.5% SDS the DNA was recovered by reverse-crosslinking for 6 h at 37°C and 65°C, respectively, in a buffer containing 0.5% SDS; 10 mM DTT and 100 μg proteinase K. The immunoprecipitated DNA was then purified by phenol-chloroform extraction and ethanol precipitation. PCR-amplification was performed by quantitative real-time PCR using primers specific for the chicken MCM4 promoter region (-0.55 kb) or primers derived from the -13 kb region of the gene.
Nascent strand abundance assay
Analysis of replication origin activity was performed according to Giacca et al. . Total genomic DNA was isolated from approximately 108 exponentially growing HD11 or BM2 cells using the Nucleobond CB kit (Macherey & Nagel) following the manufacturer's instructions. DNA was resuspended in TNE buffer (10 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA) and heat-denatured for 7 min at 95°C followed by rapid cooling on ice. Denatured DNA was layered on a linear neutral sucrose gradient (5–30% in TNE) and size fractionated by centrifugation in a Beckman SW28 rotor at 26.000 rpm for 20 h at 20°C. Nascent strand DNA fractions with an average DNA size of 1–1.5 kb were pooled, precipitated with ethanol and and treated with T4-polynucleotide kinase and λ-exonuclease as described previously [49, 50]. The purified nascent DNA strands were analyzed by quantitative real-time PCR using the following primer pairs:
Real-time PCR quantification
Q-PCR was carried out on a GeneAmp 5700 SDS (Applied Biosystems) with the qPCR Core kit for SYBR Green I (Eurogentec) following the instructions of the manufacturers. Primers were chosen with PrimerExpress 2.0 Software (Applied Biosystems). As DNA standard we used sonicated total genomic chicken DNA (average size 1 to 5 kb) serially diluted to 50.000, 10.000, 2.000, 400 and 80 genomic equivalents per reaction (1 genomic equivalent = 1.25 × 10-12 g). To produce a calibration curve, the CT value of each sample was plotted against the logarithm of its concentration. The number of genomic equivalents of each target sequence in the nascent DNA strand samples was then determined by extrapolation from the calibration curve. Several conditions were met by each assayed sample: The slope of the standard curve was around 3 to 3.5 (indicating a PCR efficiency close to 100 %). The sample was diluted such that its genomic equivalents fell within the central part of the calibration curve. The melting curve profile showed a single PCR product. The specificity of the PCR products was additionally confirmed for each primer pair by agarose gel electrophoresis.
We thank H. Arakawa and J.-M. Buerstedde for providing plasmids. This work was supported by a grant from the DFG (KL461/10-1).
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