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Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114
Received 27 February 2006/ Returned for modification 28 February 2006/ Accepted 3 March 2006
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ABSTRACT |
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INTRODUCTION |
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Two extensively characterized ORIs illustrate the complexity and elusiveness of the ORI mechanism in mammals. Analysis of the ORI in the Chinese hamster dihydrofolate reductase gene (DHFR) has been aided by the fact that the Chinese hamster ovary cell line CHOC 400 contains a 1,000-fold amplification of this region (54) and therefore enables sensitive ORI detection. Several reports find that initiation can occur at discrete loci, as determined by leading strand direction analyses (12) and nascent strand abundance analyses (41). However, other studies which used two-dimensional gel electrophoresis analysis suggest that initiation sites are chosen from a large number of potential sites within a 55-kb initiation zone (26, 72). ORI characterization within the ß-globin locus has also varied according to technique and species. In the murine locus, replication forks initiate from multiple locations within a broad zone of about 50 kb (4), but in humans, the locus instead uses a discrete ORI that is present between the two adult globin genes (40). These striking contrasts demonstrate the need for additional models in studying the nature and mechanism of DNA replication initiation in order to unify information gained using different experimental techniques.
The collective data argue that the ß-globin and DHFR ORIs can be influenced by multiple DNA elements at long range. Deletion of the ß-globin locus control region, a region that controls the stage-specific expression of genes within the cluster, results in a switch in ORI usage (3). Deletion of the DHFR promoter, located at least 30 kb from the ORI region, reduces initiation from the DHFR ORI located 3' of the gene, while introduction of a heterologous promoter restores initiation from the same ORI (63). These data clearly show a link between gene expression and ORI activity, thereby raising the question of whether ORIs may play a role in epigenetic regulation.
Indeed, recent studies reveal an interesting relationship between DNA replication and chromatin structure (53). Origin recognition complex (ORC) proteins and PCNA, an integral part of the replication fork complex, both affect gene expression and chromatin structure in the yeast mating type locus (73, 74). Moreover, histone H4 acetylation enhances the frequency of initiation in Drosophila melanogaster follicle cells (1). CpG islands, which are associated with gene promoters, are often enriched for ORI-containing sequences. Preparations of short nascent strand DNA that are enriched for ORIs are highly enriched for CpG islands (24). Further links between DNA replication and epigenetic phenomena are found in monoallelic gene regulation. For example, analyses of several imprinted murine genes show replication asynchrony between the maternally and paternally inherited alleles, with the paternal allele always replicating prior to the maternal allele even prior to the onset of monoallelic expression (39, 64). Genes in the odorant receptor clusters also show asynchronous replication (14, 65). In the immunoglobulin locus, the early-replicating 
allele almost always predetermines which allele will rearrange in B cells (56). However, it remains to be answered whether replication timing directly affects allelic choice and, if so, by what mechanism.
Here, we use X-chromosome inactivation (XCI) to interrogate potential relationships between DNA replication and epigenetic regulation in mammals. XCI equalizes X-chromosome gene dosage between the sexes in mammals by transcriptionally silencing one of two X's in the female (51). One of the earliest features of XCI is the asynchronous replication of the active (Xa) and inactive (Xi) X chromosomes (38), with the Xa chromosome replicating earlier in S phase than the Xi (68). DNA fluorescence in situ hybridization shows that replication asynchrony also occurs at the X-inactivation center (Xic) itself, although there is disagreement about whether the Xist allele on the Xa or that on the Xi replicates first (9, 33, 34, 70). One study observes that asynchrony is apparent even before the onset of XCI, but whether there is a preemptive role for DNA replication timing in determining allelic choice is not certain (33).
To investigate the link between DNA replication timing and epigenetic regulation at the Xic, we now seek to identify active ORIs within the Xic, a regulatory region of at least 80 kb (48). At the Xic, three elements associated with noncoding transcripts have been identified so far. Xist initiates silencing and produces a noncoding transcript that "coats" the entire X chromosome (10, 11, 16, 60). Xist is regulated by its antisense partner, Tsix (46), which prevents up-regulation of Xist RNA in cis and designates the future Xa. Two putative ORIs have previously been associated with CpG islands at the 5' promoter regions of Xist and Tsix (32). Tsix works in concert with the upstream Xite locus (58), which contains an enhancer and also promotes the persistence of Tsix on the future Xa (67). Jpx/Enox (15, 36) and the testes-specific gene Tsx (21) also reside at the Xic, but their functions have not been defined. Here, to determine the relationship between ORIs and known genetic elements of the Xic, we probe for ORI activity at 1.5-kb intervals across the entire 86-kb region and ask whether mutations of known Xic regulators affect origin usage.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiated mouse embryonic fibroblasts (obtained from day 13.5 [d13.5] embryos) in Dulbecco modified Eagle medium plus 15% fetal bovine serum (heat inactivated) and 500 U/ml leukemia inhibitory factor. Embryoid bodies were differentiated by use of suspension culture without leukemia inhibitory factor for 4 days and maintained thereafter under adherent conditions for 8 to 10 days. Primary fibroblasts were obtained from d13.5 embryos and maintained in Dulbecco modified Eagle medium plus 10% fetal bovine serum. The male deletion embryonic stem cell lines studied, Xite
L (58) and TsixCpG (47), were previously described. Isolation of nascent strands. For nascent strand isolation, we followed the protocol of Kumar et al. (5, 25, 28, 35, 42, 43). Total genomic DNA was prepared from 1 x 108 exponentially growing cells. The use of asynchronously growing cells in the log phase of growth minimized the effects of cell cycle and replication timing in this assay. Cells were trypsinized and washed with cold phosphate-buffered saline and cold RBS (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2) before being resuspended in RBS at approximately 2.5 x 107 cells/ml and incubated on ice for 5 min. An equal volume of RBS containing 0.4% NP-40 was added, and the volumes were mixed and incubated on ice for 10 min. Nuclei were centrifuged at 850x g for 10 min at 4°C and washed with RBS before being resuspended at approximately 5 x 107 cells/ml. An equal volume of lysis buffer (20 mM Tris-HCl [pH 8], 20 mM EDTA [pH 8], 2% sodium dodecyl sulfate [SDS], 500 µg/ml proteinase K) was added and incubation took place at 50°C overnight. DNA was phenol:chloroform extracted and ethanol precipitated prior to resuspension at approximately 1 mg/ml.
Nascent strands were isolated by size fractionation on alkaline agarose gels as described previously (66). Briefly, 300 µg genomic DNA was denatured at 85°C for 10 min with 1/6 volume of 6x alkaline loading buffer (300 mM NaOH, 6 mM EDTA [pH 8], 18% Ficoll 400, 0.15% bromocresol green, 0.25% xylene cyanol) and rapidly chilled on ice for 10 min prior to electrophoresis on a 1.2% SeaPlaque (BMA) alkaline agarose gel containing 50 mM NaOH and 1 M EDTA, pH 8. Gel electrophoresis was carried out at 0.7 V/cm for 16 h at room temperature in 1x Tris-acetate-EDTA, 50 mM NaOH, 1 mM EDTA. The gel fragment containing DNA of 0.8 to 2 kb was excised and DNA was recovered using a QIAquick gel extraction kit (QIAGEN). Cell cycle synchronization, to enrich the population of cells in S phase, was not carried out, as ES cells spend the majority of their cell cycle in S phase (27). Bromodeoxyuridine labeling of the cells, to isolate only the replicating population, was also not performed, as previous studies have shown identical results with and without the use of bromodeoxyuridine affinity purification (42).
Nascent strand length analysis (NSLA). Size-fractionated nascent strands were isolated as described above. Fractions corresponding to 0.5 kb, 1.5 kb, 3 kb, 5 kb, and 12 kb were excised and purified using a QIAquick gel extraction kit (QIAGEN). An equal amount of DNA was added to PCR mixtures (equivalent to 2.5 ng of a 12-kb fraction). PCR was carried out for 30 cycles with an annealing temperature of 60°C. Appropriate control experiments were carried out to determine that these experiments were carried out under nonsaturating conditions. PCR products were purified using MultiScreen FB plates (Millipore) according to the manufacturer's instructions. Equal volumes of eluate were then transferred to Zeta probe membrane (Bio-Rad) by slot blotting according to the manufacturer's instructions. Membranes were probed with the cognate PCR products and exposed to phosphor screens overnight.
Real-time PCR analysis. PCR amplification was carried out with iQ SYBR green Supermix (Bio-Rad) using an iCycler iQ real-time detection system (Bio-Rad). The following conditions were used: 95°C for 8.5 min and 50 cycles of 95°C for 30 s, 60 to 65°C for 30 s, and 72°C for 30 s (primer sequences are shown in Table 1). Melt peak analysis was carried out for each reaction to confirm amplicon (amp.) specificity. Each reaction was performed in triplicate and repeated with at least two independent DNA preparations. The amount of nascent DNA in each independent preparation was calculated using a standard curve generated for every reaction with each primer pair, using four 10-fold dilutions of genomic DNA.
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PCR was carried out using AmpliTaq Gold DNA polymerase (Applied Biosystems) for 34 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. This was determined to be within the linear range of amplification for each primer pair tested. Each primer pair was tested on at least three independent chromatin samples. Quantitation was carried out using AlphaImager software (Alpha Innotech).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1.5 kb across the 86-kb Xic region spanning from Jpx to Tsx (Fig. 2A). Reaction conditions for each primer pair were extensively tested for real-time quantitative PCR (QPCR) to ensure that a single amplicon product without primer dimers was generated. QPCR was performed with ORI-enriched single-stranded nascent DNA for each primer set, and a standard curve was generated from a parallel preparation of genomic DNA for each amplicon. The relative copy number of DNA was determined at each amplicon position when normalized to the non-ORI region (primer pair A) from the murine c-Myc located on chromosome 15 (31) (Fig. 2B). Normalization to an ORI region of c-Myc (primer pair C) did not significantly alter the relative copy numbers for any of the analyses presented in this work (Fig. 2C and data not shown); thus, we arbitrarily chose to show only normalization to a non-ORI region in subsequent experiments.
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4 kb away from the Xite enhancer (amplicon 35). The highest peaks were five- to sixfold above background levels, consistent with enrichment observed at other ORIs (29, 32, 37, 69). (Of note is the fact that male peaks were consistently larger than female peaks in undifferentiated cultures. We believe that this reflected lower division rates of XX ES cells due to the toxicity of two active X's. Interestingly, the peak height differences between males and females disappear in embryoid body (EB) cultures, consistent with the occurrence of dosage compensation in female EBs [see Fig. 5]). The ORI spanning Xist exon 1 was broader than what is expected for a single ORI, a result that was especially apparent in females (Fig. 2D). This implies the presence of multiple active ORIs in this region. Two ORI regions (one corresponding to a CpG island and one not) were chosen for further analysis by NSAA. In three or four independent cell preparations, both regions were found to be enriched in the nascent strand pool (Fig. 2E), further indicating the presence of origin activity at these positions. Cumulatively, these findings revealed putative ORI activity at five locations within the Xic.
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Using NSLA, we found that sequences containing the ORI located at the Tsix gene promoter were present in the shortest fragments of DNA tested (<1 kb) (Fig. 3D), consistent with the findings of Gomez and Brockdorff (32). Interestingly, this method suggested that the broad activity zone detected by NSAA at the 5' end Xist exon 1 may actually reflect the activity of two distinct ORIs at positions 13 and 17 (Fig. 3B). In addition, we detected ORI activity at the Jpx CpG island, the Xist exon 1 region, and Xist intron 7 and near Xite (Fig. 3), as indicated above, by NSAA. Thus, the nascent strand abundance and length assays yielded essentially identical results.
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However, while ORI positions were conserved upon cell differentiation, their degree of usage may vary slightly, as intensities of individual peaks did change. For example, the Jpx ORI (amp. 3) appeared to intensify during differentiation, while the Xite ORI (amp. 35) appeared to diminish in activity (Fig. 5B and C). The same QPCR analysis was then performed using primary fibroblasts, a somatic cell type in which XCI has already occurred. Similar peaks suggestive of ORIs could be observed in both male and female fibroblasts (Fig. 5D and E), although there was a trend towards less ORI activity in both fibroblast lines that was perhaps in keeping with their lower mitotic rates. Cumulatively, these results demonstrated that the five Xic ORIs are used, to differing degrees, at all stages during development.
Xic mutations affect ORI position and activity. To determine whether changes in ORI function occur in the presence of specific Xic mutations, we investigated ORI activity in cells with deletions of Tsix and Xite, two regulators of XCI initiation. In each case, we isolated the mutant X in a male background so as to restrict analyses specifically to the affected X. Because male ES cells are capable of up-regulating Xist and undergoing XCI when carrying Xic transgenes, they contain all necessary trans factors involved in XCI (49).
We first examined the effects of deleting Xite, a locus that determines X-chromosome counting choice by acting synergistically in cis with Tsix. The XiteL allele skews the ratio of XCI in favor of silencing the mutated X in the XX female (58) and contains a 12-kb deletion that includes the Xite enhancer (67), transcription start sites of the noncoding Xite transcripts, and a minor ORI (amp. 35 to 37). Nascent strand analysis by QPCR revealed that the XiteL line exhibited significant differences from wild-type male ES cells. In undifferentiated cells, the removal of this element resulted in markedly reduced activity of all the ORIs compared to that for wild-type cells (Fig. 6A). This decrease was most evident for the ORI at amplicon 21, which was greatly reduced even in comparison to the rest of the Xic in this cell line. However, the ORI located at the Xist promoter region (Fig. 2 and 3 and reference 32) becomes more apparent. Thus, deletion of the choice determinant, Xite, and of its associated minor ORI modulated the activity and position of other ORIs at a considerable distance across the Xic.
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CpG line (47) carries a 3.7-kb deletion including the Tsix promoter, the associated DXPas34 repeat element (20, 23), various enhancer elements (20, 23, 67), and the Tsix promoter ORI identified above (amp. 31). The TsixCpG mutation also had significant effects on the presence or activity of the remaining ORIs. Although the activity of the ORI located at amplicon 21 appeared to be similar to that of the wild-type undifferentiated male cells, the abundances of amplicons 3 and 35 were significantly reduced (Fig. 7A). Additionally, a new peak arose at amplicon 28, where no ORI had previously been observed; this suggested that deletion of the Tsix ORI permitted the use of a nearby ORI. The presence of this ORI was confirmed using NSLA (Fig. 7C). A clear difference was observed between the mutant cells, where short, nascent DNA fragments were present at amplicons 27 and 28, and the wild-type cells, from which these fragments were absent. While the positions of the ORIs remained unchanged upon cellular differentiation, a drastic reduction in activity at all the ORI regions was observed (Fig. 7B). Taken together, the results with XiteL and TsixCpG suggest that ORI usage at the Xic can be modulated by mutations in Xite and Tsix. Disrupting these loci results in a shift both in ORI position and in the frequency of usage of different ORIs.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results speak directly to the hypothesis that transcription may be required for origin activity. The necessity of transcription has been suggested by the coincidence of many ORIs with CpG islands (6) and by the finding that active transcription through the DHFR gene increases the efficiency of firing from the ORI located 3' to the gene, while a deletion compromises it (63). An open chromatin conformation that enables transcription factor binding may simultaneously allow ORC proteins to bind, consistent with the observation that active transcription is often associated with early replication. As shown here and elsewhere (32), however, a requirement for active transcription does not occur at Xic ORIs. The most prominent ORI identified in this study occurs within exon 1 of Xist. This ORI apparently fires regardless of the transcriptional activity of the Xist gene, as it is detected both in undifferentiated ES cells and in somatic XY cells where Xist is off. Likewise, the ORI within intron 7 of Xist can fire in the same Xist-off cells.
As observed here, an average ORI spacing of one per 15 kb is much higher than what is predicted by genome averages. However, a recent, elegant study of the IgH locus also identified origins spaced every 20 kb throughout a 300-kb region (57). In addition to the five constitutive ORIs at the Xic, we have also found at least one cryptic ORI whose activity is uncovered only when a nearby ORI is deleted. With six potential ORIs to choose from, the usage within any one cell may vary from using all to using only a small subset of the ORIs. The use of standard population-based assays precludes distinction between these possibilities. Studies with the IgH locus suggest that a small number of possible origins are used on each allele during a single duplication cycle (57).
Our results are largely consistent with those of Gomez and Brockdorff, who used NSLA and NSAA to identify two ORIs coinciding with the CpG islands of Xist and Tsix (32). However, several differences with regard to the pattern of usage should be discussed. First, while those authors reported prominent usage of the ORIs at the Xist promoter, we find that ORIs within internal regions of Xist have more intense activity. Second, the ORI usage at the Tsix promoter was reported by Gomez and Brockdorff to be XX dependent, as XY cells show minimal activity. However, we consistently observe ORI activity at the Tsix promoter in both XX and XY cells, both undifferentiated and differentiated. Finally, our data demonstrate the presence of many other ORIs at the Xic, with the activities of some ORIs clearly mutable by differentiation state and by the deletion of critical XCI regulators. These differences could have resulted from different methodologies or different cell lines used.
It is interesting that ORIs coincide with regulatory noncoding elements (Fig. 8). Xist has been shown to control both X-chromosome choice (52, 61) and the initiation of chromosome-wide silencing (60). In fact, one of the regions implicated in choice (52) roughly coincides with the exonic ORI mapped to amplicons 14 to 16. At the 5' end of Tsix, an ORI (amp. 31) maps to the prominent CpG island near the transcription start site, a region of considerable interest in terms of how Xist is blocked from initiating silencing on the future Xa chromosome. This 5' domain of Tsix has been shown to contain an imprinting center that regulates paternal allele-specific silencing of the X in placental tissues (44, 62). It also harbors elements required for X-chromosome counting and stochastic allelic choice (45, 47, 55) and includes the repeat element DXPas34 (23), a Tsix-specific bipartite enhancer (67), and binding sites for the chromatin insulator CTCF (13). In Xite, the minor ORI (amp. 35 to 37) localizes near the second of two CpG islands and transcription start sites in this locus. This domain of Xite has also been implicated in X-chromosome choice (47) and counting (45, 55). The ORI at amplicon 3 lies in the CpG island of Jpx/Enox, a gene of unknown function that is expressed at least partially from both Xa and Xi. Because Jpx/Enox is located within a 30-kb interval upstream of Xist that has been shown by genetic analysis to be required for Xist regulation (48), this noncoding RNA may also play a significant role in XCI.
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CpG), we observe an alteration in ORI location and intensity more than 6 kb away. These data are similar to those of Paixao et al., who show that the removal of a nearby CpG island from the human laminB2 ORI results in a reduction of activity of the related ORI (59). When Xite is deleted (XiteL), we observe a long-range effect more than 30 kb away (position 21). Future work will address whether these ORI modulations in turn regulate the events of XCI. It is relevant that differential replication timing of the Xic has been noted previously (9, 34, 70) and is one potential mechanism by which allelic choice might be regulated. Our findings pave the way for investigation into whether and how DNA replication influences epigenetic structure and the pattern of asymmetric gene expression during development.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant R01 (GM38895) and the MGH Fund for Medical Discovery Postdoctoral Fellowship (R.K.R.). J.T.L. is an Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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-ORC2 antibodies were added as indicated above to formaldehyde cross-linked female (EL16.7) and male (J1) undifferentiated ES cells. "Mock" denotes the control (no cells); 1:100 dilutions of starting material were used for input lanes. One representative ChIP is shown for each primer pair. Histograms show the average ORC2 or ORC4 enrichment over that of minus-antibody controls in three to six independent experiments with male cells with standard deviations shown. Similar results were gained with female cells (data not shown).
