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Human Molecular Genetics Advance Access originally published online on July 28, 2006
Human Molecular Genetics 2006 15(17):2613-2622; doi:10.1093/hmg/ddl187
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Published by Oxford University Press 2006.

Cooperative sequence modules determine replication initiation sites at the human ß-globin locus

Lixin Wang, Chii Mei Lin, Joseph O. Lopreiato and Mirit I. Aladjem*

Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, MD, USA

* To whom correspondence should be addressed at: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 37 Convent Dr, Bethesda, MD 20892, USA. Tel: +1 3014352848; Fax: +1 3014020752; Email: aladjemm{at}mail.nih.gov

Received June 3, 2006; Accepted July 20, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human beta globin locus contains two adjacent replicators, each capable of initiating DNA replication when transferred from its native locus to ectopic sites. Here, we report a detailed analysis of the sequence requirements for replication initiation from these replicators. In both replicators, initiation required a combination of an asymmetric purine:pyrimidine sequence and several AT-rich stretches. Modules from the two replicators could combine to initiate replication. AT-rich sequences were essential for replicator activity: a low frequency of initiation was observed in DNA fragments that included a short stretch of AT-rich sequences, whereas inclusion of additional AT-rich stretches increased initiation efficiency. By contrast, replication initiated at a low level without the asymmetric purine:pyrimidine modules but they were required in synergy to achieve efficient initiation. These data support a combinatorial model for replicator activity and suggest that the initiation of DNA replication requires interaction between at least two distinct sequence modules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Eukaryotic cells replicate their DNA from hundreds to thousands of chromosomal sites. Metazoan replication initiation sites, or replication origins, share some common features but do not exhibit a clear consensus sequence (1). Moreover, the location of initiation events might be affected by developmental (2,3) or metabolic (4) conditions and initiation can occur either from distinct replication origins (5–7) or from extended zones containing numerous low-frequency origins (8,9). This diversity and flexibility may reflect the coordination of numerous metabolic processes that occur simultaneously on chromatin, including DNA replication, chromatin condensation and transcription.

The replicon model postulated that DNA sequences (termed replicators) determine the location of replication initiation (10). A definitive assay for metazoan replicators involves transferring putative replicators from their native sites to ectopic sites and testing for initiation at the new locations. Replicators that exhibit ectopic initiation include sequences from the Drosophila chorion locus (11); the Chinese hamster dihydrofolate reductase (DHFR) locus (12) and the human loci encoding ß-globin (5), lamin B2 (7), c-myc (13) and possibly HPRT that can complement its murine ortholog (14). Although tandem copies of the Chinese hamster DHFR can initiate replication while they do not initiate DNA replication in loco (15), most studies observed a straightforward correspondence between replicator activity at the native locus and at ectopic sites. In the human ß-globin locus, replicators identified by ectopic assays were subsequently tested by deletion analyses at the native locus and found essential for the initiation (6).

The human ß-globin locus includes five genes that encode the beta subunit of hemoglobin. This locus replicates from a single initiation region (IR) between the two adult beta-like globin genes (16). The Lepore deletion, which removed IR, resulted in passive replication of the locus from an outside origin, suggesting that replication required genetic information uniquely supplied by the deleted region. IR can initiate DNA replication when transferred to genomic regions that lack inherent replicator activity (5). In the previous studies (5,6), we found that this region contained two redundant, independent, non-overlapping replicators, Rep-P and Rep-I, each sufficient to initiate replication at ectopic sites. We have identified three regions essential for the initiation of DNA replication: two DNA fragments from Rep-P and one DNA fragment from Rep-I (Fig. 1A). Here, we created a series of mutants and combinations of these three regions to discern the sequence requirements for the initiation of DNA replication.


Figure 1871
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  		Figure 1. Replicators and cell lines. (A) The human ß-globin locus. Gray boxes represent ß-like globin genes; arrows represent DNAse hypersensitive sites (for details, see (6)). (B) Insertion of replicator fragments using site-specific recombination. Replicator fragments were cloned in a single FLP recombination target (FRT) vector and transfected into E25B4 cells, an African green monkey kidney (CV1) derived cell line that contains an insertion of a single FRT between a promoter and a fused lacZ gene. Transfection was performed in the presence of FLP recombinase that catalyzed a recombination event between the two FRT sites (one on the plasmid and the other in the acceptor cell line), resulting in site-specific insertion of the single FRT vector into the acceptor site. Colonies that were successfully transformed were selected for hygromycin resistance and then screened for the loss of lacZ activity, which resulted from site-specific integration of the plasmid between the promoter and the lacZ gene. Filled gray arrows represent FRT sites; arrows designated P1 to P4 represent the locations of the primers used in C. Site-specific integration disrupted the fragment amplified by primers P1 and P2 but did not disrupt the fragment amplified by primers P3 and P4. Transgenes' copy numbers, integration sites and orientations were verified by PCR (C) and Southern blot analyses. (C) An example of a PCR analysis of recombinant clones. Lanes 1 and 2, DNA from colonies containing a single copy of Rep-P at the target site; lane P, DNA from the plasmid vector; lane M, molecular weight markers (100 bp ladder, Invitrogen). The left panel shows amplification products using P1 and P2; the right panel shows amplification products using P3 and P4.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We analyzed two replicators within the globin IR (Fig. 1A): Rep-P extends from the HindIII site at position 59590 to the NcoI site at position 62187, and Rep-I extends from the NcoI site at position 62188 to the XbaI site at position 65422. Sequences from each fragment were abundant in newly replicated, short exonuclease-resistant DNA strands (6).

We determined whether the initiation of DNA replication occurred from a series of clones inserted at a fixed site in the E25B4 simian cells as described (Fig. 1B and C; (6)). Insertion of all fragments into a fixed site in the simian genome was performed using site-specific recombination to neutralize position effects that might affect initiation. We measured the abundance of sequences from these inserted fragments in small (600–2500 bases) RNA-primed DNA (Fig. 2A and B). Real-time polymerase chain reaction (PCR) analysis of nascent strands used primers located within the replicator fragments and primers amplifying sequences from the adjacent hygromycin resistance gene (hyg). The lacZ marker served as a negative control. Sequences from the African green monkey ß-globin region served as positive controls. Data were normalized relative to the abundance of the lacZ primer (Fig. 2B). A high abundance of sequences from the transgenes indicated that replication initiated within the inserted fragment. Sequences from the hygromycin resistance marker were often amplified, indicating that replication initiated from the 3' end of the inserted transgene (for examples see Figs 2 and 3).


Figure 1872
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  		Figure 2. Nascent-strand abundance assay. (A) The principle of nascent-strand abundance assays. Newly replicated origin-proximal DNA is selected by size (600–2500 bp) and by resistance to lambda exonuclease (an enzyme that digests DNA with a 5'DNA tail but not DNA with a 5' RNA tail). DNA strands are depicted as solid gray lines, and 5' primer RNAs are shown as solid black boxes. As described in detail in the Materials and Methods section, DNA was isolated from asynchronously growing cell cultures, heat denatured and run on a sucrose gradient to separate template DNA from short DNA strands. Short DNA strands were then exposed to lambda exonuclease to isolate RNA-tailed DNA (which is resistant to digestion by the enzyme) from broken DNA strands. Nascent strands isolated in this way were then subjected to real-time PCR with primers encompassing the locus of interest. (B) Real-time PCR analysis of sequences from the Rep-P replicator in nascent strands from cells harboring Rep-P or Rep-P mutants in the acceptor site described in Figure 1. Arrows indicate the location of primers from Rep-P and the adjacent hyg and lacZ sequences (for primer sequences, see Table 1). Construct I includes the entire Rep-P; construct II includes Rep-P with a deletion of a 45 bp asymmetric purine:pyrimidine element (6); constructs III–V contain Rep-P variants in which the asymmetric purine:pyrimidine element was replaced with a sequence with a similar GC content that is not asymmetric, and with sequences exhibiting similar GC content and some asymmetry from the DHFR and lamin B replicators, respectively (for description of the constructs, see Tables 2 and 3). Error bars on the histogram represent standard errors of several experiments, conducted as described in (6).

 


Figure 1873
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  		Figure 3. Nascent-strand abundance of replicator variant sequences from Rep-P-2 and Rep-I-1 in the presence or absence of the asymmetric purine:pyrimidine sequence (AG). The nascent-strand abundance assay was conducted as described in Figure 2.

 


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  		Table 1. Primers and probes used in the study

 


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  		Table 2. Deleted sequences and inserted linkers

 


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  		Table 3. Fragments from the globin IR

 
We tested the role of two types of DNA sequences in the initiation of DNA replication from Rep-P and Rep-I: the first element was an asymmetric purine:pyrimidine sequence (purines on one strand and pyrimidines on the other strand); the second element was an extended AT-rich strand. Asymmetric purine:pyrimidine sequences are present in both Rep-P and Rep-I. As shown previously, the asymmetric sequence in Rep-P is essential for initiation from Rep-P (6). The asymmetric sequence in Rep-I resides near AT-rich nucleotide stretches of opposite polarity (an A-rich stretch following a T-rich stretch) and the fragment that contains both elements is essential for initiation from Rep-I (6). We created a series of cell lines harboring deletions of each of these sequences (designated AG for the asymmetric sequence and AT for the AT-rich stretch) within fragments of Rep-I and within combined Rep-I and Rep-P fragments. As indicated above, all these mutants were inserted into a constant site in E25B4 cells.

Within Rep-P, we have previously identified a 45 bp asymmetric purine:pyrimidine sequence (AG) whose removal prevented the initiation (6). We replaced AG with several linkers: a scrambled linker with a similar GC content that does not exhibit asymmetry (Fig. 2B, construct III) and two G-rich linkers from the DHFR ori-beta and the lamin B replicators (Fig. 2B, constructs IV and V). As shown in Figure 2B construct I, sequences from Rep-P were abundant in newly replicated DNA. As noted previously (6), sequences from the hyg marker were also abundant in nascent DNA, indicating that initiation events occurred within the region extending from the center to the 3' end of Rep-P. In contrast, sequences from either Rep-P or the hyg marker were absent from nascent strands derived from cells that contained Rep-P variants in which the AG sequence was deleted or replaced as described above (constructs II–V). These data indicated that all three G-rich DNA elements were unable to restore initiation.

The 921-bp fragment between the PmlI site and the EcoRI site (Rep-I-1; Fig. 1) that contains an asymmetric purine:pyrimidine sequence is required but not sufficient to initiate replication at the level observed for the entire Rep-I (6). We investigated whether initiation could be achieved by combining Rep-I-1 with fragments from Rep-P. As shown in Figure 3, low levels of Rep-I and hyg sequences were detected in newly replicated DNA strands from cells containing Rep-I-1, suggesting that Rep-I-1 initiated replication at a low frequency (Fig. 3, compare constructs I and II). The combination of Rep-P-2 and Rep-I-1 significantly increased the frequency of initiation (Fig. 3, compare constructs II and III).

We then asked whether the asymmetric AG sequences in Rep-P-2 or Rep-I-1 were required for the initiation when Rep-P-2 complemented Rep-I-1. Deletion of either region (Fig. 3, constructs IV and V) did not completely prevent initiation but had significantly decreased its frequency. By contrast, deletion of the AG sequence in the context of the entire Rep-I did not decrease initiation frequency (Fig. 4, construct II), suggesting that sequences at the 3' end of Rep-I-1 might complement the AG deletion. Interestingly, a deletion of AG elements in both Rep-P-2 and Rep-I-1 (Fig. 3, construct VI) did not further decrease the initiation frequency.


Figure 1874
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  		Figure 4. Nascent-strand abundance assays of Rep-I variants in the presence or absence of the asymmetric purine:pyrimidine sequence (AG) and the extended stretch of AT-rich sequences (AT). The nascent-strand abundance assay was conducted as described in Figure 2.

 
We have also tested whether sequences that proved essential for initiation in Rep-I-1 will be required for initiation in the context of the entire Rep-I. As noted above, a deletion of the AG sequence did not affect initiation from the entire Rep-I (Fig. 4, compare constructs I and II), but replication did not initiate when the deletion was expanded to include the AT-rich sequences of opposite polarity that resides 3' to AG (Fig. 4, construct III). A variant construct in which approximately half of the AT-rich stretch was restored (Fig. 4, construct IV) did not initiate DNA replication, but replication was able to initiate when this deletion was complemented by the AG sequence (Fig. 4, construct V). These data suggested that a minimal length of the AT-rich sequence is sufficient for initiation in the context of the entire Rep-I when complemented by either a sequence of opposite polarity or a separate asymmetric purine:pyrimidine element.

We then tested the role of AT-rich stretches in initiation of DNA replication from the combined Rep-P-2 and Rep-I-1 in the presence or absence of the AG element. As shown in Figure 5, expansion of the Rep-I-1 AG deletion to include the entire Rep-I-1 AT-rich stretch prevented initiation from the combined replicator (compare construct I with constructs II and III). Importantly, unlike the deletion of the AG sequences, which allowed a low frequency of initiation, no initiation was detected when both the AT and AG sequences were deleted. Initiation was prevented regardless of whether the asymmetric AG sequence was present in Rep-P-2 or not (Fig. 5, construct III). Some low-frequency initiation was observed when the asymmetric AG sequence and a part of the AT-rich stretch were restored (Fig. 5, compare constructs IV and V with constructs II and III). However, insertion of the asymmetric AG sequence from Rep-P-2 into the Rep-I-1 fragment was not sufficient to restore initiation when the asymmetric AG and the adjacent AT-rich region were deleted (Fig. 5, constructs VI and VII). The combined AG and AT sequences from Rep-I-1 were not sufficient to initiate replication by themselves, even when complemented by Rep-P-2 (Fig. 5, constructs VIII and IX).


Figure 1875
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  		Figure 5. Nascent-strand abundance of replicator sequences from Rep-P-2 and Rep-I-1 in the presence or absence of the asymmetric purine:pyrimidine sequence (AG) and the extended stretch of AT-rich sequences (AT). The nascent-strand abundance assay was conducted as described in Figure 2.

 
Finally, we asked whether the Rep-P-1 element, which was essential for the initiation of DNA replication from Rep-P, could complement Rep-I-1. Rep-P-1 does not contain an identifiable asymmetric purine:pyrimidine sequence but contains an extended series of AT-rich stretches. As shown in Figure 6, the combination of Rep-P-1 and Rep-I-1 exhibited a marked increase in initiation frequency (Fig. 6, constructs I and II). Deletion of the AG elements in Rep-I-1 (Fig. 6, constructs III and IV) prevented initiation of DNA replication. Inclusion of the asymmetric Rep-I-1 AG sequence in the presence of a partial deletion of the AT element (Fig. 6, construct V) exhibited a low frequency of initiation.


Figure 1876
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  		Figure 6. Nascent-strand abundance of replicator sequences from Rep-P-1 and Rep-I-1 in the presence or absence of the asymmetric purine:pyrimidine sequence (AG) and the extended stretch of AT-rich sequences (AT). The nascent-strand abundance assay was conducted as described in Figure 2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Several different combinations of DNA sequences enabled efficient initiation of DNA replication within the globin IR: stretches of AT-rich sequences were essential for the initiation, whereas asymmetric purine:pyrimidine elements enhanced the initiation frequency. Without the asymmetric purine:pyrimidine element, it is likely that in most chromosomes the region that included the AT-rich sequences was duplicated by replication forks from adjacent sites before replication could initiate. Inclusion of the asymmetric purine:pyrimidine sequence was essential to achieve efficient initiation, which also required a minimal length of AT-rich sequences.

Previous analyses had identified sequence elements necessary for replicator activity (6,7,12,13,17). Sequence comparisons yielded no consensus, but a modular structure that involves some AT-rich sequences appears to be a common theme. For example, replication from the Drosophila chorion locus requires cooperation between two distinct sequences, ori-beta and ACE3 (11); the Chinese hamster DHFR requires at least four elements, some of which are AT-rich, for ectopic initiation (12,17); the human c-myc replicator requires several elements, including an AT-rich DNA unwinding element (13); and the lamin B2 replicator absolutely requires a single AT-rich element but replicator activity is enhanced by a GC-rich second element (7). Here, we show that initiation of DNA replication in the globin locus, even at a low frequency, required a stretch of AT-rich sequences. Inclusion of longer or additional AT-rich stretches in the presence of an asymmetric purine:pyrimidine element increased initiation efficiency.

AT-rich stretches serve as sites of DNA unwinding in yeast (18) and in mammalian cells. An asymmetric AT-rich region serves as a binding site for the origin recognition complex (ORC) in budding yeast but the specificity of metazoan ORC is different: Drosophila ORC recognizes primarily DNA supercoiling, not sequence (19), consistent with a lack of consensus for Drosophila initiation sites (20); mammalian ORC does not exhibit sequence specificity beyond a preference for AT-rich sequences (21). However, sequences from the lamin B replicator were shown to bind ORC and recruit other members of the pre-replication complex (22), whereas AT-rich sequences from the c-myc replicator were shown to bind a protein, DUE-B (23). We observed that including a short stretch of AT-rich sequences, which resemble yeast ORC binding sites, was essential for a low frequency of initiation. This observation is consistent with a role for the AT-rich sequences in both ORC binding and DNA unwinding and suggests that AT-rich sequences, though essential, are not sufficient to determine where replication initiates.

Asymmetric purine:pyrimidine elements were essential for efficient initiation in either Rep-P or Rep-I, but some initiation was achieved without these sequences in the presence of AT stretches. Two asymmetric sequences or a combination of an asymmetric sequence and an extended AT-rich sequence were required to achieve maximum initiation frequency. Interestingly, although the lamin B2 AT-rich region could partially substitute the DHFR AT-rich region (17), we did not observe a complementation of the asymmetric purine:pyrimidine element by similar elements from both the DHFR and the lamin B replicators. In this context, it is noteworthy that the DHFR and lamin B2 loci replicate early during S-phase, whereas the ß-globin locus replicates at flexible times depending on the gene expression. Thus, the asymmetric purine:pyrmidine region may play a locus-specific role to dictate replication at the correct time during S-phase. Consistent with this, the asymmetric sequence is located in regions that are important for gene expression within the promoter of the human beta-like globin gene (Rep-P) and within the large intron, which is important in transcriptional regulation (Rep-I). The 45-bp Rep-P-AG sequence is not essential for transcription (Haiqing Fu and M.I.A., unpublished), but the 45-bp asymmetric region is critical for replicator-mediated maintenance of an open chromatin conformation in an environment that otherwise facilitates gene silencing (24). These observations suggest that asymmetric purine:pyrimidine sequences play a role in creating an environment conducive to initiation of DNA replication at the correct time during the cell cycle and that this role may modulate initiation from the ß-globin locus.

The apparently combinatorial organization of metazoan replicators, as shown here and in other genetic dissections of replicator activity, is reminiscent of the control of metazoan transcription: the location and efficiency of transcription are dictated by a collection of diverse modules located at varied distances from the transcription initiation site (25). The observation that several combinations of replicator modules are able to satisfy the requirement for initiation supports the notion, first proposed by DePamphilis (26), that the exact location of the initiation of DNA replication is determined anew each cell cycle and involves a choice between several potential sites. This concept is in line with recent evidence suggesting that replication initiation sites can vary with metabolic conditions (4) and with differentiation (2,3) and upholds the suggestion that the genome contains multiple sites of initiation to provide some insurance against incomplete replication that could otherwise lead to genetic instability [(27); reviewed in (28–30)].

Because several combinations of elements within the short IR can initiate replication, our data are in line with a model suggesting that the location of initiation sites is selected from multiple potential replicators by a process that might be dictated by the chromosomal environment. This model is supported by the observation that in metazoans, some loci contain distinct high-frequency initiation sites (5–7) but others exhibit extensive zones with numerous initiation sites, each initiating replication in a fraction of cell cycles (8,9,31). Within such zones, replication initiating at any site might overtake other potential initiation sites before these sites are able to start replication, causing a diffuse initiation pattern. Our data suggest that efficient initiation within the relatively short human ß-globin origin might be dictated by several sequence combination, reconciling the apparent contradictory notion that some metazoan loci contain defined replication origins, whereas others initiate from a dispersed sequence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells and culture conditions
Simian CV-1 E25B4 (B4) cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and with antibiotics as needed. DNA fragments to be tested for replicator activity were inserted downstream of the FLP recombination target (FRT) site using site-specific recombination as described previously [(5,6); Fig. 1]. Colonies were selected for hygromycin resistance and screened for insertion at the B4 site by staining for loss of beta-galactosidase activity followed by PCR and hybridization analyses to verify that single copies of clones had been inserted.

Replication initiation analyses
The primers and probes used in this study are listed in Table 1. The IR fragments tested for replicator activities are listed in Table 2. Genomic DNA and nascent-strand DNA were prepared as described previously (6). Briefly, DNA was collected from asynchronous cultures and denatured by boiling followed by rapid cooling, and short DNA strands were size-fractionated on neutral sucrose gradients. DNA strands ranging in size from 0.6 to 2.5 kb were collected and treated with lamda exonuclease as described previously. Nascent strands were further size fractionated by gel electrophoresis and amplified by real-time PCR in an ABI 7900 thermocycler using a series of primer-probe combinations surrounding the inserted replicator and adjacent sequences. The amount of DNA in each sample was quantified by pico-green analysis (Molecular Probes, Eugene, OR, USA). Genomic DNA that was not treated with exonuclease was used as a standard for calculating the number of molecules in the template. Genomic DNA from simian CV-1 cells was used as a non-template control to verify that primers used in the study were specific for the inserted DNA. The LacZ primer-probe combination, which lies nearly 5 kb from the inserted replicator candidates, was used as a standard control for sequences that did not initiate DNA replication. Data from three PCRs for each primer-probe combination were used to calculate the amount of sequence-specific nascent strands as described previously. All experiments were performed at least three times; a representative PCR analysis from a single experiment is shown.

Mutagenesis
The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to construct replicator mutants. Briefly, primer annealing was followed by extension with PfuTurbo DNA polymerase and incubation with DpnI, which digests the dam-methylated parental DNA template and selects for mutation-containing newly synthesized DNA. All mutants were verified by DNA sequencing (National Cancer Institute core sequencing facility).


   ACKNOWLEDGEMENTS
 
The authors thank their colleagues at the DNA replication group and the Laboratory of Molecular Pharmacology, NCI for helpful discussions. This study was supported by the Intramural Research Program of the NIH, Center for Cancer Research, National Cancer Institute.

Conflicts of Interest statement. The authors declare no conflicts of interests.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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