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Genome-scale identification of active DNA replication origins
Methods 57 (2012) 158–164
Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Genome-scale identification of active DNA replication origins Christelle Cayrou, Damien Grégoire 1, Philippe Coulombe, Etienne Danis 2, Marcel Méchali ⇑ Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, Montpellier 34396, France
a r t i c l e
i n f o
Article history: Available online 13 July 2012 Communicated by Kenneth Adolph Keywords: DNA replication origins Nascent strand purification Lambda-exonuclease Microarrays
a b s t r a c t Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps. Ó 2012 Published by Elsevier Inc.
1. Introduction At each cell cycle the entire genome of any living organism must be faithfully duplicated. In metazoans, DNA replication is initiated at thousands of chromosomal sites during each S phase. These DNA replication origins (Oris) should be activated only once at each cell cycle to avoid any amplification and to maintain genome integrity. In bacteria, yeast and viruses, Oris are rather well understood and are characterized by specific DNA sequence elements. In Metazoa, Oris occur at specific locations [1], and insights into their genetic characteristics begin to emerge. In bacteria, control of replication initiates by the binding of DnaA, the initiator of chromosome replication, to the DnaA boxes that form clusters of three or more elements and are located mainly in the E. Coli Ori [2]. The archaeal DNA replication machinery is a simplified form of the eukaryotic one [3]. The archaeal chromosome generally contains one Ori that has autonomously replicating sequence (ARS) activity, although some Archaea contains multiple Oris [4]. These Oris (termed ORBs) are 36 bp CG-rich sequences near an AT-rich region that might act as a melting-prone site. In budding yeast, origins are defined as ARSs on which initiation proteins are stepwise assembled. This is the only eukaryote in which ORC specifically recognizes a 17 bp AT-rich consensus sequence called ACS (for ARS Consensus Sequence) [5]. Although this motif is necessary for DNA replication, it is not sufficient for Ori function. In contrast to Saccharomyces cerevisiae, Saccharomyces ⇑ Corresponding author. Fax: +33 0434 359 920. E-mail address: [email protected] (M. Méchali). Present address: Institute of Molecular Genetics, 1919 Route de Mende, Montpellier 34293, France. 2 Present address: Department of Pharmacology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093–0636, USA. 1
1046-2023/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ymeth.2012.06.015
pombe Oris do not contain a core consensus sequence [5]. S. pombe Oris are AT-rich (from 0.5 to 3 kb in length) and contain several functionally important DNA sequence elements for their activity. In a genomic context, a 30 bp long poly-A/T tracks appears sufficient to specify replication initiation [6]. In metazoans, 30 000–60 000 Oris are activated at each cell division per cell. Oris appear to have variable features. For instance, they can be site-specific, as the human Lamin B2 or c-Myc Oris [7,8], or have a broad site specification, like the well characterized DHFR Ori [9]. Orc, a complex of 6 subunits binds to the replication origins in all known eukaryotic species. Its structure and role appears conserved, except its sequence specificity that is real in S. cerevisiae, but poor in other eukaryotes [10]. Until recently, only very few Oris were characterized in metazoans and their nature was elusive despite considerable efforts to unravel a replication origin code. Different genome-wide approaches have been developed to map Oris in metazoans. Three DNA methods have been mainly used: DNA combing, purification of replication intermediates by the bubble-trap method and nascent strands purification. The fourth one is a protein approach based on chromatin immunoprecipitation of initiation proteins. DNA combing allows visualizing replicated DNA molecules that have been stretched on silanized glasses (see Julien Bianco et al., this issue). This is a global method to determine the real spacing of Oris along DNA. However, it is not suitable to identify a given Ori DNA sequence due to the extremely low probability to detect such sequence while the replication fork is passing through the Ori. In practice, this method can work for very simple organisms or for sequences that are hundred-fold repeated in the genome. In addition, DNA combing is also limited by the DNA length that can be analyzed as it is hard to get molecules longer than 600– 800 Kb. A related method called SMARD has been developed and
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applied to the Igh locus after its purification by pulsed field gel electrophoresis [11], and recently to Nanog and Oct4 gene environment [12]. Purification of replication intermediates by the bubble-trap method [13,14] is based on the trapping of restriction fragments containing replication bubbles created at Oris. This is a good structure-based method although its resolution is limited by the size of the restriction fragments and the requirement that Oris are located preferentially at or near the center or the fragment. Chromatin IP of pre-replication complex proteins followed by identification of DNA by microarrays (ChIP-chip) or sequencing (ChIP-seq) is in principle an efficient method. It has been used in several cases to identify all potential Oris [15–18]. However, its major drawback is the low enrichment over the background. This might be partly because ORC, the main origin recognition complex, also recognizes other sites on chromosomes because of its implication in other nuclear processes [19]. Another drawback is that ORC binding to DNA is not sufficient to define an origin that is going to be activated [17]. Nevertheless, it might be improved in the future and can be a complementary additional method to the DNA-based methods (see Lubelsky et al., this issue). The RNA-primed nascent strand method is based on the purification of DNA that is first synthesized at Oris (see Fig. 1). These nascent DNA strands are primed by a 10–12 nucleotide RNA synthesized by the DNA polymerase-a primase. The purification procedure selects nascent DNA strands bigger than 400 bp to avoid the selection of Okasaki fragments synthesized at the ongoing replication forks all along the genome (see Fig. 1). This method was developed and successfully used in the nineties and quantification was carried out by competitive PCR [20]. A library of nascent DNA was also constructed [21]. The method was then significantly improved by the addition of a Lambda exonuclease digestion step [22,23] to eliminate any broken DNA fragment (RNA-primed nascent DNA strands are protected from digestion by their RNA primer). This is a crucial and obligatory step as even a very low amount of contaminating DNA can increase the background. Indeed, from 100 million exponentially growing cells (i.e., 600 lg total mouse or human DNA), no more than 20–40 ng of RNA-primed nascent DNA can be recovered in theory (0.003% of the total DNA), if no loss occurs during the purification procedure. In addition, exponentially growing cells must be used for genome-wide analysis as Oris are activated all along the S phase. Qualitatively, this method gives excellent replicates and is very precise. Quantitatively, it depends on the level and quality of the required amplification step owing to the low amount of recovered DNA. We have been upgrading this method to the level of purification and enrichment required for genome-scale analyses. By using this technique followed by identification of the sequences by microarrays, we could characterize 2412 Oris on chromosome 11 in mouse ES cells and 6184 Oris in the Drosophila genome and define their characteristics [24,25].
2. Description of the Method (Fig. 2A for a summary) 2.1. Nascent strands (NS) preparation 2.1.1. Day 1 2.1.1.1. DNA purification. 1. Dividing cells (1 108–2 108 = 2 150 mm dishes) are washed with PBS and pelleted in a 50 ml falcon tube. 2. Cells are lysed in 15 ml DNAzol (Molecular Research Center) at room temperature (RT) for 5 min. 3. 200 lg/ml proteinase K is added to the lysed sample in DNAzol and the tube is incubated on a rotating wheel at low speed at 37 °C for 2 h.
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To avoid contamination by nucleases and surrounding DNA, we used a set of pipettes exclusively reserved to the purification of nascent strands and filtered tips only. We found that combining the proteinase K treatment with DNAzol significantly improves the NS yield (Fig. 2B). 1. Insoluble material is discarded by centrifugation at 3,000 g at 4 °C for 15 min. 2. Genomic DNA present in the supernatant is precipitated by adding 15 ml 100% ethanol at RT for 5 min. 3. DNA is transferred to a new 15 ml falcon tube and washed with 5 ml 70% ethanol at RT for 5 min. DNA is collected by winding it around a drawn Pasteur pipette, to minimize DNA breaks. From this step, use only MaxyClear MAXYMum Recovery tubes (Low retention-tubes from Axygen). 1. DNA is transferred using a drawn Pasteur pipette in a new 2 ml dry tube and air-dried (at RT for 30 min). 2. DNA is resuspended in 2 ml TEN20 at 65 °C until completely dissolved (15–30 min). TEN20: 10 mM Tris, pH7.9 2 mM EDTA 20 mM NaCl 0.1% SDS 1000 U RNasin (New England BioLabs, NEB) 3. The DNA solution is boiled for 10–15 min and rapidly chilled on ice. 2.1.1.2. NS purification by sucrose gradient. 1. One or two ml of denatured genomic DNA are loaded onto a 30 ml neutral 5%–30% sucrose gradients prepared in TEN500 in ultraclear tubes. Use 1 gradient for NS from 100 million cells. TEN500: 10 mM Tris pH7.9 2 mM EDTA 500 mM NaCl 2. Gradients are centrifuged in a Beckman SW28 rotor at 25,000 RPM, at 4 °C, for 20 h. 2.1.2. Day 2 1. 1 ml-fractions are withdrawn from the top of the gradient using a wide-bore pipette tip and placed in new 1.5 ml MaxyClear tubes. 2. 50 ll of each fraction is run with the appropriate size markers on a 1.5% alkaline agarose gel at 40–50 volts, at 4 °C overnight. 2.1.3. Day 3 1. The gel is neutralized with 1x TBE and stained with Gel Red (Interchim). 2. Fractions corresponding to 0.5–1, 1–1.5 and 1.5–2 kb (Fig. 2C) are pooled separately and precipitated with 2.5 vol of 100% ethanol at 80 °C for 15–30 min. 3. Pellets are washed with 1 ml 70% ethanol and suspended in 50 ll water with 100 U RNasin (NEB). 2.1.3.1. Lambda exonuclease treatment. This is the most crucial step of the protocol. 1. After addition of 5 ll PNK reaction buffer (NEB), fractions are boiled for 5 min and chilled on ice.
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Fig. 1. Main principles of RNA-primed nascent strands isolation for mapping of replication origins. DNA synthesis starts at replication origins after the opening of DNA by a DNA helicase. DNA replication is bidirectional from the origin with the continuous synthesis of nascent strands in the 50 –30 direction on the leading strand, whereas the other strand is called the lagging strand and is copied in a discontinuous manner in short segments called Okazaki fragments. Short RNA primers initiate DNA synthesis at both the leading and lagging strands. After DNA extraction, DNA is denatured to isolate the newly synthesized DNA strands which are then fractionated by sucrose gradient centrifugation. The DNA size of each fraction is measured by agarose gel electrophoresis of samples. Fractions in the 0.5–2.5 Kb range are further treated by lambda exonuclease and first quantified by qPCR analysis before genome-scale analyses.
2. Phosphorylation with T4 PNK is performed in a volume of 100 ll at 37 °C for 1 h. T4 mix: Water to 200 ll final 10X PNK buffer (NEB) 1X ATP 50 nM final 10 U/ll T4 PNK (NEB) 40 U (i.e., 4 ll) 3. The reaction is stopped by adding 2 ll 0.5 M EDTA and samples are directly precipitated with 2.5 vol 100% ethanol/0.3 M Naacetate at 80 °C for 15–30 min.
4. Pellets are washed with 1 ml 70% ethanol and resuspended in 100 ll water with 100 U RNasin (NEB). 5. A sample after phosphorylation is digested with lambda exonuclease (L-exo) at 37 °C overnight. Lambda exo mix: Water to 200 ll final 10X L-exo buffer (Fermentas)1X 20 U/ll L-exo (Fermentas) 5 ll
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B
C
A
D
Fig. 2. RNA-primed nascent strands purification and quantification. (A) Red numbers refer to the protocol steps described in the article. 0.5–2.5 Kb nascent strands are isolated from total genomic DNA by denaturation and sucrose gradient centrifugation. RNA-primed nascent strands are enriched by lambda exonuclease digestion and hybridized against total genomic DNA on high-density tiling arrays. (B) Combining the proteinase K treatment with DNAzol significantly improves the yield of NS. (C) A sample of each fraction of the sucrose gradients was run with the appropriate size markers on a 1.5% alkaline agarose gel in order to identify the sucrose gradient fractions to be processed. (D) Two rounds of T4 PNK and lambda exonuclease treatment significantly improved the enrichment. (E) purification of the NS preparation on GFX does not affect significantly its yield.
We found that the quality of the lambda exonuclease is crucial and deserves to be always tested before use. For the experiments described here, we used a custom-made preparation by Fermentas (20 U/ll). 2.1.4. Day 4 1. NS are extracted once with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol, and precipitated after addition of 2 ll polyacryl carrier (Molecular Research Center Inc.) with 0.3 M Na-acetate (pH 5.3) 2.5 vol 100% ethanol at 80 °C for 15 min. 2. Pellets are washed with 1 ml 70% ethanol and suspended in 50 ll water with 100 U RNasin (NEB). 3. NS preparations are subjected to one or two additional cycles of T4 PNK phosphorylation and lambda exonuclease digestion (1.– 5.) in a final volume of 100 ll for each step. We observed that the second round of lambda exonuclease treatment significantly improves the NS preparations (Fig. 2D). Ideally, a third lambda exonuclease treatment should be performed, but the amount of recovered DNA might be greatly reduced. We found that two rounds are sufficient when an effective lambda exonuclease is used.
2.1.5. Day 5 1. Half of the obtained NS preparation is suspended in 50 ll 10 mM Tris pH8 and is immediately quantified by qPCR amplification of a known origin to monitor the enrichment of the last step. 2. If the qPCR quantification shows an enrichment >20, the whole remaining preparation is purified using the CyScribe GFX Purification Kit (GE Healthcare) and eluted in 50 ll water (Fig. 2E). These columns allow to purify efficiently the single-stranded DNA without affecting the level of enrichment of the nascent strands preparation (Fig. 2E). 1. Samples may be stored overnight at 4 °C or for longer time at 20 °C. Repeated freezing and thawing causes loss of single stranded nascent DNA. It is better to freeze each sample into multiple aliquotes to avoid this. We suggest testing each digestion step (from c1. to c9.) by qPCR amplification of a known origin (such as c-Myc) to monitor the NS enrichment of the preparation. One ll of samples per reaction is sufficient to quantify the level of enrichment. It is important to obtain at least a 20-fold enrichment for the tested origin by qPCR as the enrich-
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Fig. 3. Quality controls of NS preparations. (A) Nascent strands from cells that were serum-starved for 27 h and then switched, or not, to medium with serum for 24 h were analyzed. Cell growth at each step was followed by using the MTT cell proliferation assay. For each culture condition, 0.5–2.5 Kb nascent strands were isolated. (B) The nascent strand preparations were analyzed using two origins (HoxB9 and HoxB4, (2) by qPCR with different sets of primers localized along the HoxB locus. (C) Half of the DNA fraction isolated from exponentially growing cells was incubated with RNAse A before the first step of phosphorylation in order to digest the RNA primer that protects the nascent strands from lambda exonuclease digestion. Half of each fraction isolated from the sucrose gradient was treated by RNAse A (50 lg/ml, at 37 °C for 30 min). (D) The preparation was then analyzed using origins identified on mouse chromosome 15 by qPCR with different sets of primers localized along this region. (E and F) 1 lg of DNA plasmid that was digested with restriction enzymes was added to the nascent strand preparation to control the level of digestion by lambda exonuclease. The degradation of the plasmid DNA was followed after each lambda exonuclease digestion step by agarose gel electrophoresis (10% of the total fraction/well) and by qPCR for each fragment. (G) The nascent strands preparation was analyzed using the HoxB9 origin by qPCR with different sets of primers localized along the HoxB locus. Nascent strand enrichment for each step is shown.
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ment usually decreases when the analysis is then made after amplification for microarrays. Using c-Myc as a known DNA replication origin, we found reproducible enrichment values between 30 and 100fold by qPCR. While purifying NS from cycling cells, it is important to control the quality of the preparation by: - Purifying NS-like molecules from the same cells in G0 or G1 phase. This fraction should not show any enrichment (Fig. 3A and B). - Incubating half of the DNA fraction with RNase A before the first step of phosphorylation to digest the RNA primer that protects the nascent DNA strands from degradation by lambda exonuclease. Such fraction should not show enrichment as nascent DNA will be digested by the lambda exonuclease (Fig. 3C and D). - Adding to the DNA fraction 1 lg of digested plasmid to control the degree of digestion by lambda exonuclease. This may be followed by deposition of a sample on agarose gel and by qPCR (Fig. 3E–G). 2.2. NS amplification and chip data analysis 2.2.1. NS amplification with the WGA kit 1. 10 ll of purified NS are amplified using the WGAII kit (Sigma), omitting the first step of fragmentation. It is necessary to amplify several 10 ll aliquots of the preparation to obtain the amount required for microarray hybridization. Indeed, re-amplification of already amplified material gives unanalyzable results and should be strictly avoided. 1. Amplification products are purified with NucleoSpin columns (Machery Nagel). 2. Proper unbiased amplification is checked by qPCR. 3. Hybridization of the nascent DNA preparation, washing and scanning of microarrays was done in our case by Nimblegen Service Laboratory. We found that amplification may affect only the enrichment values. Therefore the microarray analyses should be considered as semiquantitative. 2.2.1.1. Microarray Design. Given the size of the fragments of purified nascent strands, we choosen to use tiling array chips that can cover the width of a potential origin by a minimum of 10 probes. Moreover, the Nimblegen procedure allows comparing on the same slide, two colors, from the sample and control DNA, and we found this to be a good approach. To characterize fly Oris [25], Drosophila melanogaster samples were hybridized using 2.1 M Nimblegen microarrays (Design ID 6262). These tiling arrays contain in total 2,164,511 oligonucleotide probes representing the non-repetitive regions of the D. melanogaster genome (chromosome 2L, 2R, 3L, 3R, 4 and X; Flybase release 4.3). To analyze the data, 1,807,015 oligonucleotide probes were selected (909,279 for the top strand and 897,736 for the bottom strand) with an average length of 50 bp for oligonucleotides and for inter-oligo spacing. All the processed data were generated using the BDGP/Flybase release 4 of the D. melanogaster genome assembly (UCSC dm2, April 2004). To map mouse Oris [25], mouse samples were hybridized using the Nimblegen 389 K tiling arrays (Design ID 4095) which cover 60.4 Mb of non-repetitive DNA sequences in chromosome 11 (56.6–117 Mb). In total, 385,496 probes were analyzed with an average coverage of one 50 bp-probe each 100 bp. All the processed data were generated using the UCSC mm8 (NCBI Build 36, February 2006) of the Mus musculus genome assembly.
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2.2.2. Data normalization and determination of significant probes Experimental (Cy5) and control (Cy3) signal intensities quantified and provided by Nimblegen were converted into log2-ratios (log2 (Cy5/Cy3)). The Lowess normalization method was applied to eliminate intensity-dependent variations in dye bias [26]. A sliding median window with a length of 5 oligonucleotide probes was used to smooth the signal. Mode (m) and s (median absolute deviation) of normalized log2-ratios were computed by assuming that the normal distribution (specified by m and s) covered the entire background noise (non-significant signals). For each probe, one p-value was computed by applying the false discovery rate (FDR) correction [27]. We suggest analyzing three independent samples for each cell type used. Normalized log2-ratios of replicate samples were combined by averaging the values at the corresponding genomic positions and the corrected p-values were combined using a Chi-Square distribution [28]. Thus, one probe was denoted as significant if the combined p-value was lower than 5% (level of significance). 2.2.3. Origin definition Oris were scored positive only when multiple consecutive positive values were detected at a given site. As the minimum size of the purified nascent strands is 0.5 kb, potential Oris should be at least 1 kb (2 0.5 kb for a bidirectional origin). We defined Oris as regions that have at least one significant probe (p < 0.05 with FDR correction) in an area containing a minimum of 10 consecutive positive probes (showing NS enrichment with a log2-ratio >0). For Drosophila cells, two significant probes (because they are twice denser in Drosophila than in mouse Chips) and at least ten consecutive positive probes should be detected. If two enriched regions are separated by <1 kb, they are merged into one. These conditions are used to minimize false positive events by excluding overhybridization signals of single probes or small regions. 3. Concluding remarks Mapping DNA replication origins has been a difficult task that hampered our understanding of their nature. Until two years ago, only 30 amongst the 30,000–60,000 origins activated in each cell at each cell cycle were convincingly identified. Recent technical improvements allowed obtaining the first genome-wide data and the corresponding bioinformatics analyses. The RNA-primed NS purification method appears at present a reliable and precise technique. The main remaining problem is inherent to the nature of Oris. Indeed, eukaryotic Oris are in large excess along the DNA and only a minority (around 20%) is used at each cell cycle. There are also cell to cell variations in the same culture due to the flexibility of Ori usage, and activation. Therefore, Ori mapping means mapping all the potential Oris in a cell population, although only a fraction of them will be activated in a given cell of that population. It is likely that different (at least quantitative) patterns will be observed in cells with different behaviors, resulting in new Ori barcodes along the genome that characterize cell identity or cells under replication stress. Acknowledgments This work was supported by the European Research Council (ERC, FP7/2007-2013 Grant Agreement no. 233339’’, by the Agence Nationale de la Recherche (ANR), the Association pour la Recherche sur le Cancer (ARC) and the ‘Ligue Nationale Contre le Cancer’ (LNCC). References [1] M. Mechali, Nat. Rev. Mol. Cell Biol. 11 (2010) 728–738. [2] M. Rajewska, K. Wegrzyn, I. Konieczny, FEMS Microbiol Rev. 36 (2012) 408– 434.
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Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Genome-scale identification of active DNA replication origins Christelle Cayrou, Damien Grégoire 1, Philippe Coulombe, Etienne Danis 2, Marcel Méchali ⇑ Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, Montpellier 34396, France
a r t i c l e
i n f o
Article history: Available online 13 July 2012 Communicated by Kenneth Adolph Keywords: DNA replication origins Nascent strand purification Lambda-exonuclease Microarrays
a b s t r a c t Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps. Ó 2012 Published by Elsevier Inc.
1. Introduction At each cell cycle the entire genome of any living organism must be faithfully duplicated. In metazoans, DNA replication is initiated at thousands of chromosomal sites during each S phase. These DNA replication origins (Oris) should be activated only once at each cell cycle to avoid any amplification and to maintain genome integrity. In bacteria, yeast and viruses, Oris are rather well understood and are characterized by specific DNA sequence elements. In Metazoa, Oris occur at specific locations [1], and insights into their genetic characteristics begin to emerge. In bacteria, control of replication initiates by the binding of DnaA, the initiator of chromosome replication, to the DnaA boxes that form clusters of three or more elements and are located mainly in the E. Coli Ori [2]. The archaeal DNA replication machinery is a simplified form of the eukaryotic one [3]. The archaeal chromosome generally contains one Ori that has autonomously replicating sequence (ARS) activity, although some Archaea contains multiple Oris [4]. These Oris (termed ORBs) are 36 bp CG-rich sequences near an AT-rich region that might act as a melting-prone site. In budding yeast, origins are defined as ARSs on which initiation proteins are stepwise assembled. This is the only eukaryote in which ORC specifically recognizes a 17 bp AT-rich consensus sequence called ACS (for ARS Consensus Sequence) [5]. Although this motif is necessary for DNA replication, it is not sufficient for Ori function. In contrast to Saccharomyces cerevisiae, Saccharomyces ⇑ Corresponding author. Fax: +33 0434 359 920. E-mail address: [email protected] (M. Méchali). Present address: Institute of Molecular Genetics, 1919 Route de Mende, Montpellier 34293, France. 2 Present address: Department of Pharmacology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093–0636, USA. 1
1046-2023/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ymeth.2012.06.015
pombe Oris do not contain a core consensus sequence [5]. S. pombe Oris are AT-rich (from 0.5 to 3 kb in length) and contain several functionally important DNA sequence elements for their activity. In a genomic context, a 30 bp long poly-A/T tracks appears sufficient to specify replication initiation [6]. In metazoans, 30 000–60 000 Oris are activated at each cell division per cell. Oris appear to have variable features. For instance, they can be site-specific, as the human Lamin B2 or c-Myc Oris [7,8], or have a broad site specification, like the well characterized DHFR Ori [9]. Orc, a complex of 6 subunits binds to the replication origins in all known eukaryotic species. Its structure and role appears conserved, except its sequence specificity that is real in S. cerevisiae, but poor in other eukaryotes [10]. Until recently, only very few Oris were characterized in metazoans and their nature was elusive despite considerable efforts to unravel a replication origin code. Different genome-wide approaches have been developed to map Oris in metazoans. Three DNA methods have been mainly used: DNA combing, purification of replication intermediates by the bubble-trap method and nascent strands purification. The fourth one is a protein approach based on chromatin immunoprecipitation of initiation proteins. DNA combing allows visualizing replicated DNA molecules that have been stretched on silanized glasses (see Julien Bianco et al., this issue). This is a global method to determine the real spacing of Oris along DNA. However, it is not suitable to identify a given Ori DNA sequence due to the extremely low probability to detect such sequence while the replication fork is passing through the Ori. In practice, this method can work for very simple organisms or for sequences that are hundred-fold repeated in the genome. In addition, DNA combing is also limited by the DNA length that can be analyzed as it is hard to get molecules longer than 600– 800 Kb. A related method called SMARD has been developed and
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applied to the Igh locus after its purification by pulsed field gel electrophoresis [11], and recently to Nanog and Oct4 gene environment [12]. Purification of replication intermediates by the bubble-trap method [13,14] is based on the trapping of restriction fragments containing replication bubbles created at Oris. This is a good structure-based method although its resolution is limited by the size of the restriction fragments and the requirement that Oris are located preferentially at or near the center or the fragment. Chromatin IP of pre-replication complex proteins followed by identification of DNA by microarrays (ChIP-chip) or sequencing (ChIP-seq) is in principle an efficient method. It has been used in several cases to identify all potential Oris [15–18]. However, its major drawback is the low enrichment over the background. This might be partly because ORC, the main origin recognition complex, also recognizes other sites on chromosomes because of its implication in other nuclear processes [19]. Another drawback is that ORC binding to DNA is not sufficient to define an origin that is going to be activated [17]. Nevertheless, it might be improved in the future and can be a complementary additional method to the DNA-based methods (see Lubelsky et al., this issue). The RNA-primed nascent strand method is based on the purification of DNA that is first synthesized at Oris (see Fig. 1). These nascent DNA strands are primed by a 10–12 nucleotide RNA synthesized by the DNA polymerase-a primase. The purification procedure selects nascent DNA strands bigger than 400 bp to avoid the selection of Okasaki fragments synthesized at the ongoing replication forks all along the genome (see Fig. 1). This method was developed and successfully used in the nineties and quantification was carried out by competitive PCR [20]. A library of nascent DNA was also constructed [21]. The method was then significantly improved by the addition of a Lambda exonuclease digestion step [22,23] to eliminate any broken DNA fragment (RNA-primed nascent DNA strands are protected from digestion by their RNA primer). This is a crucial and obligatory step as even a very low amount of contaminating DNA can increase the background. Indeed, from 100 million exponentially growing cells (i.e., 600 lg total mouse or human DNA), no more than 20–40 ng of RNA-primed nascent DNA can be recovered in theory (0.003% of the total DNA), if no loss occurs during the purification procedure. In addition, exponentially growing cells must be used for genome-wide analysis as Oris are activated all along the S phase. Qualitatively, this method gives excellent replicates and is very precise. Quantitatively, it depends on the level and quality of the required amplification step owing to the low amount of recovered DNA. We have been upgrading this method to the level of purification and enrichment required for genome-scale analyses. By using this technique followed by identification of the sequences by microarrays, we could characterize 2412 Oris on chromosome 11 in mouse ES cells and 6184 Oris in the Drosophila genome and define their characteristics [24,25].
2. Description of the Method (Fig. 2A for a summary) 2.1. Nascent strands (NS) preparation 2.1.1. Day 1 2.1.1.1. DNA purification. 1. Dividing cells (1 108–2 108 = 2 150 mm dishes) are washed with PBS and pelleted in a 50 ml falcon tube. 2. Cells are lysed in 15 ml DNAzol (Molecular Research Center) at room temperature (RT) for 5 min. 3. 200 lg/ml proteinase K is added to the lysed sample in DNAzol and the tube is incubated on a rotating wheel at low speed at 37 °C for 2 h.
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To avoid contamination by nucleases and surrounding DNA, we used a set of pipettes exclusively reserved to the purification of nascent strands and filtered tips only. We found that combining the proteinase K treatment with DNAzol significantly improves the NS yield (Fig. 2B). 1. Insoluble material is discarded by centrifugation at 3,000 g at 4 °C for 15 min. 2. Genomic DNA present in the supernatant is precipitated by adding 15 ml 100% ethanol at RT for 5 min. 3. DNA is transferred to a new 15 ml falcon tube and washed with 5 ml 70% ethanol at RT for 5 min. DNA is collected by winding it around a drawn Pasteur pipette, to minimize DNA breaks. From this step, use only MaxyClear MAXYMum Recovery tubes (Low retention-tubes from Axygen). 1. DNA is transferred using a drawn Pasteur pipette in a new 2 ml dry tube and air-dried (at RT for 30 min). 2. DNA is resuspended in 2 ml TEN20 at 65 °C until completely dissolved (15–30 min). TEN20: 10 mM Tris, pH7.9 2 mM EDTA 20 mM NaCl 0.1% SDS 1000 U RNasin (New England BioLabs, NEB) 3. The DNA solution is boiled for 10–15 min and rapidly chilled on ice. 2.1.1.2. NS purification by sucrose gradient. 1. One or two ml of denatured genomic DNA are loaded onto a 30 ml neutral 5%–30% sucrose gradients prepared in TEN500 in ultraclear tubes. Use 1 gradient for NS from 100 million cells. TEN500: 10 mM Tris pH7.9 2 mM EDTA 500 mM NaCl 2. Gradients are centrifuged in a Beckman SW28 rotor at 25,000 RPM, at 4 °C, for 20 h. 2.1.2. Day 2 1. 1 ml-fractions are withdrawn from the top of the gradient using a wide-bore pipette tip and placed in new 1.5 ml MaxyClear tubes. 2. 50 ll of each fraction is run with the appropriate size markers on a 1.5% alkaline agarose gel at 40–50 volts, at 4 °C overnight. 2.1.3. Day 3 1. The gel is neutralized with 1x TBE and stained with Gel Red (Interchim). 2. Fractions corresponding to 0.5–1, 1–1.5 and 1.5–2 kb (Fig. 2C) are pooled separately and precipitated with 2.5 vol of 100% ethanol at 80 °C for 15–30 min. 3. Pellets are washed with 1 ml 70% ethanol and suspended in 50 ll water with 100 U RNasin (NEB). 2.1.3.1. Lambda exonuclease treatment. This is the most crucial step of the protocol. 1. After addition of 5 ll PNK reaction buffer (NEB), fractions are boiled for 5 min and chilled on ice.
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Fig. 1. Main principles of RNA-primed nascent strands isolation for mapping of replication origins. DNA synthesis starts at replication origins after the opening of DNA by a DNA helicase. DNA replication is bidirectional from the origin with the continuous synthesis of nascent strands in the 50 –30 direction on the leading strand, whereas the other strand is called the lagging strand and is copied in a discontinuous manner in short segments called Okazaki fragments. Short RNA primers initiate DNA synthesis at both the leading and lagging strands. After DNA extraction, DNA is denatured to isolate the newly synthesized DNA strands which are then fractionated by sucrose gradient centrifugation. The DNA size of each fraction is measured by agarose gel electrophoresis of samples. Fractions in the 0.5–2.5 Kb range are further treated by lambda exonuclease and first quantified by qPCR analysis before genome-scale analyses.
2. Phosphorylation with T4 PNK is performed in a volume of 100 ll at 37 °C for 1 h. T4 mix: Water to 200 ll final 10X PNK buffer (NEB) 1X ATP 50 nM final 10 U/ll T4 PNK (NEB) 40 U (i.e., 4 ll) 3. The reaction is stopped by adding 2 ll 0.5 M EDTA and samples are directly precipitated with 2.5 vol 100% ethanol/0.3 M Naacetate at 80 °C for 15–30 min.
4. Pellets are washed with 1 ml 70% ethanol and resuspended in 100 ll water with 100 U RNasin (NEB). 5. A sample after phosphorylation is digested with lambda exonuclease (L-exo) at 37 °C overnight. Lambda exo mix: Water to 200 ll final 10X L-exo buffer (Fermentas)1X 20 U/ll L-exo (Fermentas) 5 ll
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B
C
A
D
Fig. 2. RNA-primed nascent strands purification and quantification. (A) Red numbers refer to the protocol steps described in the article. 0.5–2.5 Kb nascent strands are isolated from total genomic DNA by denaturation and sucrose gradient centrifugation. RNA-primed nascent strands are enriched by lambda exonuclease digestion and hybridized against total genomic DNA on high-density tiling arrays. (B) Combining the proteinase K treatment with DNAzol significantly improves the yield of NS. (C) A sample of each fraction of the sucrose gradients was run with the appropriate size markers on a 1.5% alkaline agarose gel in order to identify the sucrose gradient fractions to be processed. (D) Two rounds of T4 PNK and lambda exonuclease treatment significantly improved the enrichment. (E) purification of the NS preparation on GFX does not affect significantly its yield.
We found that the quality of the lambda exonuclease is crucial and deserves to be always tested before use. For the experiments described here, we used a custom-made preparation by Fermentas (20 U/ll). 2.1.4. Day 4 1. NS are extracted once with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol, and precipitated after addition of 2 ll polyacryl carrier (Molecular Research Center Inc.) with 0.3 M Na-acetate (pH 5.3) 2.5 vol 100% ethanol at 80 °C for 15 min. 2. Pellets are washed with 1 ml 70% ethanol and suspended in 50 ll water with 100 U RNasin (NEB). 3. NS preparations are subjected to one or two additional cycles of T4 PNK phosphorylation and lambda exonuclease digestion (1.– 5.) in a final volume of 100 ll for each step. We observed that the second round of lambda exonuclease treatment significantly improves the NS preparations (Fig. 2D). Ideally, a third lambda exonuclease treatment should be performed, but the amount of recovered DNA might be greatly reduced. We found that two rounds are sufficient when an effective lambda exonuclease is used.
2.1.5. Day 5 1. Half of the obtained NS preparation is suspended in 50 ll 10 mM Tris pH8 and is immediately quantified by qPCR amplification of a known origin to monitor the enrichment of the last step. 2. If the qPCR quantification shows an enrichment >20, the whole remaining preparation is purified using the CyScribe GFX Purification Kit (GE Healthcare) and eluted in 50 ll water (Fig. 2E). These columns allow to purify efficiently the single-stranded DNA without affecting the level of enrichment of the nascent strands preparation (Fig. 2E). 1. Samples may be stored overnight at 4 °C or for longer time at 20 °C. Repeated freezing and thawing causes loss of single stranded nascent DNA. It is better to freeze each sample into multiple aliquotes to avoid this. We suggest testing each digestion step (from c1. to c9.) by qPCR amplification of a known origin (such as c-Myc) to monitor the NS enrichment of the preparation. One ll of samples per reaction is sufficient to quantify the level of enrichment. It is important to obtain at least a 20-fold enrichment for the tested origin by qPCR as the enrich-
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Fig. 3. Quality controls of NS preparations. (A) Nascent strands from cells that were serum-starved for 27 h and then switched, or not, to medium with serum for 24 h were analyzed. Cell growth at each step was followed by using the MTT cell proliferation assay. For each culture condition, 0.5–2.5 Kb nascent strands were isolated. (B) The nascent strand preparations were analyzed using two origins (HoxB9 and HoxB4, (2) by qPCR with different sets of primers localized along the HoxB locus. (C) Half of the DNA fraction isolated from exponentially growing cells was incubated with RNAse A before the first step of phosphorylation in order to digest the RNA primer that protects the nascent strands from lambda exonuclease digestion. Half of each fraction isolated from the sucrose gradient was treated by RNAse A (50 lg/ml, at 37 °C for 30 min). (D) The preparation was then analyzed using origins identified on mouse chromosome 15 by qPCR with different sets of primers localized along this region. (E and F) 1 lg of DNA plasmid that was digested with restriction enzymes was added to the nascent strand preparation to control the level of digestion by lambda exonuclease. The degradation of the plasmid DNA was followed after each lambda exonuclease digestion step by agarose gel electrophoresis (10% of the total fraction/well) and by qPCR for each fragment. (G) The nascent strands preparation was analyzed using the HoxB9 origin by qPCR with different sets of primers localized along the HoxB locus. Nascent strand enrichment for each step is shown.
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ment usually decreases when the analysis is then made after amplification for microarrays. Using c-Myc as a known DNA replication origin, we found reproducible enrichment values between 30 and 100fold by qPCR. While purifying NS from cycling cells, it is important to control the quality of the preparation by: - Purifying NS-like molecules from the same cells in G0 or G1 phase. This fraction should not show any enrichment (Fig. 3A and B). - Incubating half of the DNA fraction with RNase A before the first step of phosphorylation to digest the RNA primer that protects the nascent DNA strands from degradation by lambda exonuclease. Such fraction should not show enrichment as nascent DNA will be digested by the lambda exonuclease (Fig. 3C and D). - Adding to the DNA fraction 1 lg of digested plasmid to control the degree of digestion by lambda exonuclease. This may be followed by deposition of a sample on agarose gel and by qPCR (Fig. 3E–G). 2.2. NS amplification and chip data analysis 2.2.1. NS amplification with the WGA kit 1. 10 ll of purified NS are amplified using the WGAII kit (Sigma), omitting the first step of fragmentation. It is necessary to amplify several 10 ll aliquots of the preparation to obtain the amount required for microarray hybridization. Indeed, re-amplification of already amplified material gives unanalyzable results and should be strictly avoided. 1. Amplification products are purified with NucleoSpin columns (Machery Nagel). 2. Proper unbiased amplification is checked by qPCR. 3. Hybridization of the nascent DNA preparation, washing and scanning of microarrays was done in our case by Nimblegen Service Laboratory. We found that amplification may affect only the enrichment values. Therefore the microarray analyses should be considered as semiquantitative. 2.2.1.1. Microarray Design. Given the size of the fragments of purified nascent strands, we choosen to use tiling array chips that can cover the width of a potential origin by a minimum of 10 probes. Moreover, the Nimblegen procedure allows comparing on the same slide, two colors, from the sample and control DNA, and we found this to be a good approach. To characterize fly Oris [25], Drosophila melanogaster samples were hybridized using 2.1 M Nimblegen microarrays (Design ID 6262). These tiling arrays contain in total 2,164,511 oligonucleotide probes representing the non-repetitive regions of the D. melanogaster genome (chromosome 2L, 2R, 3L, 3R, 4 and X; Flybase release 4.3). To analyze the data, 1,807,015 oligonucleotide probes were selected (909,279 for the top strand and 897,736 for the bottom strand) with an average length of 50 bp for oligonucleotides and for inter-oligo spacing. All the processed data were generated using the BDGP/Flybase release 4 of the D. melanogaster genome assembly (UCSC dm2, April 2004). To map mouse Oris [25], mouse samples were hybridized using the Nimblegen 389 K tiling arrays (Design ID 4095) which cover 60.4 Mb of non-repetitive DNA sequences in chromosome 11 (56.6–117 Mb). In total, 385,496 probes were analyzed with an average coverage of one 50 bp-probe each 100 bp. All the processed data were generated using the UCSC mm8 (NCBI Build 36, February 2006) of the Mus musculus genome assembly.
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2.2.2. Data normalization and determination of significant probes Experimental (Cy5) and control (Cy3) signal intensities quantified and provided by Nimblegen were converted into log2-ratios (log2 (Cy5/Cy3)). The Lowess normalization method was applied to eliminate intensity-dependent variations in dye bias [26]. A sliding median window with a length of 5 oligonucleotide probes was used to smooth the signal. Mode (m) and s (median absolute deviation) of normalized log2-ratios were computed by assuming that the normal distribution (specified by m and s) covered the entire background noise (non-significant signals). For each probe, one p-value was computed by applying the false discovery rate (FDR) correction [27]. We suggest analyzing three independent samples for each cell type used. Normalized log2-ratios of replicate samples were combined by averaging the values at the corresponding genomic positions and the corrected p-values were combined using a Chi-Square distribution [28]. Thus, one probe was denoted as significant if the combined p-value was lower than 5% (level of significance). 2.2.3. Origin definition Oris were scored positive only when multiple consecutive positive values were detected at a given site. As the minimum size of the purified nascent strands is 0.5 kb, potential Oris should be at least 1 kb (2 0.5 kb for a bidirectional origin). We defined Oris as regions that have at least one significant probe (p < 0.05 with FDR correction) in an area containing a minimum of 10 consecutive positive probes (showing NS enrichment with a log2-ratio >0). For Drosophila cells, two significant probes (because they are twice denser in Drosophila than in mouse Chips) and at least ten consecutive positive probes should be detected. If two enriched regions are separated by <1 kb, they are merged into one. These conditions are used to minimize false positive events by excluding overhybridization signals of single probes or small regions. 3. Concluding remarks Mapping DNA replication origins has been a difficult task that hampered our understanding of their nature. Until two years ago, only 30 amongst the 30,000–60,000 origins activated in each cell at each cell cycle were convincingly identified. Recent technical improvements allowed obtaining the first genome-wide data and the corresponding bioinformatics analyses. The RNA-primed NS purification method appears at present a reliable and precise technique. The main remaining problem is inherent to the nature of Oris. Indeed, eukaryotic Oris are in large excess along the DNA and only a minority (around 20%) is used at each cell cycle. There are also cell to cell variations in the same culture due to the flexibility of Ori usage, and activation. Therefore, Ori mapping means mapping all the potential Oris in a cell population, although only a fraction of them will be activated in a given cell of that population. It is likely that different (at least quantitative) patterns will be observed in cells with different behaviors, resulting in new Ori barcodes along the genome that characterize cell identity or cells under replication stress. Acknowledgments This work was supported by the European Research Council (ERC, FP7/2007-2013 Grant Agreement no. 233339’’, by the Agence Nationale de la Recherche (ANR), the Association pour la Recherche sur le Cancer (ARC) and the ‘Ligue Nationale Contre le Cancer’ (LNCC). References [1] M. Mechali, Nat. Rev. Mol. Cell Biol. 11 (2010) 728–738. [2] M. Rajewska, K. Wegrzyn, I. Konieczny, FEMS Microbiol Rev. 36 (2012) 408– 434.
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