- Methodology article
- Open Access
Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach
© Ohnesorg et al; licensee BioMed Central Ltd. 2009
- Received: 15 January 2009
- Accepted: 4 September 2009
- Published: 4 September 2009
Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.
We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 × 104 cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.
This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 105 cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.
- HeLa Cell
- Hypersensitive Site
- Encode Project
- Normalize Peak Height
- Encode Consortium
Open chromatin is a characteristic of genomic loci with regulatory functions. These regions are preferentially digested by DNaseI , and the identification of DNaseI hypersensitive sites is frequently used to identify and analyse regulatory regions such as promoters, enhancers and silencers [2, 3]. However, currently available methods have significant limitations. A commonly used approach involves Southern blotting, but this is time consuming, usually requires radioactivity and is limited to short stretches of DNA. Several PCR-based methods have been described [4, 5], but these do not readily allow multiplexing. Recent reports of large scale analysis of DNaseI hypersensitive sites have used either microarrays [6–9] or deep sequencing [9, 10]. Whilst valuable for genome wide analysis, the costs involved are a limiting factor for many applications (such as comparing different developmental stages or tissues). Another disadvantage of those methods is that they usually require many millions of cells. For ex vivo studies, this might require extensive pooling of tissues, meaning that these methods are not suitable for all applications.
Multiplex Ligation-dependent Probe Amplification (MLPA) was originally developed to detect deletions and duplications in genomic DNA , and has become popular in diagnostic settings for a range of disorders [12, 13]. It has since been modified for several other applications as well, including methylation analysis , mRNA expression analysis , identifying copy number variation in normal populations [16, 17], genotyping of mouse models [18, 19] and measuring the efficiency of Cre-mediated recombination in mouse models . The principle advantages of this method are the sensitivity and multiplexing potential. It can be used to analyse up to 50 genomic loci with as little as 20 ng genomic DNA in a single reaction. Furthermore, the only equipment that is required is a thermocycler and DNA sequencer, readily available to most researchers.
We describe here a quick and straightforward protocol for analysing DNaseI hypersensitive sites. This is demonstrated by the analysis of 20 different loci in a single reaction, based on data published by the ENCODE consortium .
Nuclei isolation and DNaseI treatment
Analysis of DNaseI hypersensitive sites in HeLa cells
We also tested the same probe mix on DNA directly isolated from HeLa cells. As can be seen in Figure 3C, there are no significant changes in normalized peak heights with increasing DNaseI digestion. This was expected as these DNA samples should have no chromatin structure, and all cuts by DNaseI should therefore occur in a random fashion.
Comparison of results using other cell lines
Determining the minimum cell number
We describe here a simple technique that allows the rapid analysis of many DNaseI hypersensitive sites using little starting material. For a proof of principle we chose loci that had been analysed by both array analysis and deep sequencing as part of the ENCODE project .
We used 20 probes in one reaction (11 sensitive, 9 non-sensitive in HeLa cells according to ENCODE data). Nine of 11 of the probes targeted to hypersensitive sites showed the expected decrease in peak height for HeLa cells. The fact that two sensitive sites did not show the expected drop in peak heights is probably due to different growth conditions, differences in passage numbers or to our batch of HeLa cells being different from the one used in the ENCODE study rather than to limitations of our method. This is supported by the high correlation observed for both technical and biological replicates (r2 > 0.9) and the fact that those two probes showed sensitivity in HEK293 cells.
We also examined three other cell lines, in order to examine the cell line specificity of our experimental approach. We could show clear differences in hypersensitivity for some loci compared to HeLa cells. Again, the conclusion that these are cell type-specific differences is supported by the concordance of replicate experiments (r2 > 0.9).
To determine suitable DNaseI digestion conditions for our method, a range of different DNaseI concentrations and incubation conditions were tested. Incubating the digestion reactions with the indicated DNaseI amounts at room temperature for 20 minutes gave more reproducible results than, for example, using less DNaseI and incubating at 37°C or incubations on ice (data not shown). By analysing the digested DNA on an agarose gel it was possible to identify the most appropriate conditions. Most of our experiments were carried out using nuclei from 2.5 × 105 cells per DNaseI digestion aliquot, providing sufficient DNA for several technical replicates. However, in the case of HeLa cells this number could be reduced to only 5 × 104 cells, still providing highly reproducible results as shown by an r2 of 0.97 when comparing the results of the experiments with both cell numbers. Furthermore, we found that two different DNaseI concentrations and an undigested control are sufficient to analyse hypersensitive sites, which reduces the amount of total starting material required to about 1.5 × 105 cells.
Although we have used 20 probes in this study, there is the potential to significantly increase the degree of multiplexing. Reducing the interprobe spacing to 3 bp would allow up to 40 loci to be examined. Indeed, by designing probes that can be labelled with different fluorophores [22, 23] it would be possible to analyse > 100 loci in a single reaction.
We describe here a rapid and accurate method for assaying DNaseI hypersensitive sites. In contrast to genome-wide approaches such as deep sequencing or microarray analysis, we consider the primary strength of this approach to be when < 100 genomic loci are being analysed. As all loci are analysed in a single reaction, relatively little starting material is required. We have shown that < 105 cells per DNaseI treatment can be used, which will allow the study of e.g. embryonic organ development without extensive pooling. The protocol is straightforward and results can be easily obtained from many samples within 48 hours. Finally, using this method makes it possible to also detect heterozygous and homozygous deletions and duplications of the examined regions, which is an artifact that is known to occur in cultured cells.
HEK293 (human embryonic kidney cells), C28 (chondrogenic cells), HeLa (cervical cancer cells) and S97 (dermal fibroblasts) were grown in DMEM containing 10% FBS and supplemented with L-glutamine in T-25 flasks and incubated at 37°C containing 5% CO2 in a humidified atmosphere.
Cell harvest and isolation of nuclei
After reaching 95-100% of confluence, cells were washed with PBS and harvested using 1 ml of 0.025% Trypsin-EDTA. After 5-10 min incubation at 37°C, cells were washed with 2 ml of 10% FBS containing DMEM and carefully resuspended in a 1.5 ml Eppendorf tube in cold PBS and placed on ice. To limit changes in chromatin structure during treatment, nuclei isolation was performed using 2.5 × 105 to 106 cells as soon as possible.
For isolation of nuclei, the NE-PER Kit (Thermo Scientific) was used with the following modifications. To prevent nuclei from excessive clumping and releasing DNA, twice the recommended volumes of the solutions were used and resuspension of cells/nuclei was carried out by carefully pipetting up and down rather than vortexing. Centrifugation steps were carried out at 250 × g at 4°C.
DNaseI treatment of nuclei
Isolated nuclei were washed in 500 μl cold DNaseI buffer containing 2% glycerol and carefully resuspended in 75 μl DNaseI buffer containing 2% glycerol. 25 μl aliquots of the nuclei suspension were added to 2 ml Eppendorf tubes containing one control without DNaseI and increasing amounts (0.5 - 2 units) of DNaseI (Promega) in 50 μl DNaseI buffer containing 2% glycerol. The solutions were mixed by carefully flicking the tubes and incubated for 20 min at room temperature (23°C).
Isolation and purification of DNaseI treated genomic DNA
DNaseI treated nuclei were lysed by adding 250 μl of nuclei lysis buffer (200 mM NaCl, 150 mM Tris HCl pH 8, 10 mM EDTA pH 8, 0.2% SDS) containing 50 μg of Proteinase K and incubated for 45 min at 55°C. 20 μg of RNaseA (Sigma) was added and incubated for 30 min at 37°C.
The DNaseI treated DNA was isolated and purified using the HighPure PCR Purification Kit (Roche) according to the manufacturer's instructions. DNA was eluted with 50 μl elution buffer provided with the kit. Purified DNA was checked for quality and degree of DNaseI digestion by agarose gel electrophoresis. DNA concentrations were measured using the NanoDrop.
MLPA and fragment analysis
Probe sets used in this study.
Fragment analysis was performed on an ABI3130 capillary sequencer. Peak data was extracted using GeneMarker software (Soft Genetics) and exported to Excel (Microsoft) for further analysis.
MLPA data analysis
Basic data analysis was performed as described . Peak data for the naked DNA experiment was normalised to the average of all probes in the examined samples, with all other reactions being normalized to all non-sensitive probes. To assist in visualisation of the results, normalized ratios of all untreated samples were set to 1.
We thank Dr. Craig Smith for providing HeLa and HEK293 cells, Dr. Peter Kannu for providing C28 cells and Dr. Laura Zamurs for providing S97 cells. This work was supported by the National Health and Medical Research Council [491293; 546478 S.W., 334314 A.S.].
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