Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique

Abstract

Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS. Advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low-cost experiments with minimal need for specialist equipment. Presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells.

Figure 1.

(A) Nuclei are isolated from cells, and aliquots are digested with a range of DNaseI as detailed in ‘Materials and methods’ section. The DNA is extracted and run on 1% agarose gels using gel electrophoresis (right panel). With the example shown, the 40-units sample gave maximal enrichment at a housekeeping promoter using the complete protocol. (B) The digested DNA is blunt-ended with T4 polymerase and ligated to the LP21–25 linker as detailed in ‘Materials and methods’ section. Following amplification with the biotinylated LP25 primer, the extracted DNA template represents a library of whole-genome DHS. When samples of this library are visualised using gel electrophoresis (bottom panel), the majority of products are between 300 and 500 bp in size.

Figure 2.

(A) The left-hand panels show the Sybr-green real-time PCR profiles (fluorescence versus cycle number) of 5-fold dilutions of quantified mouse genomic DNA standards for two primer pairs within (ppA) and 3′ (ppB) of the Stil promoter. The right-hand panel shows that DNA template derived from the 40 units DNaseI-treated sample amplified nearly six PCR cycles in advance of the 0 DNaseI-treated sample with ppA. No difference in amplification kinetics was seen between the 0 and 40 samples with ppB. (B) Quantification of samples relative to genomic standards using primers for the ‘housekeeping’ Hmbs promoter shows maximal enrichment with 40 units DNaseI treatment for primary mouse thymocytes (left panel) compared with maximal enrichment with 120 units DNaseI for the mouse T-cell line, BW5147 (right panel).

Figure 3.

The upper panel shows a Southern blot DHS map of the human STIL promoter in K562 cells using a 3′ probe (grey box labelled ‘P’) to analyse the BglII-restricted fragment (cut −4 kb and +4.2 kb relative to the STIL transcription start site). This revealed a core DHS ∼500 bp wide. The lower panel represents the mean ± SD real-time PCR quantification of enrichments from three biological replicates of K562 material relative to known genomic standards, using 10 separate primer sets that together amplify ∼1200 bp centred around the STIL transcription start site. The black bar represents the approximate location of the DHS, as identified by the Southern blot experiment in the upper panel. The real-time PCR amplification profile closely represents the Southern blot profile, although a central dip in real-time PCR signal is observed over the site of transcription factor binding.

Figure 4.

(A) Quantitative reverse transcriptase PCR quantification of STIL and TAL1 expression in hES and K562 cells. Expression was normalised to β-actin and plotted as detailed in ‘Materials and methods’ section. Both cell types express STIL, but TAL1 transcripts were not detectable (U) in hES cells. (B) Quantitative real-time PCR DNaseI profiles from hES and K562 cells using nine primer sets across the TAL1 locus. Primer set numbers refer to approximate kilobases (kb) relative to the start of TAL1 exon 1a. Primer sets corresponding to known regulatory elements are highlighted in red. Profiles represent the mean ± SD enrichment of material derived from two independent hES cell biological replicates, and two independent K562 cell cultures. Each bar represents the mean ratio of enrichment obtained from DNaseI-treated samples versus DNaseI-untreated samples for each cell type. With both cell types, no enrichment is seen at the four control sites (−16, +5, +22, +70 kb). K562 cells show maximal enrichments at the TAL1 regulatory elements and the STIL promoter, while hES cells show the highest enrichments at the STIL promoter and +50 enhancer, with minimal enrichment at the other TAL1 regulatory elements.