Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

Abstract

DNase I hypersensitive (DHS) sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×104 cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.

Unveiling the gene regulatory landscape in diseases through the identification of DNase I‑hypersensitive sites (Review)

“…Studies on DHSs help to disclose delicate gene regulation mechanisms and enable extensive annotation of the genome. The genome-wide mapping of DHSs provides a novel platform for the promising investigation of a specific molecular biological problems affected by the regulation of a given gene or a group of genes (17).…”

“…DHSs overlie CREs with parallel degrees of nuclease sensitivity and cover the main sequence of regulatory factor (42). DHSs usually contain CREs related to transcriptional activation on the reporter locus, such as enhancers, but can also contain transcription inhibition, such as silencers (17). A DHS map reveals the state and pattern of the presence of CREs, in addition to the variable and plastic states of chromatin in various cell types (25,29,33,43).…”

“…In mammalian cells, >3% of the genome is found to be DNase I-hypersensitive (17). To date, 290,000,000 DHSs have been recognized.…”