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.
Highlights
Chromatin remodeling is a characteristic of cell lineage specification and cellular response to external stimulation
One may need to know the changes in DNase I-hypersensitive (DHS) sites at a particular gene locus identified by the genome-wide approaches in response to different experimental treatments
For detecting DHS sites the nuclei should only be partially digested, so even in the DNase Idigested sample the majority of genomic DNA at a DHS site is not cut by DNase I
Summary
Chromatin remodeling is a characteristic of cell lineage specification and cellular response to external stimulation During these processes, the cells adopt an ‘‘open’’ structure at certain chromatin regions. One may need to know the changes in DHS sites at a particular gene locus identified by the genome-wide approaches in response to different experimental treatments. One may wish to determine whether certain DHS sites identified in one cell type by the genome-wide approaches are present in other cell types. For such studies, whole-genome analysis may be cost inhibitory and unnecessary. A simple reliable method is described for unambiguous determination of DHS sites in rare populations of cells
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