Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse β-globin LCR 1 1Edited by J. Karn

Abstract

We employ real-time PCR to allow us to quantify the sensitivity of chromatin to digestion by DNaseI. This approach has three clear advantages over the more conventional use of the Southern hybridization assay: the accuracy of quantification is improved; the resolution of the assay is enhanced, by designing primers to amplify small amplicons it is possible to analyze sequences both co-incident and proximal to sites of DNaseI-hypersensitivity; less material is needed, as little as 5 ng of treated genomic DNA. We applied this method in an analysis of the chromatin structure of the previously described mouse β-globin locus control region (LCR) using fetal liver cells. The four hypersensitive sites of the canonical mouse LCR, HS1 to HS4, are shown to have kinetics of digestion consistent with these sequences being nucleosome-free in vivo. A different pattern was seen for HS6, a recently described "weak" hypersensitive site. The site was also rapidly lost but more of the sites proved resistant, we interpreted this to show that this hypersensitive was only forming in a portion of the erythroid cells. This finding implies that in vivo the LCR is structurally heterogeneous. Sequences proximal to the hypersensitive sites show a third pattern of intermediate sensitivity, consistent with the chromatin being unfolded but the sites still bound by a continual nucleosomal array. Our results demonstrate that this method has the potential to achieve accurate and detailed mapping of chromatin structure from small amounts of tissue samples.

A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer

“…It has been reported that DNase I sensitivity can spread around HS sites (McArthur et al 2001). Moreover, as mentioned earlier, transcription factor-binding sites exist in the region between −1000 and −500 bp that may induce intermediate DNase I sensitivity (McArthur et al 2001). Interestingly, analysis of the conserved region downstream from CCND1 revealed a cell-type-specific HS site, which was present in MCF7 and HepG2 but not in HeLa or MDA-MB-231 cells (Fig.…”

“…The surrounding regions showed intermediate sensitivity when compared with the transcribed region (+8000 bp), which was relatively DNase I-resistant as expected. It has been reported that DNase I sensitivity can spread around HS sites (McArthur et al 2001). Moreover, as mentioned earlier, transcription factor-binding sites exist in the region between −1000 and −500 bp that may induce intermediate DNase I sensitivity (McArthur et al 2001).…”

“…To robustly quantify the DNase I sensitivity we used real-time PCR with primers designed to generate very small amplicons (<110 bp) tiled along these regions. This approach has been described and validated recently on a large-scale analysis (McArthur et al 2001;Dorschner et al 2004). DNase I HS site size ranges from 200 bp to 1 kb (Gross and Garrard 1988).…”

Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements

“…DHS assays identify short regions of approximately 200-400 bp that are sensitive to the action of nucleases because of nucleosome displacement or altered chromatin structure. These nuclease-sensitive regions are often the binding sites of transcription factors and are likely to be cis-acting regulatory elements, such as promoters and enhancers (Elgin 1988;McArthur et al 2001). Many genes regulated in a tissue-or developmental-specific fashion show changes in DNase I sensitivity coincident with transcriptional induction (Stamatoyannopoulos et al 1995), and several studies have demonstrated a functional correlation between DHS and transcription regulatory elements (Urnov 2003).…”

High-throughput localization of functional elements by quantitative chromatin profiling

“…Such differences can be readily detected using replicate quantitative real-time PCR measurements, which may be performed in a high-throughput format. The feasibility of using this protocol to measure the kinetics of DNase I digestion at known hypersensitive sites has been demonstrated previously 8 . However, accurate de novo localization of DNase I hypersensitive sites against a moving baseline of in vivo DNase I sensitivity presents a considerably greater challenge requiring the development of a quantitative methodology as outlined above.…”