Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse beta-globin LCR.
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 beta-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.
McArthur
M
M
Division of Medical Genetics, University of Washington, Seattle, 98195, USA.
Gerum
S
S
Stamatoyannopoulos
G
G
D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D008099:Liver / Q000196:embryology / Q000378:metabolism; D019901:Locus Control Region / Q000235:genetics*; D051379:Mice; D023282:Nuclease Protection Assays / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D002843:Chromatin; D005914:Globins; D004247:DNA; D003850:Deoxyribonuclease I