Nase I Hypersensitive Site Assay of Chromatin Assembled in Xenopus Oocytes

DNase I hypersensitive site assay is perhaps the most widely used assay for detection of changes in chromatin structure. Studies of many spatially or temporally regulated genes, such as P-globin, have revealed the correlation of formation of DNase I hypersensitive sites with the status of transcription (22). This assay has also been used extensively in analyses of chromatin structure in injected Xenopus oocytes (24). Although the mechanism for the formation of DNase I hypersensitive sites is still not fully understood, the detection of DNase I hypersensitive sites are widely interpreted as results of chromatin remodeling induced by the binding of transcription factors.

1. Inject 15-20 oocytes with DNA and with or without mRNA encoding TR/RXR (100 ng/ pL, 27.6 nL/oocyte) and treated with or without 50 nM T3. Incubate the oocytes overnight at 18°C incubator.

2. Collect healthy oocytes and wash the oocytes once with 500 pL of MBSH buffer.

3. To 15-20 oocytes, add 240 pL of DNase I buffer. Homogenize the oocytes by pipeting.

4. Distribute 60 pL of the lysates to four tubes labeled as A, B, C, and D, respectively. Dilute DNase I (Gibco-BRL, 133.9 U/ pL) 1/3 and1/9 in TE buffer right before use. Add 0.5 pL of undiluted DNase I to tube A, 1/3 diluted DNase I to B, and 1/9 diluted DNase I to C. No DNase I is added to tube D which will serve as a no-digestion control. Incubate at room temperature for 10 min.

5. Stop DNase I digestion by addition of 200 pL of DNase I stop buffer. Add 1 pL of RNase A (10 mg/mL ). Incubate at 37°C for 1 h.

6. Add 3 pL of proteinase K (10 mg/mL) to each tube. Incubate at 55°C for 2 to 3 h.

7. Add 30 pL of NaOAC, pH 5.4 to each tube. Extract with 300 pL of phenolchloroform once. Carefully transfer the supernatant to a new tube and extract with chloroform once more. Precipitate DNA by addition of 0.7 V of isopro-panol. Wash with 70% ethanol once.

8. Resuspend DNA in 43 pL of water. Add 2 pL of the appropriate restriction enzyme (for indirect end-labeling) and 5 pL of 10X restriction buffer according to the restriction enzyme used for digestion. Incubate at 37°C for at least 3 h to overnight.

9. Resolve DNA samples with a 1-1.2% agarose gel in 1X TBE. Transfer DNA to a Nylon membrane and carry out the standard Southern blotting using the probe one end at the restriction site and the other end within the suspected hypersensitive sites (Note 13).

Was this article helpful?

0 0

Post a comment