Figure 2.

TSA induced chromatin domain opening precedes transgene transcriptional activation. (A) DNaseI sensitivity analysis of the lacZ transgene. HeLa cells were treated with 3 μM TSA for 0–24 hours as indicated. Nuclei were collected and digested with 0–36 units/ml DNaseI for 20 min at 37°C. Genomic DNA was digested with XbaI and XhoI and was analyzed by Southern blot hybridization with a lacZ probe. XbaI and XhoI digestion produced a 3.0-kb lacZ fragment from the integrated virally transduced gene. After 2 hours of treatment with the drug, the locus became sensitive to DNaseI digestion and remained sensitive for the entire 24-hour incubation. (B) Quantitation of DNaseI sensitivity at 0, 2, and 24 hours after TSA treatment. DNaseI sensitivity was calculated as follows: the intensity of bands at each DNaseI concentration was divided by the intensity of the control bands (zero DNaseI). This value was subtracted from 100% to yield % DNaseI sensitivity. (C) Measurement of lacZ transcription by nuclear run-on after TSA treatment. lacZ transcription was first detected 4 hours after treatment; relatively high levels of transcription were observed only after 6 hours. Globin gene transcription remained inactive after TSA treatment, and the constitutively expressed APRT gene was only slightly stimulated after 24 hours. (D) Comparison of DNaseI sensitivity and run-on transcription data of lacZ. Transcription was detected 2–4 hours after the transgene domain became sensitive to DNaseI digestion.