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Figure 2 | Cell & Bioscience

Figure 2

From: Thyroid hormone receptor actions on transcription in amphibia: The roles of histone modification and chromatin disruption

Figure 2

Transcriptional activation of the HIV-1 LTR by T 3 but not histone deacetylase inhibitor trichostain A (TSA) leads to chromatin disruption. (A) Disruption of the chromatin at the HIV LTR by liganded TR requires direct binding of TR to the LTR. Xenopus oocytes were injected with the mRNAs encoding RXR and TR or mutant TR lacking the DNA binding domain (TRΔDBD) followed by the injection of a reporter plasmid into the nucleus. The reporter plasmid contained the T3-dependent promoter, the long terminal repeat (LTR), of the human immunodeficiency virus type 1 (HIV-1), directing the transcription of the reporter. The oocytes were treated with T3. After overnight incubation, the reporter plasmid minichromosome was isolated from the oocytes for micrococcal nuclease (MNase) digestion assay with increasing amounts MNase. The digested DNA was purified and analyzed by Southern blot analysis with a labeled LTR probe. (B). DNA topology analysis demonstrates T3 but not TSA induces gross alterations of the structure of the LTR minichromosome. The oocytes were injected and treated with T3 or TSA and the LTR plasmid DNA was isolated for supercoiling assay. After electrophoresis on a chloroquine-containing gel to separate the DNA with different number of negative superhelical turns (the higher the negative superhelical turns, the slower the DNA migrated on the gel), the DNA was detected by Southern blot hybridization. Note that the average number of the negative superhelical turns (indicated by a star) was reduced by 2-3 when both TR/RXR and T3 are present. As each nucleosome on the circular plasmid generates one negative superhelical turn, the liganded TR induced a structural change equivalent to the loss of 2-3 nucleosomes on the minichromosome. In contrast, TSA had little effect on the number of superhelical turns on the plasmid (compare lanes 2 to 1). See [105, 127] for details.

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