Balakrishnan Lata, Milavetz Barry
Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, ND 58203, USA.
J Mol Biol. 2007 Aug 24;371(4):1022-37. doi: 10.1016/j.jmb.2007.06.080. Epub 2007 Jul 3.
The effects of the RNA polymerase II (RNAPII) translocation inhibitors alpha amanitin and 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) and an siRNA targeting p300 on the presence of RNAPII, p300, hyperacetylated H4 and H3 and unmodified H4 and H3 in transcribing simian virus 40 (SV40) minichromosomes were determined. Following treatment with alpha amanitin we observed a profound reduction in the occupancy of the promoter by RNAPII, the loss of p300 from chromatin fragments containing RNAPII, and an increase in the amount of unmodified H4 and H3 associated with the RNAPII. Treatment with DRB had little effect on the presence of RNAPII or p300 but also resulted in a significant increase in the amount of unmodified H4 and H3 present in chromatin fragments associated with RNAPII. Following treatment with a p300 small interfering RNA (siRNA), we observed a significant decrease in late transcription and a corresponding reduction in the amounts of p300 and hyperacetylated histones associated with the transcribing SV40 minichromosomes. We conclude that in transcribing SV40 minichromosomes histone hyperacetylation and deacetylation is dependent upon the presence of p300 and an as yet unknown histone deacetylase associated with the RNAPII complex that occurs coordinately as the RNAPII complex moves through a nucleosome.
测定了RNA聚合酶II（RNAPII）易位抑制剂α鹅膏蕈碱和5,6-二氯-1-β-D-核糖苯并咪唑（DRB）以及靶向p300的小干扰RNA（siRNA）对转录猴病毒40（SV40）微型染色体中RNAPII、p300、高乙酰化H4和H3以及未修饰H4和H3存在情况的影响。用α鹅膏蕈碱处理后，我们观察到RNAPII对启动子的占据显著减少，含有RNAPII的染色质片段中p300缺失，以及与RNAPII相关的未修饰H4和H3的量增加。用DRB处理对RNAPII或p300的存在影响不大，但也导致与RNAPII相关的染色质片段中未修饰H4和H3的量显著增加。用p300小干扰RNA（siRNA）处理后，我们观察到晚期转录显著减少，并且与转录的SV40微型染色体相关的p300和高乙酰化组蛋白的量相应减少。我们得出结论，在转录SV40微型染色体时，组蛋白的高乙酰化和去乙酰化取决于p300以及与RNAPII复合物相关的一种未知组蛋白去乙酰化酶的存在，这种情况在RNAPII复合物穿过核小体时协同发生。
