Kaludov N K, Wolffe A P
Laboratory of Molecular Embryology, National Institute of Child Heath and Human Development, NIH, Building 18T, Room 106, Bethesda, MD 20892-5431, USA.
Nucleic Acids Res. 2000 May 1;28(9):1921-8. doi: 10.1093/nar/28.9.1921.
The pathways for selective transcriptional repression of methylated DNA templates by the methyl-CpG-binding protein MeCP2 have been investigated using a purified in vitro transcription system that does not assemble chromatin. MeCP2 selectively inhibits transcription complex assembly on methylated DNA but does not destabilize a pre-assembled transcription complex. MeCP2 functions to repress transcription at a distance of >500 bp from the transcription start site. The transcription repression domain (TRD) of MeCP2 will repress transcription in vitro when fused to a heterologous Gal4 DNA-binding domain. The TRD associates with TFIIB. Exogenous TFIIB does not relieve transcriptional repression established by either intact MeCP2 or a Gal4-TRD fusion protein under these in vitro conditions, nor does the addition of histone deacetylase inhibitors. We find that the transcriptional repression established by both MeCP2 and the Gal4-TRD fusion protein in vitro also correlates with selective assembly of large nucleoprotein complexes. The formation of such complexes reflects a local concentration of DNA-bound transcriptional repressor that may stabilize a state of repression even in the presence of exogenous transcriptional machinery.
利用一种不组装染色质的纯化体外转录系统，对甲基化CpG结合蛋白MeCP2选择性转录抑制甲基化DNA模板的途径进行了研究。MeCP2选择性抑制转录复合物在甲基化DNA上的组装，但不会使预先组装好的转录复合物不稳定。MeCP2在距转录起始位点>500 bp的距离处发挥抑制转录的作用。当与异源Gal4 DNA结合结构域融合时，MeCP2的转录抑制结构域（TRD）将在体外抑制转录。TRD与TFIIB相互作用。在这些体外条件下，外源性TFIIB不能解除由完整的MeCP2或Gal4-TRD融合蛋白建立的转录抑制，添加组蛋白脱乙酰酶抑制剂也不能解除。我们发现，MeCP2和Gal4-TRD融合蛋白在体外建立的转录抑制也与大型核蛋白复合物的选择性组装相关。这种复合物的形成反映了与DNA结合的转录抑制因子的局部浓度，即使在存在外源性转录机制的情况下，也可能稳定抑制状态。
