The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection
The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection
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DOI：
10.1128/jvi.01588-08
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发表时间：
2009-03-15
影响因子：
5.4
通讯作者：
Levin, Henry L.
中科院分区：
文献类型：
--
作者：
Chatterjee, Atreyi Ghatak;Leem, Young Eun;Levin, Henry L.
裂糖酵母（Schizosaccharomyces pombe）的长末端重复（LTR）反转录转座子Tf1特异地整合到pol ii转录基因的启动子中。其整合酶（IN）包含一个与组蛋白H3的n端尾部结合的c端染色体结构域相关的染色体结构域。虽然我们无法检测到组蛋白尾部与Tf1 IN的色域之间的相互作用，但色域可能在引导IN到达目标位点方面发挥作用。为了验证这一想法，我们在染色体结构域高度保守的残基上产生了单氨基酸取代的转座子，并创建了一个染色体结构域缺失的突变体。突变V1290A、Y1292A、W1305A和CHD Delta在体内显著降低了转位活性。印迹分析显示，in或cDNA水平几乎没有或没有降低。通过测量cDNA与Tf1的质粒拷贝之间的同源重组，我们发现其中两个突变没有减少cDNA进入细胞核的输入，而另一个突变导致了33%的减少。染色质免疫沉淀实验显示，CHD δ导致in与cDNA下游LTR的结合减少了大约三倍。这些数据表明，色域直接促成了整合。因此，我们测试了色域是否有助于选择插入位点。靶质粒分析结果表明，染色体结构域的缺失导致对pol II启动子的偏好急剧降低。总的来说，这些数据表明，染色体结构域促进cDNA的结合，并在有效靶向中发挥关键作用。
The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHD Delta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHD Delta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.