KRAB can repress lentivirus proviral transcription independently of integration site
KRAB can repress lentivirus proviral transcription independently of integration site
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DOI：
10.1074/jbc.m602843200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Trono, Didier
中科院分区：
文献类型：
--
作者：
Bulliard, Yannick;Wiznerowicz, Maciej;Trono, Didier
KRAB转录抑制结构域，通常存在于锌指蛋白中，通过诱导异染色质的形成起作用。我们以前利用这一特性，实现药物调控的转基因和敲低结合强力霉素可控KRAB的融合蛋白和慢病毒载体。在这里，我们询问KRAB诱导的抑制是广泛的还是仅限于基因组的特定区域。为此，我们用慢病毒载体转导细胞，所述慢病毒载体表达靶报告基因和来自双顺反子mRNA的含KRAB的转录阻遏物。我们发现大约1.4%的前病毒逃脱了抑制。然而，这种表型可以通过反式表达含KRAB的蛋白质来逆转。因此，在编码含KRAB的效应子或其上游内部核糖体进入位点的DNA序列中，不可阻遏的前病毒都含有突变或缺失，这些突变或缺失可能是由逆转录过程中的错误或重组引起的。这些结果表明，KRAB诱导的转录抑制在各种基因组背景下是稳健和活跃的，所述基因组背景至少包括慢病毒整合靶向的广泛范围的位点。
The KRAB transcriptional repressor domain, commonly found in zinc finger proteins, acts by inducing the formation of heterochromatin. We previously exploited this property to achieve drug-regulated transgenesis and knock down by combining doxycycline-controllable KRAB-containing fusion proteins and lentiviral vectors. Here, we asked whether KRAB-induced repression is widespread or limited to specific regions of the genome. For this, we transduced cells with a lentiviral vector expressing a target reporter and a KRAB-containing transcriptional repressor from a bicistronic mRNA. We found that similar to 1.4% of the resulting proviruses escaped repression. However, this phenotype could be reverted by expressing the KRAB-containing protein in trans. Accordingly, the irrepressible proviruses all contained, in the DNA sequence encoding the KRAB-containing effector or its upstream internal ribosomal entry site, mutations or deletions likely resulting from errors or recombination during reverse transcription. These results indicate that KRAB-induced transcriptional repression is robust and active over a variety of genomic contexts that include at least the wide range of sites targeted by lentiviral integration.