Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Kruppel-associated box protein
Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Kruppel-associated box protein
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DOI：
10.1128/mcb.21.3.928-939.2001
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发表时间：
2001-02-01
影响因子：
5.3
通讯作者：
Urrutia, R
中科院分区：
文献类型：
--
作者：
Gebelein, B;Urrutia, R
脊椎动物基因组包含大量与Kruppel相关的框锌指基因，这些基因编码10个或更多C-2-H-2锌指基序。这个基因家族的成员被认为是转录因子，通过其锌指区与DNA结合，并通过KRAB结构域抑制基因表达。然而，到目前为止，还没有Kruppel相关的盒锌指蛋白(KRAB-ZFP)和具有10个或更多锌指基序的蛋白质以序列特异性的方式与DNA结合。我们实验室最近鉴定出KS1是KRAB-ZFP家族的成员，它包含10个不同的C-2-H-2锌指基序，其中9个聚集在C末端，另外一个锌指被一个短连接区隔开。在这项研究中，我们使用随机寡核苷酸结合实验来鉴定一个27个碱基的KS1结合元件(KBE)。报告分析表明，KS1抑制了含有该DNA序列的启动子的表达。缺失和定点突变表明，KS1需要9个C端锌指和KRAB结构域通过KBE位点进行转录抑制，而分离的锌指和连接区是这一功能所必需的。另外的生化分析表明，KS1KRAB结构域与KAP-1辅阻遏子相互作用，而取消这种相互作用的突变减轻了KS1介导的转录抑制。因此，这项研究首次提供了KRAB-ZFP与DNA结合以调节基因表达的直接证据，并为多锌指蛋白识别DNA序列的机制提供了深入的了解。
The vertebrate genome contains a large number of Kruppel-associated box-zinc finger genes that encode 10 or more C-2-H-2 zinc finger motifs. Members of this gene family have been proposed to function as transcription factors by binding DNA through their zinc finger region and repressing gene expression via the KRAB domain. To date, however, no Kruppel-associated box-zinc finger protein (KRAB-ZFP) and few proteins with 10 or more zinc finger motifs have been shown to bind DNA in a sequence-specific manner. Our laboratory has recently identified KS1, a member of the KRAB-ZFP family that contains 10 different C-2-H-2 zinc finger motifs, 9 clustered at the C terminus with an additional zinc finger separated by a short linker region. In this study, we used a random oligonucleotide binding assay to identify a 27-bp KS1 binding element (KBE). Reporter assays demonstrate that KS1 represses the expression of promoters containing this DNA sequence. Deletion and site-directed mutagenesis reveal that KS1 requires nine C-terminal zinc fingers and the KRAB domain for transcriptional repression through the KBE site, whereas the isolated zinc finger and linker region are dispensable for this function. Additional biochemical assays demonstrate that the KS1 KRAB domain interacts with the KAP-1 corepressor, and mutations that abolish this interaction alleviate KS1-mediated transcriptional repression. Thus, this study provides the first direct evidence that a KRAB-ZFP binds DNA to regulate gene expression and provides insight into the mechanisms used by multiple-zinc-finger proteins to recognize DNA sequences.