BACKGROUND ZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the "domain of unknown function 3669" (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson's disease. RESULTS Here we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669's impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved "MLE" sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein. CONCLUSION DUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

中文翻译：

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DUF3669是ZNF746和ZNF777中的“未知功能域”，寡聚并有助于转录抑制。

背景技术ZNF746和ZNF777属于大的克鲁勃关联盒（KRAB）锌指（ZNF）转录因子家族的子集。像人类中的其他四个成员一样，它们包含一个额外的保守域，即“功能未知的域3669”（DUF3669）。先前对该亚家族成员的研究表明，参与转录调控和异常ZNF746过表达导致帕金森氏病中神经元细胞死亡。结果在这里，我们证明了ZNF746a主要同工型和ZNF777的N末端蛋白区段协同起作用，发挥了适度的转录抑制活性。完全效力取决于完整构型，该构型由DUF3669，变异KRAB结构域和相邻序列组成。虽然DUF3669具有内在的弱抑制活性，分离的KRAB-AB域没有抑制。重要的是，DUF3669提供了一种新型的蛋白质-蛋白质相互作用界面，并介导了低聚物中亚家族成员之间的直接物理相互作用。外显子5和6编码的ZNF746蛋白片段增强了阻遏子的效价，这可能是由于在K189处存在用于进行磺酰化的受体赖氨酸的缘故。有效的规范ZNF10 KRAB域的阻遏物活性并未因DUF3669的异源转移而增强，这表明背景对于DUF3669对转录的影响非常重要。ZNF746a和ZNF777蛋白片段均未与TRIM28稳定地结合在细胞内。与主要同工型不同，包含全长KRAB-A子域的同工型ZNF746b显示出显着提高的阻遏物效力。这种增加是由于KRAB域已知的规范机制引起的，因为它不在TRIM28和SETDB1的HAP1敲除模型中发生。符合KRAB-A内的真正保守的“ MLE”序列的甘氨酸到谷氨酸的替代导致阻遏物效力的进一步强劲提高，达到了与规范ZNF10 KRAB结构域相当的水平。每种抑制活性的获得都伴随着与TRIM28蛋白相互作用的增强。结论DUF3669在N末端构型的KRAB-ZNF蛋白质亚组中添加了蛋白质-蛋白质相互作用表面，其变异KRAB和邻近序列可能受sumoylation调节。