The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

中文翻译：

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哺乳动物 BEN 域蛋白序列特异性 DNA 结合的不同结构基础

BEN 结构域是最近公认的 DNA 结合模块，存在于多种后生动物和某些病毒中。几个 BEN 域因子被称为转录抑制因子，但总体而言，对于 BEN 因子如何识别其在人类中的目标知之甚少。特别是，BEN 结构域的 X 射线结构：DNA 复合物仅对果蝇已知带有单个 BEN 结构域的因子，缺乏直接的脊椎动物直系同源物。在这里，我们描述了几种哺乳动物 BEN 域 (BD) 因子，包括来自两个 NACC 家族 BTB-BEN 蛋白和来自具有四个 BD 的 BEND3。体外选择数据揭示了来自所有这些因素的分离的 BEN 结构域的序列特异性结合活性。我们对 BEND3 进行了详细的功能、基因组和结构研究。我们表明，BD4 是体内关联和抑制内源性 BEND3 靶标的主要决定因素。我们获得了与其首选结合位点结合的 BEND3-BD4 的高分辨率结构，这揭示了 BEND3 如何识别同源 DNA 靶标，并显示出与其一个非 DNA 结合 BEN 结构域 (BD1) 的差异。最后，与我们之前的无脊椎动物 BEN 结构进行比较，连同使用 AlphaFold2 和 RoseTTAFold 的其他结构预测，揭示了不同类型的 BEN 域蛋白识别目标 DNA 的不同策略。总之，这些研究扩展了 BEN 因子的 DNA 识别活性，并为哺乳动物 BEN 蛋白的序列特异性 DNA 结合提供了结构性见解。