Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the “polycomb-based transcription factor” (PcTF), a fusion protein that recognizes histone modifications through a protein–protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its binding avidity. Here, we demonstrate the activity of a modified regulator called Pc₂TF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. Pc₂TF has a smaller apparent dissociation constant value in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators.

中文翻译：

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串联组蛋白结合域增强合成染色质效应子的活性

与染色体上的生化标记特异性相互作用的融合蛋白代表了一类新的合成转录调节因子，它们解码细胞状态信息而不是 DNA 序列。在多细胞生物中，与细胞状态、组织特性和肿瘤发生相关的信息通常被编码为组蛋白的生化修饰，这些修饰与真核细胞核中的 DNA 结合并调节基因表达状态。我们之前报道了“基于多梳的转录因子”（PcTF）的开发和验证，这是一种通过其多梳染色质域（PCD）基序和组蛋白 H3 的三甲基化赖氨酸 27 之间的蛋白质-蛋白质相互作用识别组蛋白修饰的融合蛋白。 H3K27me3) 在基因组位点。我们证明了 PcTF 在癌症衍生细胞系中激活富含甲基组蛋白基因座的基因。然而，与 DNA 结合激活剂相比，PcTF 诱导甲基组蛋白相关报告基因的适度激活。因此，我们修改了 PcTF 以增强其结合亲和力。在这里，我们展示了一种名为 Pc 的改良调节器的活性₂ TF，在 N 端具有 H3K27me3 结合 PCD 的两个串联拷贝。Pc _{2 TF}在体外具有较小的表观解离常数值，并且与 PcTF 相比，在 HEK293 细胞中显示出增强的基因活化。这些结果提供了令人信服的证据，表明 PCD 基序的内在组蛋白结合活性可用于调节合成组蛋白结合转录调节剂的活性。