Köhler Anja R, Gutekunst Nicole, Harsch Annika, Bashtrykov Pavel, Jeltsch Albert
Institute of Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Genes (Basel). 2025 Apr 10;16(4):444. doi: 10.3390/genes16040444.
Dynamic changes in histone acetylation play crucial roles during cellular differentiation and disease development, but their detection in living cells is still a challenging task.
Here, we developed a Bimolecular Anchor Detector (BiAD) sensor for the detection of locus-specific changes in histone acetylation in living cells by fluorescence microscopy.
We used the BRD9 bromodomain cloned as tandem double domain (2xBRD9-BD) as a reader of histone acetylation. It was integrated into a dual-color BiAD chassis that was previously described by us.
We identified the gene body of as a potential target for our sensor, because it contains dense histone acetylation and 392 local sequence repeats. Using a binding-deficient mutant of 2xBRD9-BD as a negative control, we established a successful readout of histone acetylation at the locus. A single-domain reader did not function, indicating the requirement for the double reader to enhance the affinity and specificity of the chromatin interaction via avidity effects. With this sensor, we could detect dynamic increases in histone acetylation at the locus after the treatment of cells with the histone deacetylase inhibitor Trichostatin A for 6 h indicating the applicability of the sensor for single-cell epigenome studies.
Our data demonstrate that active chromatin modifications can be detected by BiAD sensors using 2xBRD9-BD as a reader. This complements the toolkit of the available BiAD sensors and documents the modularity of BiAD sensors.
组蛋白乙酰化的动态变化在细胞分化和疾病发展过程中起着关键作用，但其在活细胞中的检测仍然是一项具有挑战性的任务。
在此，我们开发了一种双分子锚定检测器（BiAD）传感器，用于通过荧光显微镜检测活细胞中组蛋白乙酰化的位点特异性变化。
我们使用克隆为串联双结构域（2xBRD9-BD）的BRD9溴结构域作为组蛋白乙酰化的读取器。它被整合到我们之前描述的双色BiAD框架中。
我们将 的基因体确定为传感器的潜在靶点，因为它包含密集的组蛋白乙酰化和392个局部序列重复。使用2xBRD9-BD的结合缺陷突变体作为阴性对照，我们成功读出了 位点的组蛋白乙酰化。单结构域读取器不起作用，这表明需要双读取器通过亲和力效应增强染色质相互作用的亲和力和特异性。使用该传感器，在用组蛋白去乙酰化酶抑制剂曲古抑菌素A处理细胞6小时后，我们可以检测到 位点组蛋白乙酰化的动态增加，这表明该传感器适用于单细胞表观基因组研究。
我们的数据表明，使用2xBRD9-BD作为读取器的BiAD传感器可以检测活性染色质修饰。这补充了现有BiAD传感器的工具包，并证明了BiAD传感器的模块化。
