Biophysics Graduate Program, The Ohio State University, Columbus, OH 43210, USA.
Department of Physics, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2023 Feb 22;51(3):1139-1153. doi: 10.1093/nar/gkac1267.
Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and single-molecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation.
转录因子 (TF) 需要能够访问核小体中的靶位点才能启动转录。靶位点在核小体中的位置会显著影响 TF 的占有率，但目前还没有定量理解这一现象的机制。我们使用集合和单分子荧光测量技术，研究了转录因子 Gal4 在核小体不同位置的靶向性和占有率。我们观察到，TF 占有率在延伸超过 30 个碱基对 (bp) 进入核小体的位置上急剧下降，这不能用 TF 解离速率或结合位点取向的变化来解释。相反，核小体解缠结自由能景观是 Gal4 占有率的主要决定因素，它降低了 Gal4 的结合速率。该自由能景观定义了两个具有不同可及性和动力学的区域，其边界在核小体中 30 bp 处，内部区域的可及性要低 100 多倍。由于 Gal4 在解缠结之前会多次交换，因此内部区域的 Gal4 结合速率不再取决于其浓度，而是受到核小体解缠结速率的限制，而核小体重新缠绕的频率则降低。我们的发现强调了核小体解缠结自由能景观对 TF 占有率和动力学的重要性，这最终影响了转录起始。
