Feng Xinyu A, Yamadi Maryam, Fu Yiben, Ness Kaitlin M, Liu Celina, Ahmed Ishtiyaq, Bowman Gregory D, Johnson Margaret E, Ha Taekjip, Wu Carl
Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA.
Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
bioRxiv. 2024 May 25:2023.07.14.549009. doi: 10.1101/2023.07.14.549009.
To elucidate how eukaryotic sequence-specific transcription factors (TFs) search for gene targets on chromatin, we used multi-color smFRET and single-particle imaging to track the diffusion of purified GAGA-Associated Factor (GAF) on DNA and nucleosomes. Monomeric GAF DNA-binding domain (DBD) bearing one zinc finger finds its cognate site by 1D or 3D diffusion on bare DNA and rapidly slides back-and-forth between naturally clustered motifs for seconds before escape. Multimeric, full-length GAF also finds clustered motifs on DNA by 1D-3D diffusion, but remains locked on target for longer periods. Nucleosome architecture effectively blocks GAF-DBD 1D-sliding into the histone core but favors retention of GAF-DBD when targeting solvent-exposed sites by 3D-diffusion. Despite the occlusive power of nucleosomes, 1D-3D facilitated diffusion enables GAF to effectively search for clustered cognate motifs in chromatin, providing a mechanism for navigation to nucleosome and nucleosome-free sites by a member of the largest TF family.
为了阐明真核生物序列特异性转录因子（TFs）如何在染色质上寻找基因靶点，我们使用多色单分子荧光共振能量转移（smFRET）和单颗粒成像技术来追踪纯化的GAGA相关因子（GAF）在DNA和核小体上的扩散。带有一个锌指的单体GAF DNA结合结构域（DBD）通过在裸露DNA上的一维（1D）或三维（3D）扩散找到其同源位点，并在自然聚集的基序之间快速来回滑动数秒后才逃脱。多聚体全长GAF也通过1D-3D扩散在DNA上找到聚集的基序，但在靶点上停留的时间更长。核小体结构有效地阻止了GAF-DBD一维滑动进入组蛋白核心，但当通过三维扩散靶向溶剂暴露位点时，有利于GAF-DBD的保留。尽管核小体具有封闭作用，但1D-3D促进扩散使GAF能够有效地在染色质中寻找聚集的同源基序，为最大的TF家族成员导航到核小体和无核小体位点提供了一种机制。
