Department of Physics, Drexel University, Philadelphia, PA 19104, USA.
The Wistar Institute, Philadelphia, PA 19104, USA.
J Mol Biol. 2018 Mar 16;430(6):759-776. doi: 10.1016/j.jmb.2018.01.012. Epub 2018 Jan 31.
The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.
CTCF 蛋白已成为参与基因组组织的关键结构蛋白。尽管假设 CTCF 可以启动 DNA 环化，但仍缺乏 CTCF 诱导的 DNA 环形成的直接证据。几项研究表明，CTCF 的 11 个锌指（11ZF）结构域积极参与 DNA 结合。在这里，我们使用原子力显微镜来研究人类 CTCF 的 266-579 位残基组成的 11ZF 结构域（11ZFCTCF）和 402-494 位残基组成的 3ZF 结构域（6-8ZFCTCF）对 DNA 形态的影响。我们的结果表明，这两个结构域都改变了 DNA 的结构，使其从对照 DNA 样本中观察到的松弛形态转变为紧凑的圆形复合物、网格和网络，这为 11ZFCTCF 结构域的多价性质提供了重要的见解。原子力显微镜图像显示出准圆形的 DNA/CTCF 复合物，当用 6-8ZFCTCF 结构域取代 11ZFCTCF 时，这些复合物会变得不稳定，这突出了 11ZF 基序在环形成中的作用。有趣的是，圆形 DNA/CTCF 复合物的形成主要是由非特异性结合主导的，轮廓长度和高度曲线表明单个 DNA 分子两次缠绕在蛋白质上。
