Fujioka Miki, Ke Wenfan, Schedl Paul, Jaynes James B
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Genetics. 2025 Apr 17;229(4). doi: 10.1093/genetics/iyaf032.
Chromatin insulators are major determinants of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers and promoters interact over long distances and between homologous chromosomes. Insulators can pair with copies of themselves in trans to facilitate homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops. Contrary to their canonical function of enhancer blocking, these loops can bring distant enhancers and promoters together to activate gene expression, while at the same time blocking other interactions in cis. The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3D architecture as a major functional determinant. Here, we dissect the homie insulator from the Drosophila even skipped (eve) locus, to understand its substructure. We test pairing function based on homie-carrying transgenes interacting with endogenous eve. The assay is sensitive to both pairing strength and orientation. Using this assay, we found that a Su(Hw) binding site in homie is required for efficient long-range interaction, although some activity remains without it. This binding site also contributes to the canonical insulator activities of enhancer blocking and barrier function. Based on this and other results from our functional dissection, each of the canonical insulator activities, chromosomal loop formation, enhancer blocking, and barrier activity, are partially separable. Our results show the complexity inherent in insulator functions, which can be provided by an array of different proteins with both shared and distinct properties.
染色质绝缘子是染色体结构的主要决定因素。绝缘子诱导的特定结构深刻影响核过程，包括增强子和启动子如何在远距离以及同源染色体之间相互作用。绝缘子可以与自身的反式拷贝配对以促进同源配对。它们还可以与其他绝缘子配对，有时具有高度特异性，诱导长距离染色体环化。与它们增强子阻断的经典功能相反，这些环可以将远距离的增强子和启动子聚集在一起以激活基因表达，同时在顺式中阻断其他相互作用。这些效应的细节取决于配对伙伴的选择以及配对的方向特异性，这表明三维结构是主要的功能决定因素。在这里，我们剖析了果蝇“偶数跳动”（eve）基因座中的“homie”绝缘子，以了解其亚结构。我们基于携带“homie”的转基因与内源性“eve”相互作用来测试配对功能。该检测方法对配对强度和方向都很敏感。使用这种检测方法，我们发现“homie”中的一个Su(Hw)结合位点是高效长距离相互作用所必需的，尽管没有它也仍有一些活性。这个结合位点也有助于增强子阻断和屏障功能的经典绝缘子活性。基于我们功能剖析的这一结果和其他结果，经典绝缘子活性、染色体环形成、增强子阻断和屏障活性中的每一种都是部分可分离的。我们的结果显示了绝缘子功能中固有的复杂性，这可以由一系列具有共同和不同特性的不同蛋白质提供。
