A dynamic folded hairpin conformation is associated with ??-globin activation in erythroid cells

We investigate the three-dimensional conformations of the ??-globin locus at the single-allele level in murine ES and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent FISH data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ES cells to erythroid cells, associated with the activation of the globin genes. We find that in ES cells, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, while in erythroid cells the ??-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites multiple molecular mechanisms cooperate to form an interacting domain. Overall design: ChIP-seq with indicated antibodies on chromatin isolated from primary erythroid ter119+ cells obtained from the spleens of acetylphenylhydrazine treated mice. Wild-type C57BL/6 mice were used.

本研究结合聚合物物理模型与高分辨率捕获C技术（Capture-C）数据，在单等位基因水平上探究小鼠胚胎干细胞（embryonic stem cells, ES）及红细胞系细胞中β-珠蛋白基因座（β-globin locus）的三维构象。本研究以独立获取的荧光原位杂交（Fluorescence In Situ Hybridization, FISH）数据（用于测量两两空间距离）与三向相互作用捕获技术（Tri-C）数据（用于识别三方接触）作为参照，对模型预测结果进行验证。在从胚胎干细胞向红细胞系细胞转变的过程中，该染色质构象发生重排，且与珠蛋白基因的激活密切相关。研究发现，胚胎干细胞中的空间组织呈现高度混杂的三维结构，涉及非特异性接触；而在红细胞系细胞中，β-珠蛋白基因及其增强子形成一个独立结构域，以折叠发夹构象排布，并通过微相分离的热力学机制与混杂的侧翼区域分隔开。侧翼区域富含收敛型CCCTC结合因子（CTCF）结合位点，此类位点仅少量参与红细胞系特异性的基因-增强子相互作用，这表明除CTCF位点的相互作用外，还存在多种分子机制协同作用以形成相互作用结构域。整体实验设计：对经乙酰苯肼处理的小鼠脾脏中分离得到的原代TER119阳性红细胞系细胞的染色质，使用指定抗体进行染色质免疫共沉淀测序（ChIP-seq），实验所用小鼠为野生型C57BL/6品系。