Polytene Hi-C. Drosophila melanogaster

Chemical cross-linking and high-throughput sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no such interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of chromatin folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to ten-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. Overall design: Hi-C experiments where ligation is performed on beads (tethered) on male Drosophila salivary glands from three independent biological replicates. Also one Hi-C experiment where the ligation is performed in solution (conventional).

化学交联结合高通量测序技术已在间期细胞核中鉴定出染色体内相互作用区域——这类区域被称为拓扑关联结构域（topologically associating domains, TADs），它们与几乎不存在此类相互作用的区域交替分布。本研究发现，果蝇多线染色体的带区与间带区分别对应TAD及TAD间区域。我们进一步证实了果蝇多线细胞与二倍体细胞中TAD结构的保守性。基于对多线染色体光学显微照片的直接测量，我们推导得到二倍体细胞核内染色质折叠的两种状态：一类为包含调控区域与启动子的完全伸展纤维，另一类为包含活跃基因编码区域、压缩达十倍的纤维，二者共同构成核内部的常染色质。而包含非活跃基因编码区域、压缩达三十倍的染色质纤维，则构成核外周的异染色质。综上，分子分析与直接观测的结合揭示了间期染色体的三维构象架构。实验设计：针对雄性果蝇唾腺开展三组独立生物学重复的Hi-C实验，连接反应在磁珠固定体系中进行；同时开展一组连接反应于溶液中进行的常规Hi-C实验。