Transcription rate and transcript length drive the formation of chromosomal interaction domain

Chromosomes in all organisms are highly organized and divided into multiple chromosomal interaction domains, or topological domains. Regions of active, high transcription help establish and maintain domain boundaries, but precisely how this occurs remains unclear. Here, using fluorescence microscopy and chromosome conformation capture in conjunction with deep sequencing (Hi-C), we show that in Caulobacter crescentus both transcription rate and transcript length, independent of concurrent translation, drive the formation of domain boundaries. We find that long, highly expressed genes do not form topological boundaries simply through the inhibition of supercoil diffusion. Instead, our results support a model in which long, active regions of transcription drive local decompaction of the chromosome, with these more open regions of the chromosome forming spatial gaps in vivo that diminish contacts between DNA in neighboring domains. These insights into the molecular forces responsible for domain formation in Caulobacter likely generalize to other bacteria and possibly eukaryotes. Overall design: Hi-C experiments were performed on (i) untreated wild type swarmer cells and drug-treated swarmer cells (Chloramphenicol) of Caulobacter crescentus CB15N; (ii) on swarmer cells that are starved in M2 salts for 90 minutes; (iii) on swarmer cells harboring van::PrsaA_rsaA as well as derivatives of rsaA with a cassette of 2xTAA 2x transcription terminator inserted at 60bp, 560bp, 1060 and 2060 bp from the transcription start site of the ectopic rsaA; (iv) on CB15N strain ML2000 (Plac::dnaA) at time points 90 min, 150 min, 210 min and 270 min after IPTG withdrawal; and (v) on CB15N strain overexpressing CtrAD51E_delta_omega at time point 0 min, 120 min and 180 min after addition of the inducer (xylose). RNA-seq experiment were performed on RNA extracted from (i) wild-type CB15N cells growing at exponential phase in PYE; and (ii) wild-type CB15N cells starved in M2 salts for 90 min.

所有生物的染色体均呈高度有序状态，被划分为多个染色体相互作用结构域，亦称拓扑结构域。活跃的高转录区域有助于建立并维持结构域边界，但其具体作用机制仍有待阐明。本研究结合荧光显微镜与染色体构象捕获联合深度测序（Hi-C）技术，证实新月柄杆菌（Caulobacter crescentus）中，转录速率与转录本长度可独立于伴随的翻译过程，驱动结构域边界的形成。我们发现，长片段高表达基因并非仅通过抑制超螺旋扩散来形成拓扑结构域。与之相反，本研究结果支持如下模型：活跃的长转录区域可介导染色体发生局部解压缩，此类更为开放的染色体区域在活体中形成空间间隙，进而削弱相邻结构域内DNA分子间的接触。上述关于新月柄杆菌结构域形成分子驱动力的研究见解，大概率可推广至其他细菌，甚至真核生物。

整体实验设计：
Hi-C实验的样本涵盖：(i) 未处理的野生型新月柄杆菌CB15N泳动细胞，以及经氯霉素（Chloramphenicol）处理的泳动细胞；(ii) 在M2盐溶液中饥饿培养90分钟的泳动细胞；(iii) 携带van::PrsaA_rsaA载体，且异位表达的rsaA基因转录起始位点上游60bp、560bp、1060bp及2060bp处插入2xTAA 2x转录终止子盒的rsaA突变体菌株的泳动细胞；(iv) IPTG诱导撤除后90 min、150 min、210 min及270 min时采集的CB15N菌株ML2000（Plac::dnaA）样本；(v) 诱导剂木糖（xylose）添加后0 min、120 min及180 min时采集的过表达CtrAD51E_delta_omega的CB15N菌株样本。

RNA测序（RNA-seq）实验的样本来自：(i) 在PYE培养基中指数生长期培养的野生型CB15N细胞；(ii) 在M2盐溶液中饥饿培养90分钟的野生型CB15N细胞。