Condensin connects 3D genome organization, chromosomal segregation, andgene regulation via its interactions with transcription-related factors.

总结 尽管3D基因组组织的破坏与人类疾病有关，包括发育 综合征和癌症，在我们理解特定分子如何 机器控制着基因组的组织。我们的长期目标是确定进化上保守的 分子机制连接3D基因组组织和转录调控。我们最近 发现了一个关键的基因组组织者，凝聚素和转录机制之间的分子联系 （Mol. Cell 2015; Nature Genet. 2016）。这里的目标是描述凝聚素如何连接3D基因组 组织、染色体分离和转录调控。核心假设是， 凝聚素与TBP、介体、TFIIIC和转录因子（TF）的相互作用是缔合所必需的 在高度转录（HT）基因和着丝粒之间，这促进了染色体的忠实分离 在有丝分裂过程中，凝聚素通过3D基因组组织控制基因转录（基因定位在 着丝粒）或/和局部染色体压实。我们将在非常容易处理的裂变中检验我们的假设 酵母模型（目标1和2），并利用知识描述人类细胞中更复杂的机制 从酵母系统（目的3）。在目标1下，我们将检验HT基因与 位点和着丝粒，由凝聚素-TBP相互作用介导，是一个稳定的结构，以介导适当的 染色体分离（用wt和凝聚素突变体的活细胞成像）。我们将决定 将基因位点定位在着丝粒损害转录（基于lacO的系链）。我们还将测试 另一种局部机制，其中缩合素通过DNA抑制HT基因的转录（qRT-PCR） 复性（核酸酶测定），随后进行局部压缩（FISH）。在目标2下，我们将确定如何 转录相关因子（TBP，mediator，TFIIIC和TF）有助于凝聚素加载和基因表达。 定位在着丝粒（系留系统和FISH）。我们将阐明凝聚介质 相互作用参与染色体分离和转录调节（FISH、RNA-seq和qRT-PCR 具有凝聚素突变体）。此外，我们将确定凝聚如何与TBP，介体和 TF组织全基因组关联和结构域（ChIA-PET & Hi-C与缩合蛋白和TF突变体）。 在目标3下，我们将使用来自目标1和2的技术以及人RPE 1细胞中的凝聚素突变（在 hand），并表征缩合蛋白I和hTBP之间的相互作用如何参与（1）RNA的去除 有丝分裂和基因书签过程中HT基因的聚合酶;和（2）HT基因和 域形成该项目的创新之处在于我们的新概念， 3D基因组组织，染色体分离和转录调控。拟议的研究是 重要的是，因为它有望提高对凝聚素如何建立功能的理解。 染色体结构，这仍然比粘着机制少得多的理解。

SUMMARY Although disruption of 3D genome organization is connected to human diseases, including developmental syndromes and cancer, there remain fundamental gaps in our understanding of how specific molecular machines control genome organization. Our long-term goal is to determine how evolutionarily conserved molecular mechanisms connect 3D genome organization and transcriptional regulation. We have recently discovered molecular links between a key genome organizer, condensin, and the transcriptional machinery (Mol. Cell 2015; Nature Genet. 2016). The objective here is to delineate how condensin connects 3D genome organization, chromosomal segregation, and transcriptional regulation. The central hypothesis is that the condensin interactions with TBP, mediator, TFIIIC and transcription factors (TFs) are required for associations among highly transcribed (HT) genes and centromeres, which promotes faithful segregation of chromosomes during mitosis, and that condensin controls gene transcription via 3D genome organization (gene positioning at centromeres) or/and local chromosomal compaction. We will test our hypothesis in the highly tractable fission yeast model (Aims 1 & 2) and characterize the more complex mechanisms in human cells using knowledge from the yeast system (Aim 3). Under Aim 1, we will test the hypothesis that the association between HT gene loci and centromeres, mediated by condensin-TBP interaction, is a stable structure to mediate proper chromosomal segregation (live-cell imaging with wt and condensin mutant). We will determine how merely positioning a gene locus at the centromere impairs transcription (lacO-based tethering). We will also test an alternative, local mechanism, whereby condensin represses transcription of HT genes (qRT-PCR) via DNA renaturation (nuclease assay) followed by local compaction (FISH). Under Aim 2, we will establish how transcription-related factors (TBP, mediator, TFIIIC & TFs) contribute to condensin loading and gene positioning at centromeres (tethering system & FISH). We will elucidate how the condensin-mediator interaction participates in chromosomal segregation and transcriptional regulation (FISH, RNA-seq & qRT-PCR with condensin mutant). Moreover, we will determine how the condensin interactions with TBP, mediator and TFs organize genome-wide associations and domains (ChIA-PET & Hi-C with condensin and TF mutants). Under Aim 3, we will use techniques from Aims 1 and 2 and condensin mutations in human RPE1 cells (in hand), and characterize how the interaction between condensin I and hTBP is involved in (1) removal of RNA polymerases from HT genes during mitosis and gene bookmarking; and (2) associations of HT genes and domain formation. The innovation of this project resides in our new concept connecting condensin-mediated 3D genome organization, chromosomal segregation, and transcriptional regulation. The proposed research is significant, because it is expected to improve the understanding of how condensin establishes the functional chromosomal structure, which remains much less understood than the cohesin mechanisms.