Exploring the contribution of RNA polymerases to mammalian 3D genome architecture

探索 RNA 聚合酶对哺乳动物 3D 基因组结构的贡献

• 批准号: 422389065
• 负责人: Professor Dr. Argyris Papantonis
• 金额: --
• 依托单位: Institut für Pathologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/422389065?language=en
• 关键词: Exploring; contribution; RNA; polymerases; mammalian

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 phase of the cell cycle, chromosomes reestablish their interphase organization. During the first funding round of the SPP2202, we tested the role of RNAPII in this transition, as well as in asynchronous G1 cells, by using a system allowing its auxin-mediated degradation. For the mitosis-to-G1 transition, in situ Hi-C coupled to super-resolution 3dSTORM imaging and computer simulations showed that RNAPII is required for both compartment and loop establishment upon mitotic exit. This is due to reduced and aberrant cohesin loading onto chromatin, which we can now show relies on the physical presence of RNAPII at accessible sites. Notably, the positions most affected are those bookmarked during mitosis by polymerase cofactors, which also show differential accessibility upon RNAPII depletion. In contrast, 3D folding of chromosomes in asynchronous G1-cells appeared less affected at the large scale. However, multiple new and larger CTCF /cohesin-anchored loops emerged in the absence of RNAPII. To mechanistically understand these effects, for this second funding round of the SPP2202, we will generate ultra-resolution Micro-C data and identify different scenarios affecting loop formation along chromosomes. We will combine Micro-C and super-resolution 3D-SIM imaging with epigenetic mark mapping and three new cell lines engineered to allow for the acute depletion of different factors in order to address the following questions: (1) How do loop-level changes in the 3D architecture of interphase chromatin arise in proliferating versus post-mitotic cells? (2) How does RNAPII orchestrate cohesin loading onto chromatin after mitosis? (3) Is there a role for bookmarking transcription factors in this process? In the end, we anticipate to obtain new insights into how the transcriptional apparatus acts to organize chromatin directly or indirectly. These rules of engagement would allow us to revisit the concept of transcription-based 3D chromatin organization, and thus reconcile the role of RNAPII in gene expression with that in chromosomal architecture.

哺乳动物染色体是三维实体，由聚合和相反的力量形成。有丝分裂细胞分裂诱导剧烈的染色体凝聚，但在重新进入细胞周期的G1期后，染色体重新建立其间期组织。在SPP 2202的第一轮融资期间，我们通过使用允许其生长素介导降解的系统测试了RNAPII在这一转变中以及在异步G1细胞中的作用。对于有丝分裂到G1的过渡，原位Hi-C结合超分辨率3dSTORM成像和计算机模拟表明，RNAPII是有丝分裂退出后室和环建立所必需的。这是由于减少和异常的粘附素加载到染色质上，我们现在可以证明这依赖于RNAPII在可接近位点的物理存在。值得注意的是，受影响最大的位置是在有丝分裂期间由聚合酶辅因子标记的位置，其也显示RNAPII耗尽后的差异可及性。相比之下，在异步G1-细胞中染色体的3D折叠在大尺度上受到的影响较小。然而，多个新的和更大的CTCF /粘附素锚定环出现在RNAPII的情况下。为了从机制上理解这些影响，对于SPP 2202的第二轮融资，我们将生成超分辨率Micro-C数据，并确定影响染色体沿着形成环的不同情况。我们将结合联合收割机Micro-C和超分辨率3D-SIM成像与表观遗传标记定位和三个新的细胞系工程，以允许急性耗尽不同的因素，以解决以下问题：（1）如何环水平的变化，在3D结构的间期染色质出现在增殖与有丝分裂后的细胞？(2)RNAPII如何协调有丝分裂后粘附素加载到染色质上？(3)在这个过程中，书签转录因子有作用吗？最后，我们期望获得新的见解，转录装置如何直接或间接地组织染色质。这些参与规则将使我们能够重新审视基于转录的3D染色质组织的概念，从而调和RNAPII在基因表达中的作用与染色体结构中的作用。