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

哺乳动物的染色体是由汇聚和相反的力量形成的三维实体。有丝分裂细胞分裂引起剧烈的染色体凝聚，但在重新进入细胞周期的G1期后，染色体重建其间期组织。在SPP2202的第一轮融资中，我们测试了RNAPII在这种转变中的作用，以及在异步G1细胞中，通过使用一个允许其生长素介导降解的系统。对于有丝分裂到g1的转变，原位Hi-C结合超分辨率3dSTORM成像和计算机模拟表明，RNAPII是有丝分裂退出时室和环建立所必需的。这是由于染色质上的内聚蛋白负载减少和异常，我们现在可以证明这依赖于RNAPII在可访问位点的物理存在。值得注意的是，受影响最大的位置是那些在有丝分裂期间被聚合酶辅助因子标记的位置，这些位置在RNAPII耗尽时也显示出不同的可及性。相比之下，异步g1细胞的染色体三维折叠在大尺度上受到的影响较小。然而，在缺乏RNAPII的情况下，出现了多个新的更大的CTCF /内聚蛋白锚定环。为了从机制上理解这些影响，在SPP2202的第二轮融资中，我们将生成超分辨率Micro-C数据，并确定影响染色体环形成的不同情景。我们将结合Micro-C和超分辨率3D- sim成像与表观遗传标记作图和三种新的细胞系，以允许不同因素的急性消耗，以解决以下问题：(1)增殖与有丝分裂后细胞中间期染色质3D结构的环水平变化是如何产生的？(2)有丝分裂后RNAPII如何协调内聚蛋白装载到染色质上？(3)在这个过程中是否有书签转录因子的作用？最后，我们期望对转录装置如何直接或间接地组织染色质获得新的见解。这些参与规则将使我们能够重新审视基于转录的三维染色质组织的概念，从而协调RNAPII在基因表达和染色体结构中的作用。

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.