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

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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.