The Fellow began by studying the distribution (average frequency and degree of alignment) of cohesive cohesins along the genome and investigating how this distribution influences the three-dimensional organization of sister chromatids. To achieve this, the Fellow conducted an in-depth re-analysis of scsHi-C data recently obtained by Daniel Gerlich's group, in collaboration with a PhD student co-supervised by both Gerlich’s lab and the host lab.
This investigation revealed that sister chromatids are not randomly misaligned; rather, there is a consistent genome-wide bias in the preferred direction of their misalignment. This asymmetry is attributed solely to cohesive cohesins and is not influenced by loop-extruding cohesins, CTCF, or transcription. The misalignment occurs during replication and is maintained until the onset of mitosis.
To understand how this asymmetry in misalignment is generated and to characterize its primary properties, the Fellow successfully developed a coarse-grained polymer model of two chromosomes tethered together by static links, simulating the structure of sister chromatids held together by cohesive cohesins in an equilibrium state. By exploring a wide range of simulation parameters, the Fellow was able to replicate scsHi-C experimental data and infer key features of cohesion distribution that contribute to asymmetric misalignment, such as the average frequency of cohesive links and the average misalignment in each direction, effectively reproducing the observed misalignment asymmetry.
The Fellow then aimed to elucidate the dynamic mechanisms underlying the organization of sister chromatids that could lead to the equilibrium distribution of cohesive cohesins identified above, both in the absence and presence of loop extrusion. To achieve this, the Fellow incorporated dynamic features of cohesive cohesin relocation into polymer simulations and pursued three parallel lines of research:
1. Understand the dynamic mechanisms of cohesion that could result in its asymmetric misalignment.
2. Understand the interaction between dynamic loop extrusion and cohesion, and their potential cooperation.
3. Understand the interaction between dynamic transcription and cohesion, and the influence of transcription on the distribution of cohesins along the genome.
All three lines of research yielded promising initial results; however, the investigation into the asymmetric misalignment of cohesion emerged as the most novel and impactful. Consequently, the Fellow concentrated solely on this first line of research. This focus enabled the formulation of and “in silico” testing mechanisms of cohesion establishment and relocation that could account for the observed asymmetric misalignment in G2.
Simulation results indicated that two distinct mechanisms, or a combination of both, could explain the phenomenon. The first mechanism suggests that cohesion is initially misaligned in a specific direction by the replication fork, followed by partial random relocation through diffusion or other cellular processes. The second mechanism posits that cohesion is established by the replication fork as pairs of cohesins that are oppositely semi-anchored on the two sister chromatids. In this case, the side of the cohesin that is anchored can only be partially relocated, while the non-anchored side can move freely until it reaches the anchored side of the next cohesin pair, which acts as a barrier.
The predictions of these two models diverge in the event of cofactor Sororin depletion, where the frequency of cohesion is significantly reduced; only the second model aligns with experimental data. Directional motor-driven motion was not tested due to insufficient experimental evidence.
Overall, this work resulted in the proposal of two potential models for cohesion establishment by the replication fork and subsequent relocation, which may explain the asymmetric misalignment of cohesion observed in G2. These novel insights into the establishment and dynamics of cohesion were presented at six major international conferences in the fields of chromosome organization, DNA replication, and meiosis, as well as at an invited seminar at the Institut Curie in Paris and during two internal seminars organized at the Vienna BioCenter. The manuscript summarizing these findings is currently in preparation.
