Periodic Reporting for period 1 - MisterCHROM (Modelling sister chromatids cohesion)
Période du rapport: 2022-09-01 au 2024-08-31
Over the past decade, collaborative efforts combining experimental and theoretical approaches have significantly advanced our understanding of the organization of individual chromosomes and their functional implications in various biological contexts. However, the cohesion process between pairs of replicated chromosomes has remained poorly understood, primarily due to the limitations of existing technologies in distinguishing their identical sequences. This gap has left critical questions regarding cohesion mechanisms and their biological impacts on other important cellular mechanisms unresolved.
Daniel Gerlich’s lab at the hosting institution addressed this challenge by developing a sister-chromatid-sensitive Hi-C (scsHi-C) assay, enabling the first genome-wide analysis of the native conformation of sister chromatids in human replicated chromosomes.
This project aimed to leverage the theoretical and computational expertise provided by Anton Goloborodko’s lab (the hosting lab), and the scsHi-C new experimental dataset provided by Gerlich's lab to establish a robust theoretical framework for modeling the structure and mechanisms of sister chromatid cohesion, its interaction with dynamic loop extrusion mechanisms, and its role in fundamental cellular processes such as gene expression and DNA repair.
By integrating polymer simulations of chromosomes with analyses of genomic data, the Fellow successfully modeled for the first time how cohesive linkages are distributed along sister chromatids, leading to novel hypotheses regarding the mechanisms of cohesion establishment during replication. The Fellow discovered that the cohesive linkages tethering the two sister chromatids are asymmetrically misaligned. Specifically, these linkages connect non-homologous loci, leading to misalignment, and the connected loci consistently exhibit a lateral shift biased in one direction across the entire genome. This results in a skewed alignment of one sister chromatid relative to the other. These findings support a new hypothesis that the inherent asymmetry of the replication process is transferred to the cohesion mechanism during its establishment and is maintained, to some extent, until the onset of mitosis.
Daniel Gerlich’s lab at the hosting institution addressed this challenge by developing a sister-chromatid-sensitive Hi-C (scsHi-C) assay, enabling the first genome-wide analysis of the native conformation of sister chromatids in human replicated chromosomes.
This project aimed to leverage the theoretical and computational expertise provided by Anton Goloborodko’s lab (the hosting lab), and the scsHi-C new experimental dataset provided by Gerlich's lab to establish a robust theoretical framework for modeling the structure and mechanisms of sister chromatid cohesion, its interaction with dynamic loop extrusion mechanisms, and its role in fundamental cellular processes such as gene expression and DNA repair.
By integrating polymer simulations of chromosomes with analyses of genomic data, the Fellow successfully modeled for the first time how cohesive linkages are distributed along sister chromatids, leading to novel hypotheses regarding the mechanisms of cohesion establishment during replication. The Fellow discovered that the cohesive linkages tethering the two sister chromatids are asymmetrically misaligned. Specifically, these linkages connect non-homologous loci, leading to misalignment, and the connected loci consistently exhibit a lateral shift biased in one direction across the entire genome. This results in a skewed alignment of one sister chromatid relative to the other. These findings support a new hypothesis that the inherent asymmetry of the replication process is transferred to the cohesion mechanism during its establishment and is maintained, to some extent, until the onset of mitosis.
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.
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.
This project resulted in the first observation of the asymmetric misalignment of sister chromatids and introduces a novel hypothesis that the inherent asymmetry of the replication process is transferred to cohesion during its establishment. This leads to an asymmetric misalignment of sister chromatids that is maintained, to some extent, until the onset of mitosis. Additionally, the project proposes two specific mechanisms for cohesion establishment by the replication fork, detailing expected ranges of cohesive cohesin relocation distances in G2, which differ between the two models. Both models of cohesion establishment during replication, as well as the relocation distances during G2, present opportunities for testing in future experiments.