Interdisciplinary aspects of chromatin organization and gene regulation

Alterations in nuclear and chromatin organization are hallmarks of cellular aging and many aging related diseases including cancer and neurodegeneration. However, quantitative methods to analyse subtle alterations in chromatin states to understand cell-state transitions and for early disease diagnostics are still missing. In this talk, I will first describe our AI-based chromatin imaging biomarker platform. I will then demonstrate the sensitivity of this platform to trace tumour progression and neurodegeneration using tissue biopsies. Furthermore, I will describe our ongoing clinical trials, using AI-based chromatin biomarkers in blood biopsies, for early disease diagnostics and for tracing the efficacy of therapeutic interventions in personalized and precision medicine. Finally, I will introduce a major global public health project that we have initiated to develop a chromatin imaging atlas of human blood cells in health and disease.

It is well established that chromosome territories are non-randomly organized in the interphase nucleus, with gene poor chromosome territories proximal to the nuclear periphery, while gene rich chromosome territories are closer to the nuclear center. Such an organization is conserved in evolution. We showed that the depletion of the nuclear envelope proteins i.e, lamins, not only induces chromosomal instability but also destabilizes chromosome positioning in otherwise diploid colorectal cancer cells. Remarkably, the combined loss of lamins and Emerin perturb chromosome territory and gene loci dynamics in the interphase nucleus. Furthermore, nuclear envelope proteins are essential for relaying mechanical signals into the nucleus, since cells cultured on softer substrates showed a striking change in the spatial organization of chromosome territories in an Emerin-Lamin dependent manner. Taken together, our studies reveal an overarching role for nuclear envelope proteins, in the maintenanc...

3D organization of eukaryotic genomes scales from nucleosomes to loops, chromatin domains and chromosome territories. Given the hierarchical nature of this organization we explored the dependence of higher order genome organization on nucleosome positioning across the genome. I will discuss the mechanism by which ATP-dependent chromatin remodeller, ISWI slides nucleosomes to position them regularly and what are the consequences of perturbing nucleosome positioning across the genome on higher order genome organization.

In eukaryotic cells, the nucleosome core particle (NCP) forms the basic unit of the genetic architecture. In NCPs, genomic DNA is tightly wound around an octameric core of histone proteins, much like thread wrapped around a spool. Recent experiments have shown that nucleosomes are highly dynamic, and often unwrap in an asymmetrical fashion at high ionic strengths, or in response to mechanical perturbations. By developing a sequence-specific coarse- grained model for DNA-protein complexes, which recapitulates various aspects of nucleosome structure and dynamics, we show how sequence-specificity of DNA-protein interactions is critical for nucleosome plasticity. Our force-field also captures the two-stage unwrapping observed in single-molecule pulling experiments. We further show that histone tails, which are hotspots for post-translational modifications, play a remarkable role in modulating the extent, as well as the direction of unwrapping. Our observations could set the stage for under...

Human silencers have been shown to exist and regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated, such as whether they can form “super-silencers” and whether they are linked to cancer progression. Here, through interrogating two putative silencer components of FGF18 gene, we found that two nearby silencers can cooperate via compensatory chromatin interactions to form a “super-silencer”. Furthermore, double knockout of two silencers exhibited synergistic upregulation of FGF18 expression and changes of cell identity. To perturb the “super-silencers”, we applied combinational treatment of an EZH2 inhibitor GSK343, and a REST inhibitor, X5050 (“GR”). We found that GR led to severe loss of TADs and loops, while the use of one inhibitor by itself only showed mild changes. Such changes in TADs and loops were associated with reduced CTCF and TOP2A mRNA levels. Moreover, GSK343 and X5050 synergistically upregulated...

The role of telomeres in cellular and organismal physiology including ageing and cancer is commonly appreciated. However, intriguingly, the molecular impact of telomeres in human cells has been largely limited to the subtelomeres (~10 Mb from telomeres). Questioning this paradigm we found telomere dependent molecular mechanisms affect chromatin across the genome defining functional outcomes ranging from tumor cell immunity to neurogenesis.

We investigate the role of compaction of chromatin domains in modulating search kinetics of proteins. Collapsed conformations of chromatin, characterised by long loops which bring distant regions of the genome into contact, and manifested structurally as Topologically Associated Domains (TADs) affect search kinetics of DNA associated transcription factors and other proteins. In this study, we investigate the role of the compactness of chromatin on the dynamics of proteins using a minimal model. Using analytical theory and simulations, we show that an optimal compaction exists for which the residence time of proteins on a chromatin-like polymer backbone is minimum. We show that while bulk diffusion is an advantageous search strategy for extended polymers, for highly folded polymer domains, intersegmental transfers allow optimal search. We extend these results to more detailed polymer models - using the Freely Rotating Chain model, a Lennard-Jones bead-spring polymer model, which approxi...

The three-dimensional organization of chromatin into domains and compartments leads to specific scaling of contact probability and compaction with genomic distance. However, chromatin is also dynamic, with active loop extrusion playing a crucial role. While extrusion ensures a specific spatial organization, how it affects the dynamic scaling of measurable quantities is an open question. In this work, using polymer simulations with active loop extrusion, we demonstrate that the interplay between the timescales of extrusion processes and polymer relaxation can influence the 3D organization of chromatin polymer. We point out this as a factor contributing to the experimentally observed non-trivial scaling of relaxation time with genomic separation and mean-square displacement with time. We show that the dynamic scaling exponents with loop extrusion are consistent with the experimental observations and can be very different from those predicted by existing fractal-globule models for chromat...

The organization of genome within the cell is essential for survival across all domains of life. The physical principles that govern genome organization remain elusive. Phase separation of protein and DNA has emerged as an attractive mechanism for reshaping and compacting the genome. In vitro studies have shed light on the biophysical principles of protein-DNA condensates driven by protein-protein and protein-DNA interactions. However, the role of DNA sequence and its impact on protein-DNA condensation remains elusive. Guided by experiments, we have developed a simple polymer-based model of protein-mediated DNA condensation that explicitly incorporates the influence of DNA sequence on protein binding. By employing coarse-grained Brownian dynamics simulations, we shed light on how DNA sequence affects the number, size and position of protein-DNA condensates. Comparing our simulation results with experimental data for the nucleoid-associated protein Lsr2 provides new insights into the me...

Bacterial chromosome segregation, ensuring equal distribution of replicated DNA, is crucial for cell division. During fast growth, overlapping cycles of DNA replication and segregation require efficient segregation of the origin of replication (Ori), which is known to be orchestrated by the protein families SMC and ParAB. I will discuss our approach using data-driven physical modeling to study the roles of these proteins in Ori segregation. Developing a polymer model of the Bacillus subtilis genome based on Hi-C data, we analyzed chromosome structures in wild-type cells and mutants lacking SMC or ParAB. Wild-type chromosomes showed clear Ori segregation, while the mutants were segregation deficient. The model suggests the dual role of ParB proteins, loading SMCs near the Ori and interacting with ParA enriched at the cell poles, is crucial for Ori segregation. ParB-loaded SMCs compact individual Ori and introduce an effective inter-sister repulsion. While both the ParB-bound Ori tracks ...

I will present our recent work on the role of DNA and RNA polymerases in driving the spatio-temporal dynamics of chromosome in higher eucaryotes using biophysical modeling and analysis of experimental data.