Search Talks

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.

CTCF-mediated chromatin loops play a crucial role in facilitating interactions between distal genomic regions. These loops have also been proposed to insulate enhancers from contacting with promoters in neighboring domains to prevent ectopic gene activation. However, the in vivo significance of this model has not been thoroughly tested. To test whether chromosome domains with higher density of developmental regulators are more susceptible to disruption of chromatin structure, we deleted a 25kb region containing four CTCF motifs at the boundary of a domain harboring the Fgf3, Fgf4, and Fgf15 loci. These genes with distinct spatiotemporal expression are critical for cell fate specification, patterning and organogenesis. Strikingly, heterozygous mutants showed perinatal lethality and encephalocele¬, a neural tube closure defect¬¬ caused by over-proliferation of neural tissue, abnormal cranial morphology, and skull bone hypoplasia. To confirm that these defects arise from loss of CTCF m...

Mitotic chromosomes lose interphase-specific genome organization and transcription but gain histone phosphorylation, specifically H3S10p. This phosphorylation event compacts chromosomes in early mitosis by reducing inter-nucleosomal distance before the loading of condensins. However, it is unclear if H3S10p in mitosis preserves the identity of lost chromatin domains and promoters, both physically and functionally. Here, using the pre-mitotic expression of histone H3S10 and its mutants H3S10A and H3S10D, we show that H3S10p hyper-phosphorylates active promoters and spreads into super-domains A in mitosis, causing compaction of these regions. By spreading into active domains in the absence of genome organization, H3S10p retains their identity physically. Functionally, H3S10p ensures optimal closing of promoters by stabilizing the nucleosomes, thereby protecting them from excess loading of transcription machinery post-mitosis. In the H3S10p phospho-mutants, these chromatin regions fail to...

Over the past few decades, the cancer hallmarks have been instrumental in simplifying the complexity of the disease into fundamental principles. Emerging evidence suggests that epigenetic regulation plays a pivotal role in shaping cancer phenotypes and genotypes. Epigenetic modifications are recognized by a ubiquitous class of proteins called “readers/effectors” which has become an important paradigm in chromatin biology. We have identified that chromatin readers play seminal role in regulating most of the hallmark signatures in breast cancers thereby intrinsically contributing to breast tumor heterogeneity. Their dynamic role in metabolic reprogramming in 3D-tumor core and periphery will be highlighted. Oxygen and nutrient depleted tumor core have altered metabolic programs promoting their sustenance that are epigenetically regulated by the chromatin readers. Notably, the cancer cells and their associated stromal cells can support primary tumor metastasis by reshaping extracellular ma...

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small(s)RNAs mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its paternally imprinted nature. These important features were modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines we observed defects in endosperm development and loss of around 70% of ...

Chromatin is organized hierarchically at multiple scales and this is crucial for the spatiotemporal regulation of transcription. The fundamental units of nuclear organization are the highly self-interacting regions of chromatin termed as ‘Topologically Associated Domains’ or TADs. TADs are formed by a loop-extrusion mechanism mediated by two proteins: cohesin and CTCF. The major function of these units is to limit the action of regulatory elements to genes within the same TAD. Disruption of TAD boundaries can lead to dysregulation of gene expression and accessibility with a dramatic phenotypic consequence on developmental processes and pathogenesis Given the importance of CTCF in the formation of TADs and the role of the latter in gene regulation, it is not surprising that mutation in this protein have been reported in several diseases. While CTCF is a ubiquitously expressed, essential protein, it has a paralogue; CTCFL with a similar DNA binding domain that is normally expressed only ...