ICTS

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Cohesin complexes are ring-shaped multimers that serve a multitude of functions such as regulating genome architecture, transcription, DNA repair apart from their canonical roles in chromosomal segregation fidelity during cell division. Paralogs exist for a few subunits of this complex, which if co-expressed can result in presence of multiple complexes at the same time in a cell. Meiotic chromosomes contain two distinct cohesin complexes, one specifically around the centromeres that are known to mainly aid in chromosomal segregation and the other exclusively at the chromosomal arms to facilitate genetic recombination events. However, how this distinct cohesin loading pattern is maintained on the chromosomes is not very clear. Our current work aims to dissect the molecular mechanisms mediated by chromatin receptors and epigenetic factors in dictating this preferential loading in Schizosaccharomyces pombe and some of these ideas will be discussed. Another question we are addressing is the need to have such a distinct occupancy for different complexes. Our work had previously shown that the pericentric-specific cohesin complex actively represses meiotic double-strand break formation and consequently, crossovers at the centromeres. Importantly, there is a strong correlation between centromeric crossovers and chromosomal mis-segregation events leading to infertility and developmental disorders such as Down syndrome in humans. The molecular details of how centromeric crossovers result in chromosomal mis-segregation remains largely unexplored. We are currently employing a fluorescence-based tetrad assay to categorize the different types and degrees of chromosomal segregation errors associated with aberrant recombination events as well as loss of cohesion, the two main causes for defects in oocytes with increasing age in women. In addition, we will discuss how the occurrence and distinct localization patterns of the mitotic and meiotic paralogs of cohesin protein subunits differentially affect their recombination and segregation functions during cell division.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Senescence —the endpoint of replicative lifespan for normal cells— is established via a complex sequence of molecular events. One such event is the dramatic reorganization of CTCF into senescence-induced clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine functional assays, super-resolution imaging, and 3D genomics with computational modelling to dissect SICC emergence. We establish that the competition between CTCF-bound and non-bound loci dictates clustering propensity. Upon senescence entry, cells repurpose SRRM2 —a key component of nuclear speckles— and BANF1 —a ‘molecular glue’ for chromosomes— to cluster CTCF and rewire genome architecture. This CTCF-centric reorganization in reference to nuclear speckles functionally sustains the senescence splicing program, as SICC disruption fully reverts alternative splicing patterns. We therefore uncover a new paradigm, whereby cells translate changes in nuclear biochemistry into architectural changes directing splicing choices so as to commit to the fate of senescence.

Cancer originates from normal cells due to the accumulation of genetic alterations, or mutations, caused by different mutational processes acting throughout an individual's lifetime. However, only a small subset of these mutations, known as drivers, can give somatic cells a selective growth advantage and lead to the formation of a tumour. In addition to this, heritable non-genetic changes (such as modifications to histones and chromatin architecture), due to preceding genetic alterations or other mechanisms, can help tumour cells further evolve by adapting to different stresses from the tumour microenvironment. To better understand and combat tumour evolution, it is crucial to comprehend the fundamental processes underlying the origin of genomic alterations and their mechanisms of action. Although some of these aspects have been studied independently, there is still limited understanding of their mechanisms in the context of chromatin structure, which affects DNA accessibility to various biological processes such as DNA replication, DNA damage and repair, and gene regulation. To address this gap, my lab aims to study how chromatin structure influences the origin and impact of genomic alterations in tumour evolution, by using computational and functional genomics. In this talk, I will present some of our efforts in decoding the influence of chromatin structure in somatic mutational processes and gene-regulation in cancers; and identifying prognostic markers through large-scale genome analyses.

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 mediated insulation, we replaced the 25kb boundary with a 672 bp transgene containing the four CTCF motifs, flanked by loxP sites. Re-introduction of CTCF motifs rescued the phenotypes and confirmed the role of CTCF boundaries. The Fgf3/4/15 genes were ectopically over-expressed in the midbrain and recapitulated the expression pattern of Ano1 gene, located in an upstream domain. This suggested that loss of CTCF resulted in aberrant contact of the Fgf genes with Ano1 enhancers. We performed region capture Micro-C in midbrain and visualized complete loss of loops, domain fusion and ectopic interaction of potential Ano1 enhancer with Fgf3 gene in mutant embryos. Interestingly, deletion of Ano1 enhancers along with CTCF motifs completely prevented the deleterious phenotypes. Surprisingly, among the four motifs in the boundary, deletion of one CTCF motif oriented towards Ano1 enhancer was enough to recapitulate encephalocele defect. Our work depicts how the effect of chromatin structure perturbation on gene regulation is highly dependent on developmental context, and that loss of a single CTCF motif in a gene-rich domain boundary can perturb higher-order chromatin organization and cause a detrimental effect in fetal development.

B cells undergoing physiologically programmed or aberrant genomic alterations provide an opportune system to study the causes and consequences of genome mutagenesis. Activated B cells in germinal centers express activation-induced cytidine deaminase (AID) to accomplish physiological somatic hypermutation (SHM) of their antibody-encoding genes. In attempting to diversify their immunoglobulin (Ig) heavy- and light-chain genes, several B-cell clones successfully optimize their antigen-binding affinities. However, SHM can sometimes occur at non-Ig loci, causing genetic alternations that lay the foundation for lymphomagenesis, particularly diffuse large B-cell lymphoma. Thus, SHM acts as a double-edged sword, bestowing superb humoral immunity at the potential risk of initiating disease. We refer to off-target, non-Ig AID mutations - that are often but not always associated with disease - as aberrant SHM (aSHM). A key challenge in understanding SHM and aSHM is determining how AID targets and mutates specific DNA sequences in the Ig loci to generate antibody diversity and non-Ig genes to initiate lymphomagenesis. Herein, we discuss some current advances regarding the regulation of AID's DNA mutagenesis activity in B cells.

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 all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms were dependent on OsCLSY3. We also observed many imprinted genes and seed development-associated genes under the control of CLSY3-dependent RdDM. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

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 super-silencer-controlled genes related to cell cycle, apoptosis and DNA damage, leading to anticancer effects both in vitro and in vivo. Overall, our data demonstrated the first example of a “super-silencer” and showed that combinational usage of GSK343 and X5050 to disrupt “super-silencers” could potentially lead to cancer ablation.

Experiments have long suggested a close association between intrinsically disordered proteins (IDRs) and oncogenesis. However, the precise mechanism by which IDRs contribute to cancer remains unclear. Recent studies have indicated that the phase separation ability of NUP98-HOXA9, an aberrant chimera containing a homeodomain and an IDR region, may be a key factor in the development of leukemia. In this work, we employ simple polymer physics to demonstrate that self interaction among chimeric proteins is crucial for enhancer-promoter contacts within the oncogene domain. We compare our findings with experimental data and further show that the same phase-separating property of chimeric proteins can potentially be harnessed to prevent enhancer-promoter contacts, thereby offering a potential strategy for cancer prevention.

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 chromatin.

The three-dimensional organization of the genome is fundamental to regulating gene expression, replication, and cellular function. DNA supercoiling, an integral aspect of chromatin structure, has emerged as a crucial modulator of this spatial organization. In our research, we investigate how supercoiling influences the 3D architecture of the genome in budding yeast, focusing on its impact on chromatin dynamics and the formation of functional domains and loops. We explore the interplay between supercoiling and chromatin changes, examining how these interactions drive gene regulation. By synthesizing insights from recent studies, we aim to elucidate the mechanisms through which DNA supercoiling shapes the 3D genome, offering potential insights into complex biological processes and disease mechanisms.