ICTS

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.

The DNA in a human cell which is ~2 meters long is packed in a ~10 μm nucleus. It is immersed in a condensed soup of proteins, RNA and enzymes and it is highly dynamic. Nevertheless, it must stay organized to prevent chromosome entanglement and for ensuring proper genome expression.
Studying the DNA (or chromatin) organization requires to use high spatial and temporal resolutions combined with live-cell imaging. We combine comprehensive live-cell and molecular methods in order to explore the structure. As part of the study, we use single particle tracking and perform diffusion analysis. Interesting sub-diffusion is identified with bi-modal behavior that we fit to a model that takes into account the existence of significant forces in the nucleus that acts on chromatin.
The results allowed us to identify the protein lamin A as the main player that affect the chromatin organization in the nucleus. It forms chromatin loops in the whole nuclear volume thereby restricting the chromatin dynamics and increasing its elasticity and rigidity. Together with other mechanisms, it also takes part in controlling gene expression.
It emphasizes the importance of the multi-scale organization of the genome in the nucleus.

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA Polymerase II complex assembly and productive elongation. These studies expose fundamental questions about transcription regulation. Namely, how do transient interactions (~ seconds) between transcription factors and promoters lead to specific responses? We propose an extension of kinetic proofreading to explain transcriptional regulation in eukaryotes. This model suggests active, ATP-consuming processes drive transcription factor dynamics. In this model, promoters are dwell time detectors, not occupancy detectors. We present preliminary data from live-cell imaging in support of this view.

The vast majority (~98%) of the mammalian genome is noncoding but has a vital role in gene regulation. Enhancers are noncoding DNA sequences highly enriched in binding sites for tissue-specific transcription factors. Genes with pleiotropic roles in development often have huge regulatory landscapes (~ 1Mb) with multiple enhancers driving precise gene expression.
The human genome is thought to contain 100s of thousands of enhancers with, as yet, uncharacterised activities and a large proportion of disease-causing and disease-predisposing DNA sequence variants map to potential enhancers. To advance understanding of human disease, massive efforts are currently being directed towards establishing genotype to phenotype correlations for sequence variants. A key pre-requisite to these analyses is defining the precise cell- and tissue-specific activities of the enhancers and the transcription factors that bind them.
Our research focuses on building models for understanding cell-type specific enhancer functions in the developing eye using one of the paradigm loci for enhancer- mediated gene regulation- PAX6, the master regulator of eye development. PAX6 has a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive, redundant, or distinct functions is unknown. Heterozygous loss-of function mutations in the coding region of PAX6 are the major cause of a congenital eye malformation, Aniridia. Aberrant regulation of PAX6 expression caused by re-arrangements, deletions or point mutations in the PAX6 regulatory domain has been reported as the pathogenic mutation in exome-negative Aniridia patients.
We have developed in vivo (zebrafish) and ex vivo (optic cup organoids) models enabling visualization of the precise spatial and temporal roles of PAX6 enhancers in the developing eye. Using a combination of high-resolution live imaging, single cell RNA- sequencing and synthetic biology approaches in these model systems we uncover precise cell types and regulatory activities of enhancers regulating PAX6 expression in the developing eye.

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.

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 matrix (ECM). The role of the epigenetic readers in fibrosis-mediated matrix stiffening will also be elucidated, which has a direct consequence in altered cellular invasive and metastatic properties. Thus, the chromatin readers modulate the epigenetic landscape of cancer and have a great therapeutic prospect in future.

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 condense properly during mitosis. As a result, they exhibit enhanced accessibility and transcription of active genes in the next interphase. We propose that the spreading of mitotic H3S10p into active domains preserves their identity during mitosis and, in subsequent interphase, acts as a rheostat to fine-tune transcription and chromatin domain re-formation.

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.

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.

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 the ParA gradient to move towards the nearest pole, the ParB/SMC-mediated inter-sister repulsion ensures the two Ori occupy distinct poles. The model makes testable predictions for sister-chromosome-resolved Hi-C experiments and proposes that replicated sister chromosomes are segregated via a mechanistic interplay of SMC and ParAB activity.

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.

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 maintenance of chromosomal stability, nuclear organization and genome function.