Search Talks

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 ...

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...

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 re...