Biology subjects

Saravanan, B.

Publications and source records attributed to Saravanan, B..

6 recordsLinked to original sources

bioRxiv2026

Single-molecule spatial genomics reveals the multi-scale organization and plasticity of extrachromosomal DNA in glioblastoma

Extrachromosomal DNA (ecDNA) is a major driver of intratumoral heterogeneity and is associated with poor clinical outcomes across cancers, yet how individual ecDNA molecules are organized and regulated within intact tumors remains unknown. Here, we leveraged single-molecule, multi-modal spatial genomics to resolve the three-dimensional chromatin organization and transcriptional activity of individual EGFR-containing ecDNA molecules in glioblastoma (GBM) cells in vitro, in orthotopic xenografts, and in patient-derived GBM tissue. At the larger scale, we find that distinct GBM molecular and functional states emerge depending on the local cellular environment. EGFR expression was markedly different between GBM subpopulations, and perturbations of EGFR dosage shifted GBM cellular states. ecDNA expression was modulated by multiple mechanisms, including variation in copy number, chromatin organization, DNA sequence, and chromosomal reintegration, which were simultaneously measured within the same cells. At the single-molecule scale, ecDNA adopts a physically expanded chromatin configuration with larger ecDNA molecules having higher transcriptional activity and interaction with active transcriptional machinery. ecDNA regulation was coordinated within cells and across GBM states, and ecDNA copy number, structure, and transcription were spatially organized across the tumor architecture. Co-culturing GBM cells with neurons recapitulated key features of infiltrative regions, including lower EGFR expression, reduced ecDNA copy number, and increased chromosomal reintegration, suggesting a causal role for the microenvironment in shaping ecDNA regulation. Collectively, these findings support a model in which GBM states and ecDNA are linked, plastic, and influenced by microenvironmental contexts, revealing a previously inaccessible layer of genome organization underlying tumor heterogeneity and malignant cell behavior.

genomics↗

bioRxiv2025

Context-Dependent and Gene-Specific Role of Chromatin Architecture Mediated by Histone Modifiers and Loop-extrusion Machinery

Loop-extrusion machinery, comprising the cohesin complex and CCCTC-binding factor CTCF, organizes the interphase chromosomes into topologically associating domains (TADs) and loops, but acute depletion of components of this machinery results in variable transcriptional changes in different cell types, highlighting the complex relationship between chromatin organization and gene regulation. Here, we systematically investigated the role of 3D genome architecture in gene regulation in mouse embryonic stem cells under various perturbation conditions. We found that acute depletion of cohesin or CTCF disrupts the formation of TADs, but affects gene regulation in a gene-specific and context-dependent manner. Furthermore, the loop extrusion machinery was dispensable for transcription from most genes in steady state, consistent with prior results, but became critical for a large number of genes during transition of cellular states. Through a genome-wide CRISPR screen, we uncovered multiple factors that can modulate the role of loop extrusion machinery in gene regulation in a gene-specific manner. Among them were the MORF acetyltransferase complex members (Kat6b, Ing5, Brpf1), which could antagonize the transcriptional insulation mediated by CTCF and cohesin complex at developmental genes. Interestingly, inhibition of Kat6b partially rescues the insulator defects in cells lacking the cohesin loader Nipbl, mutations of which are responsible for the developmental disorder Cornelia de Lange syndrome. Taken together, our findings uncovered interplays between the loop extrusion machinery and histone modifying complex that underscore the context-dependent and gene-specific role of the 3D genome.

genomics↗

bioRxiv2023

RNA binding limits the ligand induced transcriptional potential of estrogen receptor-alpha

Transcription factors (TFs) primarily regulate gene expression by binding to DNA through their DNA binding domains (DBD). Additionally, approximately half of these TFs also interact with RNA. However, the role of RNA in enabling TF binding on chromatin and subsequent transcription is poorly understood. Estrogen receptor- (ER) is one such TF that activates genes in response to estrogen stimulation. Here, we report that ER interacts with various types of RNAs in ligand dependent manner via its RNA binding motif in hinge region. RNA binding defects lead to a global loss of ER binding in the genome, particularly at weaker ER motifs. In the absence of RNA binding, the ER exhibits dynamic behavior in the nucleus and unexpectedly, the dynamic binding coincides with robust polymerase loading on ER bound chromatin regions. The higher occupancy of PolII was recapitulated by robust ligand induced transcription of ER-regulated genes. Collectively, our results suggest that RNA interactions strengthen ER binding to chromatin limiting the ligand-dependent transcriptional upregulation of estrogen-induced genes.

molecular biology↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗

bioRxiv2021

Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

genomics↗

bioRxiv2019

Ligand dependent gene regulation by transient ER clustered enhancers

Unliganded nuclear receptors have been implicated in ligand-dependent gene regulation. However, the underlying mechanisms are not fully understood. Here we demonstrate that unliganded ER binds to specific sites in the genome thereby pre-marking them as future functional enhancers. Upon ligand exposure, ER binds to several EREs relatively proximal to the pre-marked, or persistent, ER-bound sites. Interestingly, the persistent sites interact extensively, via chromatin looping, with the proximal transiently bound sites forming ER clustered enhancers in 3D. CRISPR-based deletion of TFF1 persistent site disrupts the formation of its clustered enhancer resulting in the loss of E2-dependent induced expression of TFF1 and its neighboring genes within the same cluster. The clustered enhancers overlap with nuclear ER puncta that coalesce in a ligand-dependent manner. Furthermore, formation of clustered enhancers, as well as puncta, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ER. Our results establish the role of persistent unliganded ER binding in priming enhancer clusters in 3D that drive transient, but robust, gene expression in a ligand-dependent fashion.

molecular biology↗