Biology subjects

Dinda, M.

Publications and source records attributed to Dinda, M..

4 recordsLinked to original sources

bioRxiv2025

Age-dependent topoisomerase I depletion alters recruitment of rDNA silencing complexes

Genomic instability and loss of proteostasis are two of the primary Hallmarks of Aging. Although these hallmarks are well-defined in the literature, the mechanisms that drive genomic instability and loss of proteostasis as cells age are still largely unknown. Using budding yeast replicative lifespan as a model for aging in actively dividing cells, we identified nuclear proteins that were depleted in the earliest stages of aging. We found that many age-depleted proteins were involved in ribosome biogenesis, specifically in ribosome processing, or in maintenance of chromatin stability. We focused on topoisomerase I (Top1) as a novel age-depleted nuclear protein and found that its depletion in the early stages of aging was not a result of transcriptional changes or changes in protein turnover. Despite the stark depletion of Top1 in early aging, rescue of this age-dependent depletion was actually harmful to replicative lifespan. We found that Top1, when overexpressed, disrupts the stoichiometry of the RENT complex by pulling Sir2 away from the ribosomal DNA (rDNA), a phenotype which is further enhanced when the overexpressed Top1 is catalytically dead. Loss of Sir2 from the rDNA via the overexpression of catalytically dead Top1 decreases RNA Pol II silencing of a reporter gene inside or adjacent to the rDNA, consistent with the lifespan defect. Finally, we found that the catalytic activity of Top1 plays an important role in the establishment of rDNA silencing, raising the possibility that rDNA secondary structure/DNA topology is important for RNA Pol I-dependent spreading of silent chromatin across the rDNA locus.

molecular biology↗

bioRxiv2025

Discovery and therapeutic exploitation of Master Regulatory miRNAs in Glioblastoma

Glioblastoma is a fatal primary malignant brain tumor, with an average survival of only 15 months despite surgical resection, chemotherapy, and radiation therapy. Due to the concurrent deregulation of numerous genes in glioblastoma, molecular monotherapies have not improved clinical outcomes. Evidence suggests that effectively targeting multiple deregulated molecules is essential for better therapies; however, this is limited by the lack of suitable drugs and the increased toxicity of combination therapies. To address this, we hypothesized that miRNAs, small gene-regulatory RNAs that suppress multiple target genes via sequence complementarity, could be developed to inhibit multiple deregulated genes simultaneously, leading to more effective treatments. We identified master regulatory miRNAs--those that target several deregulated genes in glioblastoma--using PAR-CLIP screenings in glioblastoma cells and analyzed TCGA tumor data to find which targets were deregulated. An algorithm ranked these targets based on their significance in glioblastoma malignancy. We selected two tumor suppressor master regulatory miRNAs, miR-340 and miR-382, and one oncogenic miRNA, miR-17. Validation showed that these miRNAs target critical glioblastoma pathways and significantly inhibit cell growth, survival, invasion, and tumor growth in vivo. We developed an innovative therapeutic delivery approach using Brain Penetrating Nanoparticles in combination with MRI-guided focused ultrasound and microbubbles, resulting in reduced tumor volume and extended survival in glioblastoma-bearing mice. This strategy offers a promising pathway for translating miRNA-based therapies into clinical trials for glioblastoma and other cancers. One Sentence SummaryWe developed and used new computational, experimental, and therapeutic approaches to identify and therapeutically deliver master regulatory miRNAs to inhibit the growth of glioblastoma, the most common and deadly primary brain tumor.

cancer biology↗

bioRxiv2022

Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III

Despite recent advances in single-molecule and structural analysis of condensin activity in vitro, mechanisms of functional condensin loading and loop extrusion that lead to specific chromosomal organization remain unclear. In Saccharomyces cerevisiae, the most prominent condensin loading site is the rDNA locus on chromosome XII, but its repetitiveness deters rigorous analysis of individual genes. An equally prominent non-rDNA condensin site is located on chromosome III (chrIII). It lies in the promoter of a putative non-coding RNA gene called RDT1, which is in a segment of the recombination enhancer (RE) that dictates MATa-specific chrIII organization. Here, we unexpectedly find that condensin is recruited to RDT1 through interactions with Fob1, Tof2, and cohibin (Lrs4/Csm1), a set of nucleolar factors that also recruit condensin to the rDNA. Using Micro-C XL, we uncover evidence for condensin-driven loop extrusion anchored by Fob1 and cohibin at RDT1 that extends toward MATa on the right arm of chrIII, supporting donor preference during mating-type switching. S. cerevisiae chrIII therefore provides a new platform for the study of programmed condensin-mediated chromosome conformation.

molecular biology↗

bioRxiv2019

A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MAT using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We go on to demonstrate that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses the transcription of RDT1 until it is redistributed to a dsDNA break at the MAT locus induced by the HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type switching efficiency and donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating type switching, thus providing a novel model for dissecting condensin function in vivo. Author summarySir2 is a highly conserved NAD+-dependent protein deacetylase and defining member of the sirtuin protein family. It was identified about 40 years ago in the budding yeast, Saccharomyces cerevisiae, as a gene required for silencing of the cryptic mating-type loci, HML and HMR. These heterochromatic cassettes are utilized as templates for mating-type switching, whereby a programmed DNA double-strand break at the MATa or MAT locus is repaired by gene conversion to the opposite mating type. The preference for switching to the opposite mating type is called donor preference, and in MATa cells, is driven by a cis-acting DNA element called the recombination enhancer (RE). It was believed that the only role for Sir2 in mating-type switching was silencing HML and HMR. However, in this study we show that Sir2 also regulates expression of a small gene (RDT1) in the RE that is activated during mating-type switching. The promoter of this gene is also bound by the condensin complex, and deleting this region of the RE drastically changes chromosome III structure and alters donor preference. The RE therefore appears to function as a complex locus control region (LCR) that links transcriptional control to chromatin architecture, and thus provides a new model for investigating the underlying mechanistic principles of programmed chromosome architectural dynamics.

molecular biology↗