Biology subjects

Panigrahi, L.

Publications and source records attributed to Panigrahi, L..

3 recordsLinked to original sources

bioRxiv2025

Centromeres and pericentromeric heterochromatin are accessible in living Schizosaccharomyces pombe cells

Chromatin is intrinsically repressive, limiting access to DNA, implying a major regulatory role. Studies with nuclei support this model. However, we have shown previously that genomic DNA is completely accessible in living budding yeast and human cells, except for centromeric chromatin. The fission yeast, Schizosaccharomyces pombe, provides a tractable model for heterochromatin. As in mammalian cells, S. pombe heterochromatin is marked by histone H3K9 di- and tri-methylation (H3K9me2/3), introduced by the Clr4 histone methylase, and heterochromatin protein 1 (HP1/Swi6). Here, we developed a copper-inducible DNA methyltransferase system to measure heterochromatin accessibility in living S. pombe cells. We find that euchromatin and heterochromatin are similarly and generally accessible in vivo, indicating that S. pombe chromatin is globally dynamic. S. pombe centromeres are also fully accessible, unlike budding yeast and human centromeres. In contrast to living cells, S. pombe chromatin is mostly inaccessible in isolated nuclei, primarily due to tight nucleosome spacing with very little linker DNA. Loss of Clr4 or Swi6 has little effect on genome accessibility in live cells or nuclei, suggesting that the S. pombe H3K9me/HP1 system does not repress transcription by preventing access to DNA.

genomics↗

bioRxiv2023

Wat1/mLst8, a TOR complex protein regulates mitochondrial integrity and calcium ion homeostasis in fission yeast S. pombe

The mTOR complexes play a fundamental role in mitochondrial biogenesis and cellular homeostasis. Wat1, an ortholog of mammalian Lst8 is an important component of TOR complex and is essential for the regulation of downstream signaling. Earlier we reported the role of Wat1 in oxidative stress response. Here, we show that the inactivation of wat1 leads to respiratory defects and mitochondrial depolarization leading to decrease in ATP production. The confocal and electron microscopy in wat1{Delta} cells revealed the fragmented mitochondrial morphology implying its role in mitochondrial fission. Furthermore, we also showed its role in autophagy and the maintenance of calcium ion homeostasis. Additionally, tor2-287 mutant cells also exhibit defects in mitochondrial integrity indicating the TORC1-dependent involvement of Wat1 in the maintenance of mitochondrial homeostasis. The interaction studies of Wat1 and Tor2 with Por1 and Mmm1 proteins revealed a cross-talk between mitochondria and endoplasmic reticulum through the Mitochondria-associated membranes (MAM) and endoplasmic reticulum-mitochondria encounter structure (ERMES) complex, involving TORC1. Taken together, this study demonstrates involvement of Wat1/mLst8 in harmonizing various mitochondrial functions, redox status, and Ca2+ homeostasis.

cell biology↗

bioRxiv2022

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

{beta}-thalassemia and HbE result from mutations in the {beta}-globin locus that impedes the production of functional {beta}-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of {beta}-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

genomics↗