Biology subjects

Misra, C. S.

Publications and source records attributed to Misra, C. S..

6 recordsLinked to original sources

bioRxiv2025

Histone H2A Ubiquitination Mediates the Establishment of Reactivation-Competent HSV-1 Latent Infection

Herpes simplex virus 1 (HSV-1) establishes a latent infection of neurons, where it persists for life with the capability to reactivate. During latent infection, heterochromatin is deposited onto the viral genome to presumably permit viral gene silencing. Repressive histone modifications associated with the latent genome include the constitutive heterochromatin modifications, H3K9me2/3, and the facultative heterochromatin modification, H3K27me3. The role of these different types during entry into latency and as substrates for reactivation is unknown. H3K27me3 is associated with Polycomb silencing, and a second modification also associated with Polycomb silencing, especially in pluripotent cells and during early development, is H2AK119ub1. Here we found that H2AK119ub1 is enriched on a sub-population of latent HSV genomes. We examined the contribution of both H2AK119ub1 and H3K7me3 deposition to HSV-1 gene silencing during entry into latency. We found that H2AK119ub1 was deposited prior to H3K27me3 and that only inhibition of H2AK119ub1 deposition plays a subtle role in latency establishment. Importantly, we found that inhibiting the enzymatic activity of Polycomb Repressive Complex 1 (PRC1) that deposits H2AK119ub1 during latency establishment prevented later reactivation. In contrast, inhibiting the activity of PRC2, which deposits H3K27me3, did not impact reactivation. Together, these data demonstrate the heterogenic nature of the epigenetic structure of latent HSV genomes and provide evidence that those associated with H2AK119ub1 are the template for reactivation.

microbiology↗

bioRxiv2023

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster and show that knockdown of most of the genes in this cluster leads to growth defects. We further characterised one of the genes, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and show its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

molecular biology↗

bioRxiv2022

CRISPRi in Deinococcus radiodurans

The extremely radiation resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting. In view of this, the CRISPR-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A Cas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post irradiation. Targeting the Radiation and Desiccation Resistance Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPRi tool developed in this study will facilitate study of essential genes, hypothetical genes, cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further the tool is amenable for implementing high-throughput approaches for such studies.

molecular biology↗

bioRxiv2022

The conserved transcriptional program of metazoan male germ cells uncovers ancient origins of human infertility

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.

systems biology↗

bioRxiv2020

Comparative transcriptomic analysis reveals conserved transcriptional programs underpinning organogenesis and reproduction in land plants

The evolution of plant organs, including leaves, stems, roots, and flowers, mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. Furthermore, the fertilization products of angiosperms, seeds serve as the basis for most of our food. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes, and the development of novel regulatory programs. However, our knowledge of these events is limited, as no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we have generated gene expression atlases for various organs and gametes of 10 plant species comprising bryophytes, vascular plants, gymnosperms, and flowering plants. Comparative analysis of the atlases identified hundreds of organ- and gamete-specific gene families and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, the appearance of organ-specific gene families does not coincide with the corresponding organs appearance, suggesting that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, suggesting that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function. To provide easy access to the expression atlases and these comparative analyses, we provide an online database, www.evorepro.plant.tools, that allows the exploration of expression profiles, organ-specific genes, phylogenetic trees, co-expression networks, and others.

evolutionary biology↗

bioRxiv2019

Determination of Cas9/dCas9 associated toxicity in microbes

The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for various applications. In case of the latter, a couple of previous studies had shown Cas9 protein expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results showed differential levels of Cas9 toxicity among the bacteria and lower transformation efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, Cas9/dCas9 associated toxicity was acute.

microbiology↗