Biology subjects

Bisht, D.

Publications and source records attributed to Bisht, D..

8 recordsLinked to original sources

bioRxiv2025

BRG1 cooperates with PLK1 to regulate dormant origin activation during S phase.

The ATP-dependent chromatin remodeler BRG1 is well-established in the regulation of gene expression, DNA replication, homologous recombination, DNA damage repair, and apoptosis. While its genome-wide occupancy has primarily been linked to transcriptional regulation, its potential role in DNA replication control remains less well defined. In this study, we investigated BRG1s involvement in replication origin regulation by integrating BRG1 ChIP-seq data with replication origin mapping and chromatin state analyses. Our results revealed that BRG1 is preferentially enriched at genomic regions characterized by DNase I hypersensitivity, low GC content, and a capacity to form G-quadruplex structures hallmarks of dormant replication origins. Notably, BRG1-bound sites were also enriched in late-replicating, heterochromatic regions. These findings suggest a specific association of BRG1 with dormant replication origins. To validate this association, we performed functional assays in HeLa cells. BRG1 depletion via siRNA led to reduced replication fork progression and increased activation of dormant origins, consistent with replication stress. Mechanistically, we found that BRG1 physically interacts with the mitotic kinase PLK1. Loss of BRG1 reduced chromatin-bound levels of both BRG1 and PLK1, implicating BRG1 in PLK1 recruitment or stability. Importantly, overexpression of PLK1 in BRG1-depleted cells rescued replication fork progression, supporting a cooperative role for BRG1 and PLK1 in regulating replication through dormant origins. Together, these data uncover a previously unrecognized function for BRG1 in the selective regulation of dormant replication origin firing, acting in concert with PLK1.

molecular biology↗

bioRxiv2025

Expanding the DNA Motif Lexicon of the Transcriptional Regulatory Code

Transcriptional regulatory sequences in metazoans contain intricate combinations of transcription factor (TF) motifs. Stereospecific arrangements of simple motifs constitute composite elements (CEs) that enhance DNA-protein interaction specificity and enable combinatorial regulatory logic. Despite their importance, CEs remain underexplored. We advance CE discovery and functional characterization by developing an integrated framework that combines computational prediction, experimental testing and deep learning. The extended TF motif catalog comprises both synergistic and counteracting CEs, which are supported by evidence of TF binding in vivo and in vitro. A deep learning model GRACE trained on customized massively parallel reporter assays learns the lexicon of CEs at single-nucleotide resolution. Comparative analysis with a neural network model trained on chromatin accessibility demonstrates striking convergence and distinctions within the expanded regulatory lexicon, enabling joint predictions of motif contributions and the impact of variants on chromatin structure and transcriptional activity in diverse cellular contexts.

genomics↗

bioRxiv2025

Titin as a mechanical damper: Balancing Stability and longevity through inter-domain linker design

Titin, a giant protein ([~]3-4 MDa), functions as a molecular-spring to regulate muscle elasticity. More than 90% of Titin is composed of domains that absorb mechanical energy and undergo stochastic unfolding-refolding under tension ([~]tens of pN). These domains are connected in tandem by interdomain linkers (IDLs), which constitute less than 10% of the total mass. Despite their small genomic footprint, bioinformatics mapping suggests that IDLs have an outsized impact on protein mechanics, potentially contributing to disease pathology. Using magnetic tweezers, here we examine how linkers influence mechano-response of domains to constant and oscillatory forces. We found that short linkers limit interdomain movement and promote first-order cooperative folding transitions of domains. In contrast, long flexible linkers induce creep-like deformations interspersed with sharp, stepwise transitions. Surprisingly, linkers that improve domain-stability resist unfolding under constant pulling forces, but lose power retention faster under oscillatory forces. Our findings reveal a trade-off between mechanical stability and energy retention in titin, a key muscle protein. These insights offer new design principles for mechano-responsive protein engineering. TeaserTiny linkers fine-tune how bulky domains in titin respond to force.

biophysics↗

bioRxiv2024

Understanding genetic diversity and phylogeography of Common Teal and its phylogenetic relationship with other water bird species in the wetlands of Kashmir Himalayas.

Understanding the genetic diversity and phylogeography of migratory species is critical for biodiversity conservation and the effective management of wetland ecosystems. The Kashmir Himalayas, an integral part of the Central Asian Flyway, host several key wetlands that provide critical wintering grounds for a variety of migratory birds. This study focuses on assessing the genetic characteristics and phylogenetic relationships of the Common Teal (Anas crecca) in comparison to other species within the families Anatidae and Rallidae. We analysed 149 blood samples, including 71 from A. crecca and 78 from other species in the two families, collected from wetlands in the Kashmir region. Using four mitochondrial markers--cytochrome oxidase subunit I (COI), cytochrome b (Cyt b), 16S rRNA, and the control region--we evaluated the genetic diversity and lineage connectivity of these species. Our findings reveal that the mitochondrial DNA haplotypes of A. crecca in the Kashmir Himalayas are shared with European populations, indicating strong maternal gene flow and connectivity between distant populations. A minimum spanning haplotype network analysis showed minimal nucleotide differences among haplotypes, particularly in the Cyt b and control regions, suggesting low genetic differentiation and a high degree of similarity among individuals. Notably, we identified at least four distinct maternal lineages of A. crecca in the Kashmir wetlands, reflecting diverse migratory sources. Our results also highlight that DNA barcoding using COI exhibited both high and low species resolution, with significant intraspecific variation, making it a valuable tool for further phylogeographic studies. The observed genetic diversity and haplotype sharing with distant populations underscore the ecological importance of Kashmirs wetlands as crucial habitats for migratory species. Our study emphasizes the need for targeted conservation and management strategies to preserve these vital ecosystems and the biodiversity they support.

genetics↗

bioRxiv2024

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

systems biology↗

bioRxiv2021

PICH, an ATP-dependent chromatin remodeling protein, transcriptionally co-regulates oxidative stress response.

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation often implemented by chromatin remodeling proteins. The study presented in this paper shows that the expression of PICH, an ATP-dependent chromatin remodeler, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response, both in the absence and presence of oxidative stress. In turn, Nrf2 regulates the expression of PICH in the presence of oxidative stress. Both PICH and Nrf2 together regulate the expression of antioxidant genes and this transcriptional regulation is dependent on the ATPase activity of PICH. In addition, H3K27ac modification also plays a role in activating transcription in the presence of oxidative stress. Co-immunoprecipitation experiments show that PICH and Nrf2 interact with H3K27ac in the presence of oxidative stress. Mechanistically, PICH recognizes ARE sequences present on its target genes and introduces a conformational change to the DNA sequences leading us to hypothesize that PICH regulates transcription by remodeling DNA. PICH ablation leads to reduced expression of Nrf2 and impaired antioxidant response leading to increased ROS content, thus, showing PICH is essential for the cell to respond to oxidative stress.

molecular biology↗

bioRxiv2021

Comparative evaluation of plasma protein purification and 2-D gel electrophoresis protocols for analysis of HIV-1 infected human plasma proteins

Purification of proteins from human plasma is a herculean task to perform 2-D gel electrophoresis. Human plasma contains nearly 70% albumin and globulin. The removal of such high abundance high molecular weight proteins is very difficult before performing 2-D gel electrophoresis. It becomes more difficult when we intent to investigate in infectious diseases like HIV/AIDS. We tried to the best of our efforts adopting various organic and non-organic based protocols based on various published papers. After failure of these protocols in results of 2-D gel-electrophoresis Aurum serum mini kit (Bio-Rad, USA) was adopted for plasma protein purification for performing 2-D gel electrophoresis. The low-abundance proteins were better resolved by 10% SDS-PAGE in 2-D gel-electrophoresis. Then,we extended the MALDI-TOF/TOF analysis of low-abundance proteins in human plasma by adopting the Aurum serum mini kit (Bio-Rad, USA) for 2-D gel electrophoresis. Thus, we concluded that, depletion of high abundant proteins like albumin and globulin, the use of the Aurum serum mini kit (Bio-Rad, USA) is the protocol of choice to perform the 2-D gel electrophoresis of HIV-1 infected human plasma.

biochemistry↗

bioRxiv2019

Altering mammalian transcription networking with ADAADi: An inhibitor of ATP-dependent chromatin remodeling

Transcriptional control has been earnestly pursued for the regulation of cellular proliferation associated with cancer progression. The foundational paradigm of targeting transcription factors has yielded exquisite specificity, but many factors cannot yet be targeted. In contrast, targeting epigenetic factors to control chromatin structure and consequential gene expression generally yields more global effects on transcription. Our working paradigm targets neither specific transcription factors nor global epigenetic factors but ATP-dependent chromatin remodeling factors that regulate expression of a limited set of genes. Active DNA-dependent ATPase A Domain inhibitor (ADAADi) synthesized by aminoglycoside phosphotransferases is the first-in-class inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. Mammalian cells are sensitive to ADAADi but cell lines are variable in their individual responses to the inhibitor. The ADAADi product can be generated from a variety of aminoglycoside substrates with cells showing differential responses to ADAADi depending on the starting aminoglycoside. RNA seq analysis demonstrated that targeting the chromatin remodeling by treatment with a sub-lethal concentration of ADAADi yields alterations to the transcriptional network of the cell. Predominantly, the tumor-promoting genes were repressed while pro-apoptotic and tumor suppressors genes were upregulated on treatment with ADAADi, leading to apoptotic-type cell death. Treatment with ADAADi reversed the EMT process as well as inhibited migration of cells and their colony forming ability. In conjunction with the previous report that treatment with ADAADi regresses tumors in mouse model, this chromatin remodeling inhibitor shows promising anti-tumor properties by targeting the main hallmarks of cancer.

molecular biology↗