Biology subjects

Mazumder, A.

Publications and source records attributed to Mazumder, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Reverse-engineering amyloid strains with generative protein design

Amyloid fibrils are intrinsically polymorphic protein assemblies that form distinct structural strains linked to diverse biological and pathological outcomes. Yet, the principles governing how sequence encodes diverse fibril architectures, and the extent to which a given fold constrains underlying amino-acid sequence compatibility, remain poorly understood. Here, we apply generative protein design to directly interrogate the sequence-structure relationship of defined fibril architectures, using -synuclein (S), a protein known to form highly polymorphic amyloid fibrils, as a model system. Sampling sequence space under structural constraints reveals a continuous compatibility manifold in which diverse sequences encode a common amyloid architecture. De novo designed sequences assemble into fibrils, often with enhanced aggregation efficiency relative to S. A subset exhibits strain-like behaviour, including similar morphologies, efficient cross-templating, and induction of S cellular propagation, thereby functionally validating structural compatibility with the native fibril fold. Energetic analysis shows that stability is achieved through distinct but compensatory interactions, supporting a non-unique mapping between sequence and structure. Together, our results define a continuous and constrained compatibility landscape underlying amyloid strains, providing a framework for understanding the determinants of polymorphism and establishing generative protein design as a strategy to access this space, interrogate amyloid sequence-structure relationships, and engineer fibrillar protein assemblies and functional biomaterials.

biophysics↗

bioRxiv2026

Nucleosome remodeling by a CHD enzyme promotes H3K9 methylation establishment and spreading via remodeler-writer feedback

In Schizosaccharomyces pombe, the conserved CHD remodeler Mit1 function within the SHREC remodeler-deacetylase complex (a homolog of the metazoan Mi-2/NuRD complex), which is essential for H3K9 methylation-dependent heterochromatin establishment. However, the mechanism by which remodeler activity promotes silencing is unknown. Current models posit a hierarchical relationship between histone modifications and remodeler activity, with Mit1 acting exclusively downstream of H3K9 methylation. Here, we challenge this model by showing that tethering Mit1 at an ectopic site within euchromatin is sufficient to initiate heterochromatin assembly and generate extended domains of de novo H3K9 methylation. This process requires the Mit1 catalytic activity but does not involve direct physical interaction with Clr4, suggesting Mit1-mediated nucleosome remodeling creates a chromatin context that enhances Clr4 function. Using a genome-wide deletion screen, we determined that Mit1-initiated silencing requires all core heterochromatin factors and is critically dependent on Clr4 dosage. Furthermore, Mit1 activity facilitates heterochromatin spreading at subtelomeric regions and promotes H3K9 methylation at novel genomic sites implicated in cellular adaptation. Together, our findings support a model in which remodeler-writer pairs, analogous to reader-writer pairs, constitute conserved regulatory modules through which nucleosome organization directs the establishment of heritable epigenetic states.

genetics↗

bioRxiv2026

Nucleolar reorganization on stress depends on physicochemical changes due to nascent rRNA synthesis

Successive maturation of ribosomal subunits occurs through multilayered phase-separated structures of the cell nucleolus. The spatio-functional relationship between transcription of rRNA and nucleolar substructures and how this adapts to cellular stress remain incompletely understood. In this study, we resolve the sub nucleolar structures using expansion microscopy to reveal ordered structures of fibrillar center (FC) and dense fibrillar component (DFC) domains as nested shells, which is reorganized upon cellular stress like DNA damage or RNA polymerase I (RNAPI) inhibition. Direct visualization of nascent (5 ETS) and mature (28S) rRNA suggested that rRNA synthesis is the critical regulator of nucleolar size, and organization. Nucleolar reorganization upon stress emerges to be a direct function of nascent rRNA levels. Stress-induced transcription inhibition remodels the sub-nucleolar compartments from a viscoelastic state into solid-like condensates thereby perturbing the nucleolar pH gradient due to the missing rRNA scaffold. We show, that rather than signaling to mediate rDNA repair, nucleolar reorganization naturally arises primarily from reduced rRNA levels and the resultant biophysical restructuring of the nucleolus under cellular stress.

cell biology↗

bioRxiv2025

QTLIT: A user-friendly Shiny app for Quantitative Trait Loci (QTL) analysis

Identification of Quantitative Trait Loci from DNA sequence data is a multistep process, which is difficult for the plant breeders. To make the QTL identification breeders friendly, we developed QTLIT, a tool comprised of popular R packages and open sources software under a single umbrella for QTL analysis that enables intuitive exploration, multidimensional analyses, and visualization of results generated from any kind of DNA sequence data that deals with Single Nucleotide Polymorphism (SNP) variation. This tool requires raw or processed FASTQ files and proceed it for SNP identification to further perform QTL analysis on genomic regions of interest along with phenotyping data of various pedigree-based mapping populations. QTLIT generates a full range of plots such as VCF quality, linkage map, LOD score etc. It has broad range of applications and options for different parameters which the user can adjust as per their requirements. Moreover, it has two separate interfaces where one can utilize this tool to generate alignment files and/or variant calling for other downstream analysis. QTLIT is a fast and intuitive QTL analysis tool suitable for a wide range of users due to its architecture and its user-friendly graphical interface. QTLIT source code is available at https://github.com/TKM-Lab/QTLIT and the user interface version is available on http://14.139.229.202:8080/QTLIT.

genomics↗

bioRxiv2025

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

biophysics↗

bioRxiv2025

Mechanics-dependent Global Nuclear Eviction and Site-Specific Recruitment of YAP Regulates DNA Damage Responses

Yes-associated protein (YAP), a transcriptional coactivator, plays key roles in cell growth, proliferation and apoptosis, and its levels are frequently dysregulated in cancers. YAP levels in the nucleus are highly sensitive to nuclear mechanical cues, and such cues are also parallelly emerging to be a key modulator of DNA Damage Responses (DDR). However, whether DNA-damage can induce mechanical changes that regulate downstream events such as YAP localization and that in turn feeds back onto DDR activation, remains unknown. In this study, we report that YAP translocates in a nuclear mechanics-dependent manner upon induction of Double Strand Breaks (DSBs). This translocation is not a mere epiphenomenon, and we find that: first, global nuclear eviction of YAP enhances DDR signaling; second, local enrichment of YAP at DNA damage sites promotes recruitment of DNA repair proteins previously identified as potential interactors of YAP or its partner TEAD1. Together, these findings indicate that YAP is not only a transcriptional coactivator, but also plays an under-appreciated role in regulating DDR.

cell biology↗

bioRxiv2025

Discrete Subdomains Establish Epigenetic Diversity in Subtelomeric Heterochromatin

Subtelomeres are imperfect repeats adjacent to telomeres that are transcriptionally repressed by heterochromatin. Although essential for genome integrity, their repetitive nature has thwarted dissection of local heterochromatin assembly and maintenance mechanisms. By engineering Schizosaccharomyces pombe strains carrying fluorescent reporters at a single subtelomere, we uncovered distinct subdomains. These subdomains have different silencing requirements: Telomere-proximal regions rely on canonical shelterin- or RNAi-dependent nucleation pathways, whereas telomere-distal regions involve nucleosome remodelers, histone chaperones, and boundary-associated factors. Subdomains also exhibit discrete epigenetic states emerging both at homologous loci on different chromosome arms and along the same subtelomeric sequence. We document these epigenetic states using multi-generational live imaging and targeted perturbations. These analyses show that subtelomeric subdomains display position-specific, clonally variable silencing across a spectrum from robust to fragile epigenetic states. Interestingly, structural variants, common in subtelomeric sequences across eukaryotes, dictate local epigenetic stability. These findings reveal that subtelomeres, long recognized for their sequence variability, form a dynamic mosaic of coexisting epigenetic domains. This implies a wide range of gene-repressive regulatory logic at the chromosome ends, from environmental responsiveness to silencing stability.

genomics↗

bioRxiv2025

Heterotypic Protein Interactions Modulate the Condensate Dynamics and Aggregation of α-Synuclein

Multicomponent biomolecular condensates formed by diverse multivalent proteins underlie numerous cellular processes, from ribosome biogenesis to stress response regulation, and have recently been implicated in disease-related protein aggregation. Understanding how heterotypic protein interactions modulate condensate dynamics and transitions to amyloid states remains a major challenge. Here, we combine fluorescence -based ensemble and single-molecule approaches, molecular simulations, and systematic domain deletions to investigate how charge patterning and domain structure influence co-condensation between -synuclein, a disordered neuronal protein linked to Parkinsons disease, and the SARS-CoV-2 nucleocapsid protein (NP), a structured viral RNA-binding protein. We find that co-condensation is driven by multivalent electrostatic interactions and occurs when the heterotypic affinity exceeds a threshold, resulting in restricted protein dynamics and altered condensate material properties. These changes promote the formation of dense amyloid fibrils, as confirmed by atomic force microscopy and fluorescence assays. Our results elucidate how heterotypic interactions within multiphasic condensates can modulate phase behavior and aggregation, providing insight into broader mechanisms linking condensate dysregulation with neurodegenerative diseases.

biophysics↗

bioRxiv2024

HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage

Double-strand breaks (DSBs) pose significant threat to genomic stability and need immediate attention from DNA Damage Response (DDR) machinery involved in Homologous Recombination (HR) or Non-homologous end joining (NHEJ). DDR in heterochromatin is challenging owing to the distinct chromatin organization. Heterochromatin Protein 1 (HP1) isoforms that contribute significantly to the organization of heterochromatin, have been shown to be involved in DDR. Mammalian HP1 has three isoforms, HP1, HP1{beta}, and HP1{gamma}, which possess significant homology and yet have distinct functions. HP1 is the only isoform known to undergo liquid-liquid phase separation. We show that the minute-scale dynamics of HP1 and HP1{beta} differ dramatically and they promote differential recruitment of HR vs. NHEJ factors at the sites of laser-induced clustered DSBs. Perturbing HP1 phase-separation abrogates both the recruitment of HR factors and readouts of HR. Our study provides a link between phase-separation and DDR-centric roles of HP1 and hints at spatial partitioning of repair pathways in response to damage in heterochromatin.

cell biology↗

bioRxiv2024

Acute Activation of Genes Through Transcriptional Condensates Impact Non-target Genes in a Chromatin Domain

Transcription activation of genes by estrogen is driven by enhancers, which are often located within the same Topologically Associating Domain (TAD) as non-targeted promoters. We investigated how acute enhancer-driven activation affects neighbouring non-target genes within the same TAD. Using single-molecule RNA FISH (smFISH), we tracked the transcription of TFF1 (enhancer-target gene) and TFF3 (non-target gene) during estrogen stimulation. We observed mutually exclusive expression patterns: TFF1 expression peaked at 1 hour, while TFF3 reached its peak at 3 hours, after TFF1 activation had diminished. Chromatin looping data indicated that the enhancer loops with TFF1 but not TFF3, suggesting that TFF3 upregulation is not due to direct enhancer-promoter interactions. CRISPR deletion of the enhancer, affected TFF1 transcription more acutely than TFF3. 1,6-hexanediol (HD) exposure suggested that the TFF1 enhancer:promoter undergo a potential ER-mediated condensate formation, which sequesters the transcriptional machinery and inhibits TFF3 expression. As estrogen signalling fades at 3h, TFF1 expression declines while TFF3 expression increases. Our findings reveal that enhancer-driven activation can indirectly repress neighbouring genes within the same TAD, highlighting a dynamic shift in gene expression as signalling progresses.

molecular biology↗

bioRxiv2024

Aberrant subcellular localization of mismatch repair protein MLH1 dysregulates the cell cycle to create new therapeutic opportunities

Estrogen receptor positive (ER+) breast cancer is one of the most common causes of cancer-related death in women. Mortality is largely driven by recurrence of treatment-resistant disease after many years of apparent response, making the molecular events that cause recurrence a critical area of investigation. Loss of expression of MLH1, a tumor suppressor best studied in its role in mismatch repair, induces resistance of ER+ breast cancer cells to standard estrogen-targeting therapies. It does so by delinking cell cycle progression from estrogen regulation, a role distinct from its function in mismatch repair. MLH1 loss, as currently clinically diagnosed by detecting genomic instability or by immunohistochemistry for absence of protein, occurs in 12-15% of all cancers. Here, we demonstrate that sub-clonal, patient-derived mutations in MLH1, which neither impact protein abundance nor contribute sufficiently to genomic instability to be detected diagnostically, seed endocrine treatment resistance by enabling estrogen-independent growth in vitro, ex vivo in patient-derived organoids (p=0.005) and in vivo (p=0.0001). The mechanism underlying this endocrine treatment resistance is aberrant localization of MLH1 to the cytoplasm in vitro and in vivo (p=0.04), which precludes cell cycle arrest in response to endocrine therapy while simultaneously rendering cells acutely dependent on CDK4/6 activity. Consequently, administration of CDK4/6 inhibitors causes extreme regression in cells with cytoplasmic MLH1 compared to control cell populations with nuclear localization of MLH1 in vitro (p=0.00000009), ex vivo (p=0.01) and in vivo (p=0.01). As aberrant cytoplasmic localization occurs in an additional [~]12% of ER+ breast cancer patients, it constitutes a new, major contributor to MLH1 dysregulation. The potential applicability of cytoplasmic MLH1 as a predictor of responsiveness to existing targeted therapies in a hard-to-treat breast cancer subtype posits an update of current clinical diagnostic criteria and therapeutic strategies. This is particularly important in the adjuvant setting where identification of biomarkers predicting responsiveness to CDK4/6 inhibitors remains an urgent, unmet clinical need.

cancer biology↗

bioRxiv2024

A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres, and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening, and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate domain-specific functions. For example, decreased mating type silencing was linked to mutations in heterochromatin maintenance genes, while compromised subtelomere silencing was associated with metabolic pathways. Furthermore, similar phenotypic profiles revealed shared functions for subunits within complexes. We also discovered that the uncharacterized protein Dhm2 plays a crucial role in maintaining constitutive and facultative heterochromatin, while its absence caused phenotypes akin to DNA replication-deficient mutants. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity.

genomics↗

bioRxiv2022

Strategic in vitro bioaugmentation of indigenous Actinobacteria isolated from east Kolkata wetlands (Ramsar site) to degrade highly toxic waste engine oil in free and emulsified state

The present study investigates the bioremediation of waste engine oil at both dissolved or being suspended in oily wastewater using an actinobacterium, Gordonia terrae DSM 43249 strain isolated from East Kolkata wetlands. The isolated strain was found capable of sustaining in highly toxic oil contaminated wastewater and simultaneously can efficiently biodegrade the pollutants in both simulated fresh and marine water system at optimized environmental conditions. Moreover, in order to understand the effect of physical presence of oil in oily wastewater on the bioremediation process, three types of simulated oil-water forms were studied: water with free waste engine oil, oil-water mixture in the form of coarse emulsion and microemulsion. It was observed that the percentage degradation became maximum with the microemulsion form (72.73%) followed by the coarse one (65.45%). The minimum percentage degradation of 39.74% was seen with the free oil. Statistical interpretation also revalidates the experimental observations, showing that the oil percentage degradation is much sensitive to the oil and water composition in an oily-water system (F=772.64> Fcritical =5.143). Hence, it is presumed from the present study that such a high percentage degradation of oil especially, when oil is thoroughly mixed with water, can be considered as one of the potential applications for oil treatment such as during oil spillage using G.terrae DSM 43249. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/503898v3_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@154b649org.highwire.dtl.DTLVardef@8b86cborg.highwire.dtl.DTLVardef@3e8f3forg.highwire.dtl.DTLVardef@12e88d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Bleaching-resistant single-molecule fluorescence and FRET monitoring based on fluorophore exchange via transient DNA binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Forster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach should enable capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

biophysics↗

bioRxiv2022

Nuclear inhibitor of protein phosphatase 1 (NIPP1) regulates CNS tau phosphorylation and myelination during development

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and has been shown to play roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model, in which the nipp1 gene is excised from neural precursor cells. These mice demonstrate severe developmental impairments that lead to premature lethality within the first few postnatal weeks. To delineate some of the neurological changes occurring in these animals, we first assessed microtubule associated protein tau, a known target of NIPP1 activity. Furthermore, observed tremors prompted exploration of myelin integrity, an integral structure for CNS function, whose disruption is associated with various neurological disorders and neurodegenerative diseases. First, immunoblotting demonstrated increased phospho-tau and altered AKT and PP1 activity in NIPP1 cKO mice, suggesting increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Second, immunoblots, electron microscopy, and electrophysiology demonstrated a myelin deficit within the brain and optic nerve. Our study suggests that NIPP1 in neural precursors regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Evolution of Delta variant by non-Spike signature co-appearing mutations: trailblazer of COVID-19 disease outcome

The high transmissibility and infectivity of a SARS-CoV-2 variant is usually ascribed to the Spike mutations, while emerging non-spike mutations might be a serious threat to the current Spike-recombinant vaccines. In addition to mutations in structural Spike glycoprotein, rapid accumulation of mutations across non-structural genes is leading to continuous virus evolution, altering its pathogenicity. We performed whole genome sequencing of SARS-CoV-2 positive samples collected from different clinical groups from eastern India, during the second pandemic wave (April-May, 2021). In addition to the several common spike mutations in Delta variant, two mutually explicit signature constellations of non-spike co-appearing mutations were identified, driving symptomatic and asymptomatic infections. We attempted to correlate these unique signatures of non-Spike co-appearing mutations to COVID-19 disease outcome. Results revealed that the Delta strains harboring a unique constellation of 9 non-spike co-appearing mutations could be the wheeler and dealer of symptomatic infection, even post vaccination. The strains predominantly driving asymptomatic infection possessed 7 non-spike co-appearing mutations, which were mutually exclusive in contrast to the set of mutations causing symptomatic disease. Phylodynamic analysis depicted high probability of emergence of these unique sub-clusters within India, with subsequent spread worldwide. Interestingly, some mutations of this signature were selected in Omicron and IHU variants, which suggest that gradual accumulation of such co-existing mutations may lead to emergence of more "vaccine-evading variants" in future. Hence, unfaltering genome sequencing and tracking of non-Spike mutations might be significant in formulation of any future vaccines against emerging SARS-CoV-2 variants that might evade the current vaccine-induced immunity.

genomics↗

bioRxiv2022

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

biophysics↗

bioRxiv2021

Buffered EGFR signaling regulated by spitz to argos expression ratio is critical for patterning the Drosophila eye

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR (DER) signaling achieves specificity through multiple ligands and feedback loops to finetune signaling spatiotemporally. The principal Drosophila EGF, cleaved Spitz, and the negative feedback molecule, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The relative expression dose of Spitz and Argos early in development has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eyedisc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos in the Drosophila third instar eye imaginal disc indicative of directional DER signaling. By genetically tuning DER signaling, we show that rather than absolute levels of expression, the ratio of expression to be critical for determining the adult eye phenotype. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype ratio, and breaks down beyond. This provides a powerful instance of developmental buffering.

developmental biology↗