Biology subjects

Naik, H.

Publications and source records attributed to Naik, H..

7 recordsLinked to original sources

bioRxiv2026

From planktonic to sedentary lifestyle: Molecular dissection of the establishment and maintenance of mycobacterial biofilm

Biofilm represents a complex aggregation of bacteria embedded within a self-produced extracellular polymeric substance (EPS). We investigated the characteristics of mycobacterial biofilm using Mycobacterium smegmatis (Msm) as model organism. By combining transcriptomic (RNA-seq) and proteomic (LC-MS) analyses, the research captures dynamic changes during the establishment and maturation of the biofilm. Transcriptomics analysis showed a distinct gene expression profile as compared to its planktonic form. Interestingly, clear differences were seen between initial ([~]2-day old) and mature ([~]5-day old) biofilm stages, highlighting phasic gene expression throughout biofilm development. Marked alteration in oxidative stress-related genes and energy metabolism from ATP to NADH was observed. Furthermore, quantitative mass spectrometry-based proteome examination of EPS showed an abundance of cytoplasmic proteins present differentially between initial and mature biofilm stages. Pathway enrichment revealed enhanced oxidative stress responses and metabolic shifts in mature biofilms, including upregulation of NADH dehydrogenase and downregulation of ATP synthase, indicating altered energy metabolism. Our findings thus provide insights into the molecular adaptations, including production of mycofactocin, occurring during mycobacterial biofilm establishment and maturation, and advance our understanding of mycobacterial biofilm physiology.

microbiology↗

bioRxiv2023

Gene-specific reactivation of X-linked genes upon Xist loss is linked to the chromatin states in extraembryonic endoderm and epiblast stem cells

In eutherian mammals, X-chromosome dosage between sexes is balanced through the inactivation of one of the two X-chromosomes in female cells. In mouse, X-inactivation initiates at [~]4-8 cell stages of embryogenesis, where paternal-X undergoes imprinted X-inactivation. Subsequently, it switches to random X-inactivation in post-iplantation epiblast. The initiation of XCI is orchestrated by Xist. However, the role of Xist in the maintenance of X-chromosome inactivation remains underexplored. Here, we have explored the role of Xist in the maintenance of X-inactivation in extraembryonic endoderm stem cells (XEN) and epiblast stem cells (EpiSC), which undergo imprinted and random form of X-inactivation respectively. We show that removal of Xist leads to the partial reactivation of inactive-X chromosome. Intriguingly, many reactivated genes were found to be common between XEN and EpiSC, indicating these genes require Xist to maintain their silent state irrespective of the lineages or forms of X-inactivation. Notably, despite Xist ablation and the subsequent removal of DNA methylation, several X-linked genes remained resistant to reactivation, indicating the involvement of other factors in maintaining the silencing of these genes. On the other hand, we show that genes on the inactive-X with low levels of H3K9me3 and high levels of H3K27me3 are more susceptible to reactivation upon the loss of Xist. Interestingly, active-X homolog of the reactivated genes was found to be enriched with H3K4me3 and H3K27ac. Taken together, our study sheds light on the role of chromatin states in the reactivation of X-linked genes following the loss of Xist in XEN and EpiSC.

genetics↗

bioRxiv2023

Deletion of Xist upstream sequences alters TAD interactions and leads to defects in Xist coating and expression

The topological organization of the genome plays an important role in regulating gene expression. However, the connection between the two remains poorly understood. X-chromosome inactivation is a unique model system to explore the interlink between topologically associated domains (TADs) and gene expression. TADs are largely lost upon X-inactivation, and the inactive-X gets bipartitely reorganized into two large mega domains. However, the X-inactivation center (XIC) harbors two TADs - at the locus of long non-coding RNA Xist (Xist-TAD) and Tsix (Tsix-TAD). Xist is the master regulator of X-inactivation, which coat the inactive-X and facilitates heterochromatinization. Here, we deleted Xist upstream sequences ([~]6 kb) near the Xist TADs boundary in extraembryonic endoderm stem cells (XEN), which undergo imprinted X-inactivation. This deletion led to the major rearrangement of TADs and affected the expression of genes located within Xist and Tsix TAD, specially the expression of Xist was upregulated, suggesting TADs are essential for proper transcriptional regulation. On the other hand, Xist-upstream deletion on the inactive-X resulted in dispersal of Xist coating and loss of enrichment of repressive chromatin marks on the inactive-X but no effect on X-linked gene silencing. However, we found that autosomal genes were dysregulated in Xist-upstream deleted cells, probably because of misregulation of genes located in Xist and Tsix-TAD, specially Xist. We conclude that Xist upstream sequences are necessary for proper organization of the TADs at the XIC, maintenance of Xist coating/expression and autosomal gene expression.

genetics↗

bioRxiv2023

Chromatin states contribute to coordinated allelic transcriptional bursting to drive iPSC reprogramming

Molecular mechanisms behind the reprogramming of somatic cells to induced pluripotent stem cells (iPSC) remain poorly understood. While dynamic changes in gene expression are considered to drive reprogramming, the contribution of individual alleles of genes to reprogramming remains unexplored. It is thought that two alleles of a gene can transcribe independently or coordinatedly, which in turn can lead to temporal expression heterogeneity with potentially distinct impacts on cell fate. Here, we profiled genome-wide transcriptional burst kinetics with an allelic resolution during the reprogramming of mouse embryonic fibroblast (MEF) to iPSC. We show that many genes involved in iPSC reprogramming pathways exhibit bursty expression and contribute to dynamic autosomal random monoallelic expression (aRME). Moreover, we find that the degree of coordination of allelic bursting differs among genes and changes dynamically during iPSC reprogramming. Importantly, we show that alleles of many reprogramming-related genes burst in a highly coordinated fashion. ATAC-seq analysis revealed that coordination of allelic bursting is linked to allelic chromatin accessibility. Consistently, we show that highly coordinated genes are enriched with chromatin accessibility regulators such as H3K36me3, H3K27ac, histone variant H3.3 and BRD4. Collectively, our study demonstrates that chromatin states contribute to coordinated allelic bursting to fine-tune the expression of genes involved in iPSC reprogramming and provides insights into the implications of allelic bursting coordination in cell fate specification. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/548864v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@162ec20org.highwire.dtl.DTLVardef@17c2db0org.highwire.dtl.DTLVardef@19b9d59org.highwire.dtl.DTLVardef@1775052_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2021

Single cell analysis reveals X chromosome upregulation is not global and primarily belongs to ancestral genes in pre-gastrulation embryos

Evolution of sex chromosome dosage compensation in mammals remains poorly understood. Ohnos hypothesis state that evolution of dosage compensation occurred through two steps: first, to compensate the dosage imbalance created due to the degradation of Y chromosome in male, upregulation of X-chromosome happened. Subsequently, transmission of X-chromosome upregulation (XCU) in female led to the evolution of X-chromosome inactivation (XCI) to counteract extra dosage of X-linked genes in female cells. Here, we have profiled gene-wise dynamics of XCU in pre-gastrulation mouse embryos at single cell level and found that XCU is dynamically linked with XCI, however, XCU is not global or chromosome-wide like XCI. Therefore, our result raises question whether XCU driven the evolution of XCI. If so, then why XCI is chromosome wide while XCU is not. We propose that XCI might have evolved independent of XCU and therefore refining the current model is necessary. Separately, we show that higher occupancy of different activating factors at upregulated X-linked genes leads to enhanced transcriptional burst frequency and thereby leads to upregulation. On the other hand, our analysis indicates that extent of upregulation, enrichment of different activating marks differs between ancestral and newly acquired X-linked genes. Altogether, our study provides significant insight into the dynamics and mechanistic basis of evolution of sex chromosome dosage compensation. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

genetics↗

bioRxiv2020

Dynamic erasure of X chromosome upregulation during iPSC reprogramming and in the inner cell mass

In mammals, sex chromosome dosage is compensated through X-chromosome inactivation and active-X upregulation. It is believed that during early development, X-chromosome inactivation and active X upregulation happen in a highly coordinated fashion. However, such coordination between two X-chromosomes in other developmental contexts remains unexplored. Here, we have profiled the coordination between two X-chromosomes in female cells in different developmental contexts and cell types: pre-implantation embryos, embryonic epiblast cells, iPSC reprogramming, germ cell reprogramming, B-cell, and extra-embryonic endoderm stem (XEN) cells. Interestingly, we found that two X-chromosomes in female cells are not always coordinated; instead, it happens in a lineage-specific manner. Specially, while embryonic mouse epiblast cells, iPSC undergo erasure of X-upregulation upon reactivation of the inactive X, germ cells do not. Importantly, we show that the erasure of X-upregulation in epiblast or iPSC is potentially mediated via undifferentiated embryonic transcription Factor 1 (UTF1), which is absent or lowly expressed in late germ cells and therefore, germ cells are unable to erase upregulation. Moreover, we found that partial reactivation of the inactive X is insufficient to drive the erasure of upregulation globally, nor from their counterparts on the active X in XEN and B-cells. Finally, through a phenomenological mathematical model, we show that cross-inhibition between two X-chromosomes can reproduce the dynamics of reactivation and erasure of upregulation. Altogether, our study provides insight into the coordination between two X-chromosomes in female cells in different developmental contexts and related mechanistic aspects. O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/424181v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f01b7borg.highwire.dtl.DTLVardef@9c586borg.highwire.dtl.DTLVardef@70f8eaorg.highwire.dtl.DTLVardef@e5d000_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

Fast and robust animal pose estimation

Quantitative behavioral measurements are important for answering questions across scientific disciplines--from neuroscience to ecology. State-of-the-art deep-learning methods offer major advances in data quality and detail by allowing researchers to automatically estimate locations of an animals body parts directly from images or videos. However, currently-available animal pose estimation methods have limitations in speed and robustness. Here we introduce a new easy-to-use software toolkit, DeepPoseKit, that addresses these problems using an eZcient multi-scale deep-learning model, called Stacked DenseNet, and a fast GPU-based peak-detection algorithm for estimating keypoint locations with subpixel precision. These advances improve processing speed >2x with no loss in accuracy compared to currently-available methods. We demonstrate the versatility of our methods with multiple challenging animal pose estimation tasks in laboratory and field settings--including groups of interacting individuals. Our work reduces barriers to using advanced tools for measuring behavior and has broad applicability across the behavioral sciences.

animal behavior and cognition↗