12 results on '"Ahilya N. Sawh"'
Search Results
2. Multiplexed Sequential DNA FISH in Caenorhabditis elegans Embryos
- Author
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Ahilya N. Sawh and Susan E. Mango
- Subjects
Science (General) ,Q1-390 - Abstract
Summary: This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization).For complete details on the use and execution of this protocol, please refer to Sawh et al. (2020).
- Published
- 2020
- Full Text
- View/download PDF
3. A Truncated Form of Dicer Tilts the Balance of RNA Interference Pathways
- Author
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Ahilya N. Sawh and Thomas F. Duchaine
- Subjects
Biology (General) ,QH301-705.5 - Abstract
The RNase III enzyme Dicer is responsible for key steps in the biogenesis of small RNA species in multiple RNA interference pathways. Here, we show that, in the adult C. elegans soma, half of the total DCR-1 protein is expressed as a truncated, stable C-terminal fragment named small DCR-1 (sDCR-1). sDCR-1 operates independently of full-length DCR-1 in two distinct RNAi pathways; it enhances exogenous RNAi (exoRNAi) and concurrently acts as a negative regulator of microRNA (miRNA) biogenesis. Enhancement of exoRNAi relies on sDCR-1 catalytic activity, whereas impinging on miRNA processing does not. Instead, sDCR-1 competes with pre-miRNA processing by interacting with the miRNA-dedicated Argonautes ALG-1 and ALG-2. Finally, triggering a strong exoRNAi response in the presence of elevated levels of sDCR-1 exacerbates the miRNA processing defect. Our results unveil a surprising role for a truncated form of DCR-1 in the modulation of multiple RNAi activities and in the regulation of mechanistic boundaries between pathways.
- Published
- 2013
- Full Text
- View/download PDF
4. Gene family evolution underlies cell-type diversification in the hypothalamus of teleosts
- Author
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Maxwell E.R. Shafer, Ahilya N. Sawh, and Alexander F. Schier
- Subjects
Cell type ,Ecology ,Characidae ,Hypothalamus ,Biology ,biology.organism_classification ,Biological Evolution ,Transcriptome ,Caves ,Evolutionary biology ,Gene duplication ,Animals ,Subfunctionalization ,Gene family ,Neofunctionalization ,Zebrafish ,Gene ,Ecology, Evolution, Behavior and Systematics - Abstract
Hundreds of cell types form the vertebrate brain but it is largely unknown how similar cellular repertoires are between or within species or how cell-type diversity evolves. To examine cell-type diversity across and within species, we performed single-cell RNA sequencing of ~130,000 hypothalamic cells from zebrafish (Danio rerio) and surface and cave morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which diverged from a common ancestor over 150 million years ago. Shared cell types displayed shifts in paralogue expression that were generated by subfunctionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes and characterized by the neofunctionalization of expression patterns of members of recently expanded or contracted gene families. Comparisons between surface and cave morphs revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types and reveal how gene family evolution and shifts in paralogue expression contribute to cellular diversity. Single-cell transcriptomics of hypothalamic cells from zebrafish and surface and cave morphs of Mexican tetra shows conservation of cell types and that cellular novelty is associated with genetic novelty and the species-specific expression of paralogous genes.
- Published
- 2021
5. Chromosome organization in 4D: insights from C. elegans development
- Author
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Ahilya N Sawh and Susan E Mango
- Subjects
Genome ,Genetics ,Animals ,Caenorhabditis elegans ,Promoter Regions, Genetic ,Chromatin ,Nucleosomes ,Developmental Biology - Abstract
Eukaryotic genome organization is ordered and multilayered, from the nucleosome to chromosomal scales. These layers are not static during development, but are remodeled over time and between tissues. Thus, animal model studies with high spatiotemporal resolution are necessary to understand the various forms and functions of genome organization in vivo. In C. elegans, sequencing- and imaging-based advances have provided insight on how histone modifications, regulatory elements, and large-scale chromosome conformations are established and changed. Recent observations include unexpected physiological roles for topologically associating domains, different roles for the nuclear lamina at different chromatin scales, cell-type-specific enhancer and promoter regulatory grammars, and prevalent compartment variability in early development. Here, we summarize these and other recent findings in C. elegans, and suggest future avenues of research to enrich our in vivo knowledge of the forms and functions of nuclear organization.
- Published
- 2022
- Full Text
- View/download PDF
6. Gene family evolution underlies cell type diversification in the hypothalamus of teleosts
- Author
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Ahilya N. Sawh, Alexander F. Schier, and Maxwell E.R. Shafer
- Subjects
Cell type ,biology ,Effector ,Evolutionary biology ,Gene duplication ,Danio ,Gene family ,biology.organism_classification ,Gene ,Zebrafish ,Genome - Abstract
Hundreds of cell types form the vertebrate brain, but it is largely unknown how similar these cellular repertoires are between or within species, or how cell type diversity evolves. To examine cell type diversity across and within species, we performed single-cell RNA sequencing of ∼130,000 hypothalamic cells from zebrafish (Danio rerio) and surface- and cave-morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which last shared a common ancestor over 150 million years ago. Orthologous cell types displayed differential paralogue expression that was generated by sub-functionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes, and characterized by the neo-functionalization of members of recently expanded or contracted gene families. Within species comparisons revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences between surface- and cave-morphs. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types, and reveal how gene family evolution and the neo- and sub-functionalization of paralogs contribute to cellular diversity.
- Published
- 2020
7. A Family of Argonaute-Interacting Proteins Gates Nuclear RNAi
- Author
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Ahmet C. Berkyurek, Eric A. Miska, Mathieu N. Flamand, Mihail Sarov, Stephanie Merrett, Ajay A. Vashisht, Alexandra Lewis, James A. Wohlschlegel, Ahilya N. Sawh, Thomas F. Duchaine, Andre Greiner, Miska, Eric [0000-0002-4450-576X], and Apollo - University of Cambridge Repository
- Subjects
Piwi-interacting RNA ,piRNA ,trans-generational epigenetic inheritance ,22G-RNA ,Article ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Gene expression ,Gene silencing ,Animals ,Epigenetics ,Gene Silencing ,RNA, Small Interfering ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,030304 developmental biology ,RNA, Double-Stranded ,RNA, Nuclear ,NRDE-3 ,Cell Nucleus ,0303 health sciences ,biology ,RNA ,Nuclear Proteins ,RNA-Binding Proteins ,Cell Biology ,Argonaute ,Cell biology ,Histone ,Germ Cells ,RNAi ,Argonaute Proteins ,biology.protein ,030217 neurology & neurosurgery - Abstract
Summary Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.
- Published
- 2020
8. A Family of Nuclear Argonaute Interacting Proteins Gates Nuclear RNAi
- Author
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James A. Wohlschlegel, Stephanie Merrett, Ahilya N. Sawh, Alexandra Lewis, Mathieu N. Flamand, Ahmet C. Berkyurek, Thomas F. Duchaine, Ajay A. Vashisht, Eric A. Miska, Mihail Sarov, and Andre Greiner
- Subjects
RNA interference ,fungi ,Gene expression ,Gene silencing ,Piwi-interacting RNA ,RNA ,Epigenetics ,Argonaute ,Biology ,Germline ,Cell biology - Abstract
Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational inheritance (TEI) of gene silencing. How the nuclear activities of RNAi are physiologically controlled remains poorly understood. Here, we identify and characterize a family of nuclear Argonaute-interacting proteins (NIPs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. NIP-1 and NIP-2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, NIP-1 and NIP-2 are negative regulators of nuclear RNAi triggered from exogenous sources (exo-RNAi). Loss of NIP-3, an unstable protein mostly expressed in the male germline, misdirects the RNAi response to transposable elements and impairs TEI.Our data support a model wherein NIPs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes in both somatic and germline tissues.
- Published
- 2019
9. Multiplexed Sequential DNA FISH in Caenorhabditis elegans Embryos
- Author
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Susan E. Mango and Ahilya N. Sawh
- Subjects
Molecular Conformation ,Computational biology ,Biology ,Chromosomes ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Chromosome (genetic algorithm) ,Protocol ,medicine ,Animals ,lcsh:Science (General) ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,In Situ Hybridization, Fluorescence ,DNA Primers ,Genome ,General Immunology and Microbiology ,medicine.diagnostic_test ,General Neuroscience ,Embryo ,DNA ,Genomics ,biology.organism_classification ,chemistry ,%22">Fish ,Single-Cell Analysis ,lcsh:Q1-390 ,Fluorescence in situ hybridization - Abstract
Summary This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization). For complete details on the use and execution of this protocol, please refer to Sawh et al. (2020)., Graphical Abstract, Highlights • An optimized FISH protocol to map chromosome conformation in C. elegans embryos • Removes limitations on the maximum number of targets visualized in a single sample • Customizable to interrogate conformation from the kb to Mb to whole-genome scale • Unsupervised clustering of single chromosomes can define similar populations in vivo, This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization).
- Published
- 2020
10. Lamina-Dependent Stretching and Unconventional Chromosome Compartments in Early C. elegans Embryos
- Author
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Maxwell E.R. Shafer, Xiaowei Zhuang, Ahilya N. Sawh, Susan E. Mango, Siyuan Wang, and Jun-Han Su
- Subjects
Lamina ,Embryo, Nonmammalian ,Molecular Conformation ,Context (language use) ,Biology ,Chromosomes ,Article ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Compartment (development) ,Caenorhabditis elegans ,Molecular Biology ,In Situ Hybridization, Fluorescence ,030304 developmental biology ,0303 health sciences ,Nuclear Lamina ,Autosome ,Gastrulation ,Chromosome ,Cell Biology ,Chromatin ,Cell biology ,Nuclear lamina ,030217 neurology & neurosurgery - Abstract
Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.
- Published
- 2020
11. Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1
- Author
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Magdalena M. Szewczyk, Kanwaldeep K. Mallhi, Ahilya N. Sawh, Jyoti Pande, Fiona Simpson, and Ashok K. Grover
- Subjects
Gene isoform ,Male ,Endothelium ,Physiology ,Calcium-Transporting ATPases ,Biology ,Rats, Inbred WKY ,Muscle, Smooth, Vascular ,Structure-Activity Relationship ,Smooth muscle ,medicine ,Extracellular ,Ca2 pump ,Animals ,Humans ,Protein Isoforms ,Gene ,Aorta ,Messenger RNA ,Dose-Response Relationship, Drug ,Cell Membrane ,Cell Biology ,Cell biology ,Rats ,medicine.anatomical_structure ,Membrane ,Biochemistry ,Endothelium, Vascular ,Peptides ,Muscle Contraction - Abstract
Plasma membrane Ca2+pumps (PMCA) that expel Ca2+from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca2+-Mg2+-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a Kivalue of 46 ± 5 μM, making it 10× more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca2+-Mg2+-ATPase in human embryonic kidney-293 cells with a higher Kivalue (105 ± 11 μM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 μM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca2+] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.
- Published
- 2006
12. Turning Dicer on its head
- Author
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Ahilya N. Sawh and Thomas F. Duchaine
- Subjects
enzymes and coenzymes (carbohydrates) ,biology ,Structural Biology ,Head (linguistics) ,Computer science ,fungi ,genetic processes ,biology.protein ,food and beverages ,Computational biology ,Molecular Biology ,Domain (software engineering) ,Dicer - Abstract
Recent structural analysis of full-length human Dicer supports a new model of the enzyme's domain arrangement and provides a structural basis for many of Dicer's biochemical attributes.
- Published
- 2012
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