Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique

Methods ◽  
2017 ◽  
Vol 126 ◽  
pp. 130-137 ◽  
Author(s):  
Xicotencatl Gracida ◽  
John A. Calarco
Keyword(s):  
Affinity Purification ◽  
Transcriptome Profiling ◽  
Cell Type ◽  
C Elegans ◽  
Purification Technique ◽  
Cell Type Specific

A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans.

2021 ◽  
Author(s):  
Laura E Wester ◽  
Anne Lanjuin ◽  
Emanuel H W Bruckisch ◽  
Maria C Perez Matos ◽  
Caroline Heintz ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Single Copy ◽  
Specific Cell ◽  
Cell Type ◽  
Specific Expression ◽  
Tissue Specific ◽  
C Elegans ◽  
Cell Type Specific

Translating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types. Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome. Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging.

scholarly journals Molecular and cellular adaptations in hippocampal parvalbumin neurons mediate behavioral responses to chronic social stress

2021 ◽  
Author(s):  
Dionnet L Bhatti ◽  
Lucian Medrihan ◽  
Michelle X Chen ◽  
Junghee Jin ◽  
Kathryn McCabe ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Affinity Purification ◽  
Behavioral Outcomes ◽  
Behavioral Responses ◽  
Specific Gene ◽  
Mrna Levels ◽  
Cell Type ◽  
Stress Resilience ◽  
Cell Type Specific ◽  
Stress Susceptibility

BACKGROUND: Behavioral responses to stress are, in part, mediated by the hippocampus and Parvalbumin (PV)-expressing neurons. However, whether chronic stress induces molecular and cellular adaptations in hippocampal PV neurons contribute to stress-induced behavioral outcomes remains elusive. METHOD: Using chronic social defeat stress (CSDS), we investigated the role of neuronal activity and gene expression in hippocampal PV neurons in mediating stress-resilience and -susceptibility. We first used in vivo high-density silicon probe recordings and chemogenetics to test whether the activity of PV neurons in ventral dentate gyrus (PVvDG) is associated with particular behavioral outcomes. To find critical molecular pathways associated with stress-resilience and -susceptibility, we used PV-neuron-selective translating ribosome affinity purification and RNAseq. We used immunoblotting, RNAscope, and region- or cell type-specific gene deletion to determine whether Ahnak, a molecule regulating depression-like behavior, was necessary for behavioral divergence after CSDS. RESULTS: We find CSDS modulates neuronal activity in vDG. Notably, stress-susceptibility is associated with an increase of PVvDG firing, which we find is necessary and sufficient for susceptibility. Additionally, genes involved in mitochondrial function, protein synthesis and synaptogenesis are differentially expressed in hippocampal PV neurons of stress-resilient and -susceptible mice. Interestingly, protein and mRNA levels of Ahnak, an endogenous regulator of L-type calcium channels are associated with susceptibility after CSDS. vDG- and PV cell type-specific deletions reveal that Ahnak is required for stress-susceptibility to CSDS. CONCLUSIONS: These findings indicate that CSDS-induced molecular and cellular adaptations in hippocampal PV neurons mediate behavioral consequences, proposing a mechanism underlying individual differences in stress vulnerability.

scholarly journals Cell type‐specific structural plasticity of the ciliary transition zone in C. elegans

2019 ◽  
Vol 111 (4) ◽  
pp. 95-107 ◽  
Author(s):  
Jyothi S. Akella ◽  
Malan Silva ◽  
Natalia S. Morsci ◽  
Ken C. Nguyen ◽  
William J. Rice ◽  
...  
Keyword(s):  
Transition Zone ◽  
Structural Plasticity ◽  
Cell Type ◽  
C Elegans ◽  
Cell Type Specific

scholarly journals Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Ana J. Chucair-Elliott ◽  
Sarah R. Ocañas ◽  
David R. Stanford ◽  
Victor A. Ansere ◽  
Kyla B. Buettner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Tissue Level ◽  
Cell Phenotype ◽  
Specific Gene ◽  
Cell Type ◽  
A Cell ◽  
Cell Type Specific

AbstractEpigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.

scholarly journals Cell-Type Specific Responses to DNA Replication Stress in Early C. elegans Embryos

PLoS ONE ◽  
2016 ◽  
Vol 11 (10) ◽  
pp. e0164601 ◽  
Author(s):  
Holly Stevens ◽  
Ashley B. Williams ◽  
W. Matthew Michael
Keyword(s):  
Dna Replication ◽  
Replication Stress ◽  
Cell Type ◽  
C Elegans ◽  
Dna Replication Stress ◽  
Cell Type Specific

scholarly journals Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP)

2014 ◽  
Vol 9 (6) ◽  
pp. 1282-1291 ◽  
Author(s):  
Myriam Heiman ◽  
Ruth Kulicke ◽  
Robert J Fenster ◽  
Paul Greengard ◽  
Nathaniel Heintz
Keyword(s):  
Affinity Purification ◽  
Cell Type ◽  
Mrna Purification ◽  
Cell Type Specific ◽  
Specific Mrna

scholarly journals An in vivo, neuron-specific approach for pairing translational and epigenetic signatures of early-life exercise

2021 ◽  
Author(s):  
Anthony Mark Raus ◽  
Tyson D Fuller ◽  
Nellie E Nelson ◽  
David A Valientes ◽  
Anita Bayat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Early Life ◽  
Affinity Purification ◽  
Cell Types ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Cell Type Specific ◽  
Induced Changes ◽  
Epigenetic Signatures

Aerobic exercise promotes physiological and molecular adaptations in neurons to influence brain function and behavior. The most well studied neurobiological consequences of exercise are those which underlie exercise-induced improvements in hippocampal memory, including the expression and regulation of the neurotrophic factor Bdnf. Whether aerobic exercise taking place during early-life periods of postnatal brain maturation has similar impacts on gene expression and its regulation remains to be investigated. Using unbiased next-generation sequencing we characterize gene expression programs and their regulation by specific, memory-associated histone modifications during juvenile-adolescent voluntary exercise (ELE). Traditional transcriptomic and epigenomic sequencing approaches have either used heterogeneous cell populations from whole tissue homogenates or flow cytometry for single cell isolation to distinguish cell types / subtypes. These methods fall short in providing cell-type specificity without compromising sequencing depth or procedure-induced changes to cellular phenotype. In this study, we use simultaneous isolation of translating mRNA and nuclear chromatin from a neuron-enriched cell population to more accurately pair ELE-induced changes in gene expression with epigenetic modifications. We employ a line of transgenic mice expressing the NuTRAP (Nuclear Tagging and Translating Ribosome Affinity Purification) cassette under the Emx1 promoter allowing for brain cell-type specificity. We then developed a technique that combines nuclear isolation using Isolation of Nuclei TAgged in Specific Cell Types (INTACT) with Translating Ribosomal Affinity Purification (TRAP) methods to determine cell type-specific epigenetic modifications influencing gene expression programs from a population of Emx1 expressing hippocampal neurons. Data from RNA-seq and CUT&RUN-seq were coupled to evaluate histone modifications influencing the expression of translating mRNA in neurons after early-life exercise (ELE). We also performed separate INTACT and TRAP isolations for validation of our protocol and demonstrate similar molecular functions and biological processes implicated by gene ontology (GO) analysis. Finally, as prior studies use tissue from opposite brain hemispheres to pair transcriptomic and epigenomic data from the same rodent, we take a bioinformatics approach to compare hemispheric differences in gene expression programs and histone modifications altered by by ELE. Our data reveal transcriptional and epigenetic signatures of ELE exposure and identify novel candidate gene-histone modification interactions for further investigation. Importantly, our novel approach of combined INTACT/TRAP methods from the same cell suspension allows for simultaneous transcriptomic and epigenomic sequencing in a cell-type specific manner.

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