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CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside ...nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.
Support or Funding Information
This project has been supported by CABRI Undergraduate Research Fellowship
Calcium/ calmodulin-dependent serine protein kinase (CASK) is a protein highly expressed in neuronal cells and associated to the etiology of neurodevelopmental disorders, such as intellectual ...disability and microcephaly. CASK is known to regulate the trafficking of membrane receptors at pre- and post-synaptic terminals. Accumulating evidence also suggests a role of CASK in transcriptional regulation; however, the molecular mechanism by which CASK modulate transcription in neurons is currently unknown. To investigate the underlying mechanism, I manipulated the level of CASK by infecting primary cortical neurons with a lentiviral particle carrying short hairpin (sh)RNA targeting CASK. Following CASK knockdown, I showed that the activation of neuronal activity-dependent genes induced by KCl treatment is significantly reduced in the absence of CASK. Based on the analysis of a protein-protein interactions database, I found that CASK can bind to mediator complex, which is required for transcription initiation and long-range chromatin interactions. Thus, I used chromatin immunoprecipitation followed by quantitative PCR to study the binding of mediator complex on CASK targets after the neurons were depolarized and CASK is knocked down. This study showed that the recruitment of the mediator on the regulatory regions of CASK targets is induced by neuronal activity, but it’s reduced in the absence of CASK. Such results suggest that CASK may regulate transcriptional initiation or long-range chromatin interactions via its interaction with the mediator complex. My findings provide novel insights into the mechanism by which CASK modulates activity-regulated gene expression in neuronal cells.
Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of ...microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates.
Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.
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•The potential of Paramecium multimicronucleatum in heavy metal bioremediation.•The first proteome profile of Paramecium in response to cadmium exposure.•Proteome analysis revealed 6 and 23 proteins with higher and lower levels, respectively.•The differentially abundant proteins are associated with stress tolerance, viability, and homeostasis.
The ability to rapidly change gene expression patterns is essential for differentiation, development, and functioning of the brain. Throughout development, or in response to environmental stimuli, ...gene expression patterns are tightly regulated by the dynamic interplay between transcription activators and repressors. Nuclear receptor corepressor 1 (NCoR1) and silencing mediator for retinoid or thyroid‐hormone receptors (SMRT) are the best characterized transcriptional co‐repressors from a molecular point of view. They mediate epigenetic silencing of gene expression in a wide range of developmental and homeostatic processes in many tissues, including the brain. For instance, NCoR1 and SMRT regulate neuronal stem cell proliferation and differentiation during brain development and they have been implicated in learning and memory. However, we still have a limited understanding of their regional and cell type‐specific expression in the brain. In this study, we used fluorescent immunohistochemistry to map their expression patterns throughout the adult mouse brain. Our findings reveal that NCoR1 and SMRT share an overall neuroanatomical distribution, and are detected in both excitatory and inhibitory neurons. However, we observed striking differences in their cell type‐specific expression in glial cells. Specifically, all oligodendrocytes express NCoR1, but only a subset express SMRT. In addition, NCoR1, but not SMRT, was detected in a subset of astrocytes and in the microglia. These novel observations are corroborated by single cell transcriptomics and emphasize how NCoR1 and SMRT may contribute to distinct biological functions, suggesting an exclusive role of NCoR1 in innate immune responses in the brain.
Schematic representation of NCoR1 and SMRT distribution in neuronal and non‐neuronal cell types. Five major types of brain cells (glutamatergic and GABAergic neurons, astrocytes, oligodendrocytes, and microglia) are depicted within a representative coronal slice of the mouse brain. Both NCor1 and SMRT broadly localize within the nuclei of most neuronal cells. However, a diverse pattern of expression is detected in non‐neuronal cells. While NCoR1 is expressed in a small fraction of astrocytes, SMRT is not expressed in GFAP+ astrocytes or in Iba1+ microglia.
Algal technology offers enormous potential as a source of renewable biomass and bioproducts for fuels, biochemicals, nutrients, and numerous other applications. While tremendous opportunities exist, ...significant challenges are also present in harnessing and utilizing this valuable and potentially sustainable bioproduction platform. Funded by a joint partnership between the Bioenergy Technologies Office (BETO) at the Department of Energy (DOE) and the National Science Foundation (NSF), the recent virtual workshop, “Understanding the Rules of Life: Complexity in Algal Systems Workshop,” was organized to bring together thought leaders from academia, national laboratories and other government agencies, and industry to identify key challenges and propose research strategies to overcome these barriers. Two parallel sessions were undertaken: 1) algae strain and toolkit development and 2) algal ecosystem and microbiome dynamics, in order to advance algae engineering and cultivation efforts. In each session, attendees discussed the most pressing challenges from the perspective of model systems and omics analysis. Themes such as genetic engineering toolboxes, translation from the laboratory to outdoor cultivation, functional genomics, metabolomics, algal ecology, consortial design, microbiome dynamics, mathematical models, and data analytics emerged from the discussion. Overall, this workshop has served to elucidate the key hurdles and many opportunities present as we continue to advance algal science and technology from the laboratory and into the commercial realm.
Algal technology offers enormous potential as a source of renewable biomass and bioproducts for fuels, biochemicals, nutrients, and numerous other applications. While tremendous opportunities exist, ...significant challenges are also present in harnessing and utilizing this valuable and potentially sustainable bioproduction platform. Funded by a joint partnership between the Bioenergy Technologies Office (BETO) at the Department of Energy (DOE) and the National Science Foundation (NSF), the recent virtual workshop, "Understanding the Rules of Life: Complexity in Algal Systems Workshop," was organized to bring together thought leaders from academia, national laboratories and other government agencies, and industry to identify key challenges and propose research strategies to overcome these barriers. Two parallel sessions were undertaken: 1) algae strain and toolkit development and 2) algal ecosystem and microbiome dynamics, in order to advance algae engineering and cultivation efforts. In each session, attendees discussed the most pressing challenges from the perspective of model systems and omics analysis. Themes such as genetic engineering toolboxes, translation from the laboratory to outdoor cultivation, functional genomics, metabolomics, algal ecology, consortial design, microbiome dynamics, mathematical models, and data analytics emerged from the discussion. Overall, this workshop has served to elucidate the key hurdles and many opportunities present as we continue to advance algal science and technology from the laboratory and into the commercial realm.
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