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Visualizing Live Chromatin Dynamics through CRISPR-Based Imaging Techniques
Chaudhary, Narendra,Im, Jae-Kyeong,Nho, Si-Hyeong,Kim, Hajin Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.9
The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.
Bhardwaj, Jyoti,Chaudhary, Narendra,Seo, Hyo-Jin,Kim, Min-Yong,Shin, Tai-Sun,Kim, Jong-Deog Informa Healthcare USA, Inc. 2014 Immunopharmacology and immunotoxicology Vol.36 No.3
<P>The anti-cancer activity of saponins and phenolic compounds present in green tea was previously reported. However, the immunomodulatory and adjuvanticity activity of tea saponin has never been studied. In this study, we investigated the immunomodulatory effect of tea saponin in T-lymphocytes and EL4 cells <I>via</I> regulation of cytokine response and mitogen-activated protein kinases (MAPK) signaling pathway. Quantitative analysis of mRNA expression level of cytokines were performed by reverse transcription polymerase chain reaction following stimulation with tea saponin, ovalbumin (OVA) alone or tea saponin in combination with OVA. Tea saponin inhibited the proliferation of EL4 cells measured in a dose-dependent manner. No cytotoxicity effect of tea saponin was detected in T-lymphocytes; rather, tea saponin enhanced the proliferation of T-lymphocytes. Tea saponin with OVA increased the expression of interleukin (IL)-1, IL-2, IL-12, interferon-γ and tumor necrosis factor (TNF)-α and decreased the expression level of IL-10 and IL-8 in T-lymphocytes. Furthermore, tea saponin, in the presence of OVA, downregulated the MAPK signaling pathway <I>via</I> inhibition of IL-4, IL-8 and nuclear factor kappaB (NF-κB) in EL4 cells. Th1 cytokines enhancer and Th2 cytokines and NF-κB inhibitor, tea saponin can markedly inhibit the proliferation and invasiveness of T-lymphoma (EL4) cells, possibly due to TNF-α- and NF-κB-mediated regulation of MAPK signaling pathway.</P>