Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors

Qin, Peng; Lu, Shaoying; Shi, Yuxin; Pan, Yijia; Limsakul, Praopim; Chernov, Andrei V.; Qiu, Juhui; Chai, Xiaoqi; Shi, Yiwen; Wang, Pengzhi; Ji, Yanmin; Li, Yi-Shuan J.; Strongin, Alex Y.; Verkhusha, Vladislav V.; Belmonte, Juan Carlos Izpisúa; Ren, Bing; Wang, Yuanliang; Chien, Shu; Wang, Yingxiao

doi:10.1073/pnas.1811818115

Cited by 48 publications

(43 citation statements)

References 58 publications

(71 reference statements)

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“…Third, we employed a FRET-based heterochromatin sensor to investigate the effects of HP1α and uSTAT3 on global heterochromatin levels. The heterochromatin FRET sensor consists of peptides of H3 and HP1 fused to YFP and CFP, respectively, which has been previously used as a robust readout of H3K9me3 levels when expressed by transfection [48][49][50]. We found that, as expected, altering HP1α levels, by overexpressing HP1α or expressing a small hairpin (sh) HP1α RNA, affect global heterochromatin levels, as indicated by the FRET sensor ( Fig.…”

Section: Ustat3 Promotes Heterochromatin Formationsupporting

confidence: 55%

Unphosphorylated STAT3 in heterochromatin formation and tumor suppression in lung cancer

Dutta

Zhang

et al. 2020

BMC Cancer

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Background: Aberrant JAK/STAT activation has been detected in many types of human cancers. The role of JAK/ STAT activation in cancer has been mostly attributed to direct transcriptional regulation of target genes by phosphorylated STAT (pSTAT), while the unphosphorylated STAT (uSTAT) is believed to be dormant and reside in the cytoplasm. However, several studies have shown that uSTATs can be found in the nucleus. In addition, it has been shown that tissue-specific loss of STAT3 or STAT5 in mice promotes cancer growth in certain tissues, and thus these STAT proteins can act as tumor suppressors. However, no unifying mechanism has been shown for the tumor suppressor function of STATs to date. We have previously demonstrated a non-canonical mode of JAK/STAT signaling for Drosophila STAT and human STAT5A, where a fraction of uSTAT is in the nucleus and associated with Heterochromatin Protein 1 (HP1); STAT activation (by phosphorylation) causes its dispersal, leading to HP1 delocalization and heterochromatin loss. Methods: We used a combination of imaging, cell biological assays, and mouse xenografts to investigate the role of STAT3 in lung cancer development. Results: We found that uSTAT3 has a function in promoting heterochromatin formation in lung cancer cells, suppressing cell proliferation in vitro, and suppressing tumor growth in mouse xenografts. Conclusions: Thus, uSTAT3 possesses noncanonical function in promoting heterochromatin formation, and the tumor suppressor function of STAT3 is likely attributable to the heterochromatin-promoting activity of uSTAT3 in the non-canonical JAK/STAT pathway.

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Section: Ustat3 Promotes Heterochromatin Formationsupporting

confidence: 55%

Unphosphorylated STAT3 in heterochromatin formation and tumor suppression in lung cancer

Dutta

Zhang

et al. 2020

BMC Cancer

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“…In the past two decades, genetically-encoded FRET biosensors have served as a critical tool in revealing spatially and temporally dynamic signalling networks regulated by GPCRs in cultured cells. Many FRET-based sensors have now been created, allowing the optical detection of a wide range of signalling processes including second messengers, ion flux, protein kinases, monomeric GTPases, post-translational modifications and protein-protein interactions 12–15,53 . In contrast, the in vivo application of intracellular signalling-related FRET biosensors is much less developed, especially in freely-moving animals.…”

Section: Discussionmentioning

confidence: 99%

“…Among these technologies, fiber photometry has emerged as the least invasive, most accessible and affordable approach for recording neural activity in freely moving rodents 7–10 . Many intracellular signalling biosensors have been developed which utilize changes in Förster resonance energy transfer (FRET) between two fluorescent proteins 11 , in order to report levels or activity of second messengers, protein kinases, GTPases, post-translational modifications and protein-protein interactions 12–15 . These tools have been widely used to dissect signaling pathways in cultured cells and have recently begun to be applied in vivo , aided by recent technological developments such as intravital imaging 14,16 , microendoscopy 3 , 2-photon microscopy 2,5,6,17 and fluorescence lifetime measurement 4 .…”

Section: Introductionmentioning

confidence: 99%

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Jones-Tabah¹,

Mohammad²,

Hadj-Youssef³

et al. 2020

Preprint

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Like many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell-type specific, and can change in response to disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson’s disease (PD), alterations in this pathway lead to activation of extracellular regulated kinases (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

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“…The intensity images were pre-processed and deconvoluted in external software packages such as MetaMorph and MetaFluo (Figure S7). The 3D snapshot is colored by the FRET/ECFP ratio values of a new histone-localized histone 3 lysine 9 tri-methylation (H3K9me3) FRET biosensor at the intensity isosurface of the histone 3 [21]. The color represents level of epigenetic H3K9 tri-methylation at the surface of condensed histone in a dividing HeLa cell.…”

Section: Movie and 3d Visualizationmentioning

confidence: 99%

“…The source code, documentation and example data are freely available at our group website and GitHub. The ratiometric image analysis functionalities of Fluocell have been extensively tested by us and other groups [15][16][17][18][19][20][21]. In addition, Fluocell also contains a previously published Diffusion module for image-based FRAP analysis, as well as intensity-based polarity analysis functions which have been used to quantify the spatial distribution of PI3K and Rac1 in polarized cells seeded on micropatterns [22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Fluocell for Ratiometric and High-Throughput Live-Cell Image Visualization and Quantitation

et al. 2019

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Spatiotemporal regulation of molecular activities dictates cellular function and fate. Investigation of dynamic molecular activities in live cells often requires the visualization and quantitation of fluorescent ratio image sequences with subcellular resolution and in high throughput. Hence, there is a great need for convenient software tools specifically designed with these capabilities. Here we describe a well-characterized open-source software package, Fluocell, customized to visualize pixelwise ratiometric images and calculate ratio time courses with subcellular resolution and in high throughput. Fluocell also provides group statistics and kinetic analysis functions for the quantified time courses, as well as 3D structure and function visualization for ratio images. The application of Fluocell is demonstrated by the ratiometric analysis of intensity images for several single-chain Förster (or fluorescence) resonance energy transfer (FRET)-based biosensors, allowing efficient quantification of dynamic molecular activities in a heterogeneous population of single live cells. Our analysis revealed distinct activation kinetics of Fyn kinase in the cytosolic and membrane compartments, and visualized a 4D spatiotemporal distribution of epigenetic signals in mitotic cells. Therefore, Fluocell provides an integrated environment for ratiometric live-cell image visualization and analysis, which generates high-quality single-cell dynamic data and allows the quantitative machine-learning of biophysical and biochemical computational models for molecular regulations in cells and tissues.

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Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors

Cited by 48 publications

References 58 publications

Unphosphorylated STAT3 in heterochromatin formation and tumor suppression in lung cancer

Unphosphorylated STAT3 in heterochromatin formation and tumor suppression in lung cancer

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Fluocell for Ratiometric and High-Throughput Live-Cell Image Visualization and Quantitation

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