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Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

Kimura H, Hayashi-Takanaka Y, Stasevich TJ, Sato Y - Histochem. Cell Biol. (2015)

Bottom Line: As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein.In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation.Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, hkimura@bio.titech.ac.jp.

ABSTRACT
Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

No MeSH data available.


Related in: MedlinePlus

RNAP2 activation kinetics revealed by FabLEM. Fluorescence images (top) and schematic illustration (bottom) showing how RNAP2 activation by FabLEM is tracked. After addition of dexamethasone (Dex), a steroid hormone that binds to the glucocorticoid receptor (GR), to cell culture medium, GFP-GR enters the nucleus and accumulates at the gene array (top, arrowheads). FabLEM using RNAP2 phosphorylation-specific Fabs (Cy5-labeled Rpb1-CTD-Ser5ph and Cy3-labeled Rpb1-CTD-Ser2ph) revealed the timing of initiation and elongation of RNAP2 at the array
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Fig6: RNAP2 activation kinetics revealed by FabLEM. Fluorescence images (top) and schematic illustration (bottom) showing how RNAP2 activation by FabLEM is tracked. After addition of dexamethasone (Dex), a steroid hormone that binds to the glucocorticoid receptor (GR), to cell culture medium, GFP-GR enters the nucleus and accumulates at the gene array (top, arrowheads). FabLEM using RNAP2 phosphorylation-specific Fabs (Cy5-labeled Rpb1-CTD-Ser5ph and Cy3-labeled Rpb1-CTD-Ser2ph) revealed the timing of initiation and elongation of RNAP2 at the array

Mentions: We have recently applied the FabLEM technique to measure the kinetics of RNAP2 in living cells and to quantify the effects of histone modifications on transcription (Stasevich et al. 2014a, b). As a model system, we used a mouse cell line that stably expresses GFP-tagged glucocorticoid receptor (GFP-GR) and harbors a genome-integrated gene array consisting of ~200 copies of glucocorticoid-responsive promoter (the mouse mammary tumor virus long-terminal repeat). Transcription of the gene array can be activated by addition of glucocorticoid hormone, which induces nuclear translocation of GR for gene activation. After loading Cy3- and Cy5-labeled Fabs that recognize differentially phosphorylated forms of RNAP2 and treating cells with the hormone, the kinetics of RNAP2 recruitment (unphosphorylated), initiation (S5 phosphorylated), and elongation (S2 phosphorylated) at the array after GFP-GR accumulation were determined (Fig. 6). Quantitative measurements and fitting to mathematical models revealed that the transition from initiation to elongation is quite efficient at the array. This high elongation efficiency was correlated with the level of preexisting histone H3K27ac, which appeared to be controlled by a balance between p300 histone acetyl transferase and histone deacetylase 4 or 7. Thus, H3K27ac can alter downstream transcription kinetics by indirectly recruiting P-TEFb kinase, in addition to enhancing the binding of GFP-GR. This study indicates a mechanism for how a histone modification can contribute to the regulation of transcription by RNAP2 in living cells (Stasevich et al. 2014a).Fig. 6


Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

Kimura H, Hayashi-Takanaka Y, Stasevich TJ, Sato Y - Histochem. Cell Biol. (2015)

RNAP2 activation kinetics revealed by FabLEM. Fluorescence images (top) and schematic illustration (bottom) showing how RNAP2 activation by FabLEM is tracked. After addition of dexamethasone (Dex), a steroid hormone that binds to the glucocorticoid receptor (GR), to cell culture medium, GFP-GR enters the nucleus and accumulates at the gene array (top, arrowheads). FabLEM using RNAP2 phosphorylation-specific Fabs (Cy5-labeled Rpb1-CTD-Ser5ph and Cy3-labeled Rpb1-CTD-Ser2ph) revealed the timing of initiation and elongation of RNAP2 at the array
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522274&req=5

Fig6: RNAP2 activation kinetics revealed by FabLEM. Fluorescence images (top) and schematic illustration (bottom) showing how RNAP2 activation by FabLEM is tracked. After addition of dexamethasone (Dex), a steroid hormone that binds to the glucocorticoid receptor (GR), to cell culture medium, GFP-GR enters the nucleus and accumulates at the gene array (top, arrowheads). FabLEM using RNAP2 phosphorylation-specific Fabs (Cy5-labeled Rpb1-CTD-Ser5ph and Cy3-labeled Rpb1-CTD-Ser2ph) revealed the timing of initiation and elongation of RNAP2 at the array
Mentions: We have recently applied the FabLEM technique to measure the kinetics of RNAP2 in living cells and to quantify the effects of histone modifications on transcription (Stasevich et al. 2014a, b). As a model system, we used a mouse cell line that stably expresses GFP-tagged glucocorticoid receptor (GFP-GR) and harbors a genome-integrated gene array consisting of ~200 copies of glucocorticoid-responsive promoter (the mouse mammary tumor virus long-terminal repeat). Transcription of the gene array can be activated by addition of glucocorticoid hormone, which induces nuclear translocation of GR for gene activation. After loading Cy3- and Cy5-labeled Fabs that recognize differentially phosphorylated forms of RNAP2 and treating cells with the hormone, the kinetics of RNAP2 recruitment (unphosphorylated), initiation (S5 phosphorylated), and elongation (S2 phosphorylated) at the array after GFP-GR accumulation were determined (Fig. 6). Quantitative measurements and fitting to mathematical models revealed that the transition from initiation to elongation is quite efficient at the array. This high elongation efficiency was correlated with the level of preexisting histone H3K27ac, which appeared to be controlled by a balance between p300 histone acetyl transferase and histone deacetylase 4 or 7. Thus, H3K27ac can alter downstream transcription kinetics by indirectly recruiting P-TEFb kinase, in addition to enhancing the binding of GFP-GR. This study indicates a mechanism for how a histone modification can contribute to the regulation of transcription by RNAP2 in living cells (Stasevich et al. 2014a).Fig. 6

Bottom Line: As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein.In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation.Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, hkimura@bio.titech.ac.jp.

ABSTRACT
Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

No MeSH data available.


Related in: MedlinePlus