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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

H3K9-mintbody in zebrafish. Zebrafish expressing H3K9-mintbody developed normally. A fluorescence image of a zebrafish expressing H3K9-mintbody (30 h post fertilization) was collected using a confocal microscope. Bar 100 μm
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Fig5: H3K9-mintbody in zebrafish. Zebrafish expressing H3K9-mintbody developed normally. A fluorescence image of a zebrafish expressing H3K9-mintbody (30 h post fertilization) was collected using a confocal microscope. Bar 100 μm

Mentions: Although FabLEM is a powerful technique, it requires purified Fabs and direct loading into cells, which may prevent long-term and high-throughput assays. In vivo analysis using model organisms is also limited except just after fertilization during which microinjection and imaging are relatively easy. To overcome these limitations, we have also developed a genetically encoded system using single-chain variable fragments (scFv; Ahmad et al. 2012) tagged with the enhanced green fluorescent protein (EGFP). We cloned the scFv coding sequence from hybridoma cells producing the specific antibody against histone H3 Lys9 acetylation (H3K9ac) and then genetically fused the scFv with EGFP (Fig. 2, bottom). We named the scFv-EGFP probe a modification-specific intracellular antibody, or “mintbody” (Sato et al. 2013). The H3K9ac-specific mintbody (H3K9ac-mintbody) bound to the target acetylation in living cells, and the changes in acetylation levels in response to a histone deacetylase inhibitor could be monitored by FRAP or the nuclear/cytoplasmic intensity ratio, just like FabLEM. Making use of the genetically encoded system, we have generated transgenic fruit fly and zebrafish that express the H3K9ac-mintbody. Importantly, those transgenic Drosophila and zebrafish developed normally and remain fertile, indicating that the expression of mintbody at a certain level does not affect development and differentiation (Fig. 5).Fig. 5


Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

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

H3K9-mintbody in zebrafish. Zebrafish expressing H3K9-mintbody developed normally. A fluorescence image of a zebrafish expressing H3K9-mintbody (30 h post fertilization) was collected using a confocal microscope. Bar 100 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522274&req=5

Fig5: H3K9-mintbody in zebrafish. Zebrafish expressing H3K9-mintbody developed normally. A fluorescence image of a zebrafish expressing H3K9-mintbody (30 h post fertilization) was collected using a confocal microscope. Bar 100 μm
Mentions: Although FabLEM is a powerful technique, it requires purified Fabs and direct loading into cells, which may prevent long-term and high-throughput assays. In vivo analysis using model organisms is also limited except just after fertilization during which microinjection and imaging are relatively easy. To overcome these limitations, we have also developed a genetically encoded system using single-chain variable fragments (scFv; Ahmad et al. 2012) tagged with the enhanced green fluorescent protein (EGFP). We cloned the scFv coding sequence from hybridoma cells producing the specific antibody against histone H3 Lys9 acetylation (H3K9ac) and then genetically fused the scFv with EGFP (Fig. 2, bottom). We named the scFv-EGFP probe a modification-specific intracellular antibody, or “mintbody” (Sato et al. 2013). The H3K9ac-specific mintbody (H3K9ac-mintbody) bound to the target acetylation in living cells, and the changes in acetylation levels in response to a histone deacetylase inhibitor could be monitored by FRAP or the nuclear/cytoplasmic intensity ratio, just like FabLEM. Making use of the genetically encoded system, we have generated transgenic fruit fly and zebrafish that express the H3K9ac-mintbody. Importantly, those transgenic Drosophila and zebrafish developed normally and remain fertile, indicating that the expression of mintbody at a certain level does not affect development and differentiation (Fig. 5).Fig. 5

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