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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

Schematic illustration of histone and RNAP2 modifications. Transcriptionally inactive condensed chromatin harbors inactive histone marks, such as H3K9me2, H3K9me3, and H3K27me3. In contrast, transcriptionally active promoter regions are associated with active marks, such as H3K4me3 and H3K27ac. RNAP2 phosphorylation is associated with the transcription cycle. Ser5 and Ser2 along the Rpb1-CTD repeat are phosphorylated during the initiation and elongation, respectively
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Fig1: Schematic illustration of histone and RNAP2 modifications. Transcriptionally inactive condensed chromatin harbors inactive histone marks, such as H3K9me2, H3K9me3, and H3K27me3. In contrast, transcriptionally active promoter regions are associated with active marks, such as H3K4me3 and H3K27ac. RNAP2 phosphorylation is associated with the transcription cycle. Ser5 and Ser2 along the Rpb1-CTD repeat are phosphorylated during the initiation and elongation, respectively

Mentions: Posttranslational protein modification plays a critical role in gene regulation, as it does in many other important biological events, such as signal transduction. Both the template chromatin for transcription and the transcribing enzyme, RNA polymerase, can be heavily modified (Fig. 1) (Bannister and Kouzarides 2011; Eick and Geyer 2013; Kimura 2013; Jeronimo et al. 2013), and our approach to tracking their modifications in living cells has been a breakthrough to fully understand how genes are regulated in vivo.Fig. 1


Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

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

Schematic illustration of histone and RNAP2 modifications. Transcriptionally inactive condensed chromatin harbors inactive histone marks, such as H3K9me2, H3K9me3, and H3K27me3. In contrast, transcriptionally active promoter regions are associated with active marks, such as H3K4me3 and H3K27ac. RNAP2 phosphorylation is associated with the transcription cycle. Ser5 and Ser2 along the Rpb1-CTD repeat are phosphorylated during the initiation and elongation, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic illustration of histone and RNAP2 modifications. Transcriptionally inactive condensed chromatin harbors inactive histone marks, such as H3K9me2, H3K9me3, and H3K27me3. In contrast, transcriptionally active promoter regions are associated with active marks, such as H3K4me3 and H3K27ac. RNAP2 phosphorylation is associated with the transcription cycle. Ser5 and Ser2 along the Rpb1-CTD repeat are phosphorylated during the initiation and elongation, respectively
Mentions: Posttranslational protein modification plays a critical role in gene regulation, as it does in many other important biological events, such as signal transduction. Both the template chromatin for transcription and the transcribing enzyme, RNA polymerase, can be heavily modified (Fig. 1) (Bannister and Kouzarides 2011; Eick and Geyer 2013; Kimura 2013; Jeronimo et al. 2013), and our approach to tracking their modifications in living cells has been a breakthrough to fully understand how genes are regulated in vivo.Fig. 1

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