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Direct observation of dimerization between different CREB1 isoforms in a living cell.

Sadamoto H, Saito K, Muto H, Kinjo M, Ito E - PLoS ONE (2011)

Bottom Line: In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor.Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells.As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan. sadamotoh@kph.bunri-u.ac.jp

ABSTRACT
Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

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The quantitative evaluation of FCCS data for interaction between CREB1 isoforms.A, Relative cross-correlation [(Gc(τ)−1)/(Gr(0)−1)] calculated from FCCS measurement. Typical curves of cross-correlation were shown for EGFP- mRFP chimera (a), the EGFP-CREB1 activator isoform protein and the tandem mRFP-CREB1 repressor isoform protein (b), the EGFP-truncated CREB1 activator isoform protein and the tandem mRFP-truncated CREB1 repressor protein (c), and the EGFP-CREB1 activator protein and the tandem mRFP-truncated CREB1 repressor protein (d). B, Relative cross amplitudes [(Gc(0)−1)/(Gr(0)−1)] calculated from each FCCS measurement that corresponds to the fraction of the associated molecules (Nc/Ng). Here Nc is the average number of particles that have both green and red fluorescence in the excitation-detection volume, and Ng is that of the green fluorescent particles. Each relative cross amplitude: EGFP-Act and mRFP-Rep (black bar), EGFP-Act mut and mRFP-Rep mut (white bar), EGFP-Act and mRFP-Rep mut (hatched bar), EGFP-mRFP chimera proteins (cross-hatched bar).
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pone-0020285-g003: The quantitative evaluation of FCCS data for interaction between CREB1 isoforms.A, Relative cross-correlation [(Gc(τ)−1)/(Gr(0)−1)] calculated from FCCS measurement. Typical curves of cross-correlation were shown for EGFP- mRFP chimera (a), the EGFP-CREB1 activator isoform protein and the tandem mRFP-CREB1 repressor isoform protein (b), the EGFP-truncated CREB1 activator isoform protein and the tandem mRFP-truncated CREB1 repressor protein (c), and the EGFP-CREB1 activator protein and the tandem mRFP-truncated CREB1 repressor protein (d). B, Relative cross amplitudes [(Gc(0)−1)/(Gr(0)−1)] calculated from each FCCS measurement that corresponds to the fraction of the associated molecules (Nc/Ng). Here Nc is the average number of particles that have both green and red fluorescence in the excitation-detection volume, and Ng is that of the green fluorescent particles. Each relative cross amplitude: EGFP-Act and mRFP-Rep (black bar), EGFP-Act mut and mRFP-Rep mut (white bar), EGFP-Act and mRFP-Rep mut (hatched bar), EGFP-mRFP chimera proteins (cross-hatched bar).

Mentions: We next performed the normalization of cross-correlation data among various samples. Following the previous report [16], the cross-correlation [Gc(τ)−1] was normalized by [Gr(0)−1] as the relative cross-correlation [Gc(τ)−1]/[Gr(0)−1] (Fig. 3A). As a result, we observed clear cross-correlation in the measurements of positive control experiments (Fig. 2A, 3Aa) and differently labeled CREB1 isoforms (EGFP-Act and mRFP-Rep, Fig. 2B, 3Ab), whereas, almost none of cross-correlation in the negative control experiments using truncated CREB1 isoform proteins (EGFP-Act mut and mRFP-Rep mut, and EGFP-Act and mRFP-Rep mut, Fig. 2C, 2D, 3Ac, 3Ad). The fluorescence auto-correlation functions of the green and red fluorescent molecules, Gg(τ) and Gr(τ) (τ: the time delay), and the fluorescence cross-correlation function, Gc(τ), were calculated by equation (1) in Materials and Methods.


Direct observation of dimerization between different CREB1 isoforms in a living cell.

Sadamoto H, Saito K, Muto H, Kinjo M, Ito E - PLoS ONE (2011)

The quantitative evaluation of FCCS data for interaction between CREB1 isoforms.A, Relative cross-correlation [(Gc(τ)−1)/(Gr(0)−1)] calculated from FCCS measurement. Typical curves of cross-correlation were shown for EGFP- mRFP chimera (a), the EGFP-CREB1 activator isoform protein and the tandem mRFP-CREB1 repressor isoform protein (b), the EGFP-truncated CREB1 activator isoform protein and the tandem mRFP-truncated CREB1 repressor protein (c), and the EGFP-CREB1 activator protein and the tandem mRFP-truncated CREB1 repressor protein (d). B, Relative cross amplitudes [(Gc(0)−1)/(Gr(0)−1)] calculated from each FCCS measurement that corresponds to the fraction of the associated molecules (Nc/Ng). Here Nc is the average number of particles that have both green and red fluorescence in the excitation-detection volume, and Ng is that of the green fluorescent particles. Each relative cross amplitude: EGFP-Act and mRFP-Rep (black bar), EGFP-Act mut and mRFP-Rep mut (white bar), EGFP-Act and mRFP-Rep mut (hatched bar), EGFP-mRFP chimera proteins (cross-hatched bar).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105992&req=5

pone-0020285-g003: The quantitative evaluation of FCCS data for interaction between CREB1 isoforms.A, Relative cross-correlation [(Gc(τ)−1)/(Gr(0)−1)] calculated from FCCS measurement. Typical curves of cross-correlation were shown for EGFP- mRFP chimera (a), the EGFP-CREB1 activator isoform protein and the tandem mRFP-CREB1 repressor isoform protein (b), the EGFP-truncated CREB1 activator isoform protein and the tandem mRFP-truncated CREB1 repressor protein (c), and the EGFP-CREB1 activator protein and the tandem mRFP-truncated CREB1 repressor protein (d). B, Relative cross amplitudes [(Gc(0)−1)/(Gr(0)−1)] calculated from each FCCS measurement that corresponds to the fraction of the associated molecules (Nc/Ng). Here Nc is the average number of particles that have both green and red fluorescence in the excitation-detection volume, and Ng is that of the green fluorescent particles. Each relative cross amplitude: EGFP-Act and mRFP-Rep (black bar), EGFP-Act mut and mRFP-Rep mut (white bar), EGFP-Act and mRFP-Rep mut (hatched bar), EGFP-mRFP chimera proteins (cross-hatched bar).
Mentions: We next performed the normalization of cross-correlation data among various samples. Following the previous report [16], the cross-correlation [Gc(τ)−1] was normalized by [Gr(0)−1] as the relative cross-correlation [Gc(τ)−1]/[Gr(0)−1] (Fig. 3A). As a result, we observed clear cross-correlation in the measurements of positive control experiments (Fig. 2A, 3Aa) and differently labeled CREB1 isoforms (EGFP-Act and mRFP-Rep, Fig. 2B, 3Ab), whereas, almost none of cross-correlation in the negative control experiments using truncated CREB1 isoform proteins (EGFP-Act mut and mRFP-Rep mut, and EGFP-Act and mRFP-Rep mut, Fig. 2C, 2D, 3Ac, 3Ad). The fluorescence auto-correlation functions of the green and red fluorescent molecules, Gg(τ) and Gr(τ) (τ: the time delay), and the fluorescence cross-correlation function, Gc(τ), were calculated by equation (1) in Materials and Methods.

Bottom Line: In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor.Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells.As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan. sadamotoh@kph.bunri-u.ac.jp

ABSTRACT
Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

Show MeSH
Related in: MedlinePlus