The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

Dutko-Gwóźdź, Joanna; Gwóźdź, Tomasz; Orłowski, Marek; Greb‐Markiewicz, Beata; Duś, Danuta; Dobrucki, Jurek; Ożyhar, Andrzej

doi:10.1016/j.mce.2008.07.021

Cited by 12 publications

(8 citation statements)

References 36 publications

(51 reference statements)

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“…Although members of the NGFI-B family are thought to function exclusively as monomers, DHR38 in fact interacts strongly with Usp, and this interaction is evolutionarily conserved (Dutko-Gwozdz et al, 2008;Saiki et al, 1985;Sutherland et al, 1995). DHR38 can repress transcription of a reporter gene under the control of the hsp27 element as revealed in transfection experiments with Drosophila Schneider cells.…”

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confidence: 99%

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

Pakuła

Orłowski

Rymarczyk

et al. 2012

Journal of Biomolecular Structure and Dynamics

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The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.

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confidence: 99%

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

Pakuła

Orłowski

Rymarczyk

et al. 2012

Journal of Biomolecular Structure and Dynamics

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“…We used CHO-K1 cells, which were routinely used for heterologous studies of the EcR/USP complex [26]. The rationale for using heterologous cell cultures to test EcR and USP function is discussed extensively by Henrich et al [27].…”

Section: Resultsmentioning

confidence: 99%

The N-terminus of ecdysteroid receptor isoforms and ultraspiracle interacts with different ecdysteroid response elements in a sequence specific manner to modulate transcriptional activity

Schauer

Callender

Henrich

et al. 2011

The Journal of Steroid Biochemistry and Molecular Biology

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“…For instance, the reported rates of movement of mitochondria in neurons are 0.3–0.7 μm/s (6), chloroplasts 3 μm/s (7), and migration of fibroblasts and keratocytes in culture can reach 10–15 μm/h (8, 9). The influence of object translocations can be minimized using a small bleached area and a large field of view, as in (10), where the measured t 1/2 values were independent of object translocation (not shown).…”

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Conditions for using FRAP as a quantitative technique—Influence of the bleaching protocol

2010

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Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent method of data analysis. ' 2010 International Society for Advancement of Cytometry Key termsFRAP; fluorescence recovery after photobleaching; nuclear proteins; histone; mobile fraction; protein dynamics FLUORESCENCE recovery after photobleaching (FRAP) is a quantitative technique capable of measuring rates of exchange of proteins at their binding sites in a cell, and proportions between their mobile and immobile fractions. With an intention of comparing the protocols and the dynamic behavior of various nuclear proteins, we analyzed 47 original papers describing FRAP studies (protein dynamics data and FRAP technical information extracted from these papers has been compiled into Supp. Info. Table 1), as well as nine theoretical and 43 review papers on the subject. A broad analysis was rendered difficult, as the description of FRAP protocols was often incomplete or even lacking. Only three papers provided a full description of their FRAP protocol and stated if correction for movements of the cell or the nucleus was required and applied. Only eight papers provided information about the pixel size, or experimental conditions that enabled the reader to calculate this parameter. In 27 papers there was no information about the area of the bleached region, 29 papers provided only a general description (half of the nucleus or a strip across the nucleus). Eleven papers had no information about intensities of the bleaching light, 16 papers gave no information about the objective lens.In principle, the lack of technical information need not prevent interexperiment analysis of protein dynamics data, so long as the fluorescence recovery times are independent of the bleaching protocol. To establish if recovery times are indeed independent of the bleaching conditions, we performed FRAP experiments using GFPtagged histone H1 in HeLa cells. Histone H1 is highly dynamic; it has a large bound and a smaller unbound fraction (1,2). Tagging H1 ...

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The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

Abstract: The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptor superfamily, is considered to be functional receptor for the ecdysteroids that

Cited by 12 publications

References 36 publications

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

The N-terminus of ecdysteroid receptor isoforms and ultraspiracle interacts with different ecdysteroid response elements in a sequence specific manner to modulate transcriptional activity

Conditions for using FRAP as a quantitative technique—Influence of the bleaching protocol

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