FRAP and FRET Methods to Study Nuclear Receptors in Living Cells

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARγ) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor α. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARγ but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARγ in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

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Integrating nuclear receptor mobility in models of gene regulation

Nuclear Receptor Signaling ◽

10.1621/nrs.04010 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04010 ◽

Cited By ~ 4

Author(s):

Laurent Gelman ◽

Jerome N. Feige ◽

Cicerone Tudor ◽

Yves Engelborghs ◽

Walter Wahli ◽

...

Keyword(s):

Gene Regulation ◽

Fluorescence Microscopy ◽

Nuclear Receptors ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Living Cells ◽

Chromatin Interaction ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptors

The mode of action of nuclear receptors in living cells is an actively investigated field but much remains hypothetical due to the lack, until recently, of methods allowing the assessment of molecular mechanisms in vivo. However, these last years, the development of fluorescence microscopy methods has allowed initiating the dissection of the molecular mechanisms underlying gene regulation by nuclear receptors directly in living cells or organisms. Following our analyses on peroxisome proliferator activated receptors (PPARs) in living cells, we discuss here the different models arising from the use of these tools, that attempt to link mobility, DNA binding or chromatin interaction, and transcriptional activity.

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Diffusion-Time Distribution Analysis Reveals Characteristic Ligand-Dependent Interaction Patterns of Nuclear Receptors in Living Cells†

Biochemistry ◽

10.1021/bi050744v ◽

2005 ◽

Vol 44 (35) ◽

pp. 11676-11683 ◽

Cited By ~ 27

Author(s):

Hanna Jankevics ◽

Michael Prummer ◽

Paulina Izewska ◽

Horst Pick ◽

Kirsten Leufgen ◽

...

Keyword(s):

Nuclear Receptors ◽

Time Distribution ◽

Living Cells ◽

Diffusion Time ◽

Distribution Analysis ◽

Interaction Patterns ◽

Dependent Interaction

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Using Inducible Vectors to Study Intracellular Trafficking of GFP-Tagged Steroid/Nuclear Receptors in Living Cells

Methods ◽

10.1006/meth.1999.0874 ◽

1999 ◽

Vol 19 (3) ◽

pp. 386-393 ◽

Cited By ~ 67

Author(s):

Dawn Walker ◽

Han Htun ◽

Gordon L. Hager

Keyword(s):

Nuclear Receptors ◽

Intracellular Trafficking ◽

Living Cells

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

Molecular and Cellular Endocrinology ◽

10.1016/j.mce.2008.07.021 ◽

2008 ◽

Vol 294 (1-2) ◽

pp. 45-51 ◽

Cited By ~ 10

Author(s):

Joanna Dutko-Gwóźdź ◽

Tomasz Gwóźdź ◽

Marek Orłowski ◽

Beata Greb-Markiewicz ◽

Danuta Duś ◽

...

Keyword(s):

Nuclear Receptors ◽

Living Cells

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Imaging of heme/hemeproteins in nucleus of the living cells expressing heme-binding nuclear receptors

FEBS Letters ◽

10.1016/j.febslet.2013.05.036 ◽

2013 ◽

Vol 587 (14) ◽

pp. 2131-2136 ◽

Cited By ~ 5

Author(s):

Ryuhei Itoh ◽

Ken-ichi Fujita ◽

Anfeng Mu ◽

Dao Hoang Thien Kim ◽

Tran Tien Tai ◽

...

Keyword(s):

Nuclear Receptors ◽

Living Cells ◽

Heme Binding

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The composite nature of the interaction between nuclear receptors EcR and DHR38

Biological Chemistry ◽

10.1515/hsz-2011-0283 ◽

2012 ◽

Vol 393 (6) ◽

pp. 457-471 ◽

Cited By ~ 8

Author(s):

Anna Zoglowek ◽

Marek Orłowski ◽

Szymon Pakuła ◽

Joanna Dutko-Gwóźdź ◽

Dorota Pajdzik ◽

...

Keyword(s):

Nuclear Receptors ◽

Recombinant Dna ◽

Living Cells ◽

Biological Processes ◽

Ecdysteroid Receptor ◽

Dna Binding Domains ◽

Intrinsically Disordered ◽

Binding Domains ◽

Composite Nature

Abstract Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.

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Fluorescence ratio imaging of dynamic intracellular ionic signals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102298 ◽

1988 ◽

Vol 46 ◽

pp. 42-43

Author(s):

R. Y. Tsien ◽

A. Minta ◽

M. Poenie ◽

J.P.Y. Kao ◽

A. Harootunian

Keyword(s):

Binding Sites ◽

Living Cells ◽

Molecular Engineering ◽

Ph Indicators ◽

Highly Sensitive ◽

Fluorescence Ratio Imaging ◽

Ratio Imaging ◽

Technical Advances ◽

Indicator Dyes ◽

Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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