Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells

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Reference

Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells

CREFCOEUR, Remco Paul, et al.

Abstract

Light-sensitive proteins are useful tools to control protein localization, activation and gene expression, but are currently limited to excitation with red or blue light. Here we report a novel optogenetic system based on the ultraviolet-B-dependent interaction of the Arabidopsis ultraviolet-B photoreceptor UVR8 with COP1 that can be performed in visible light background. We use this system to induce nuclear accumulation of cytoplasmic green fluorescent protein fused to UVR8 in cells expressing nuclear COP1, and to recruit a nucleoplasmic red fluorescent protein fused to COP1 to chromatin in cells expressing UVR8-H2B. We also show that ultraviolet-B-dependent interactions between DNA-binding and transcription activation domains result in a linear induction of gene expression. The UVR8-COP1 interactions in mammalian cells can be induced using subsecond pulses of ultraviolet-B light and last several hours. As UVR8 photoperception is based on intrinsic tryptophan residues, these interactions do not depend on the addition of an exogenous chromophore.

CREFCOEUR, Remco Paul,

. Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells.

, 2013, vol. 4, p. 1779

DOI : 10.1038/ncomms2800 PMID : 23653191

Available at:

http://archive-ouverte.unige.ch/unige:29083

Disclaimer: layout of this document may differ from the published version.

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Supplementary Figures S1-S7 for

UV-B-mediated induction of protein-protein interactions in mammalian cells

Remco P. Crefcoeur

, Ruohe Yin

, Roman Ulm

and Thanos D. Halazonetis

1

Department of Molecular Biology,

Department of Botany and Plant Biology, and

Department of Biochemistry, University of Geneva, 30, Quai Ernest-Ansermet CH-1211, Geneva, Switzerland.

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Supplementary Figure S1 | UVR8 and COP1 domain maps and constructs. UVR8 contains a seven bladed β-propeller type domain. Trp285 (W285) is the main UV-B chromophore. COP1 contains a RING domain, a coiled-coil domain and a WD40 repeat domain. The constructs used in this study are shown below the full-length proteins. All domains are drawn to scale. Numbers indicate the amino acid positions that correspond to the wild-type UVR8 and COP1 proteins.

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Supplementary Figure S2 | No nuclear retention of UVR8 monomers in the absence of NLS-COP1. (a) U2OS cells expressing GFP-UVR8 before (-) and 2 hours after (+) exposure to a UV-B pulse. Scale bar, 20 µm. (b) U2OS cells expressing GFP-UVR8^W285A before (-) and 2 hours after (+) exposure to UV-B. Scale bar, 20 µm.

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Supplementary Figure S3 | Constitutive interaction of H2B-GFP- UVR8 with mCh-NLS-COP1^C340. U2OS cells expressing H2B-GFP- UVR8 and mCh-NLS-COP1^C340 (top row), H2B-GFP-UVR8^W285A and mCh-NLS-COP1^C340 (middle row) or H2B-GFP-UVR8^W285F and mCh- NLS-COP1^C340 (bottom row) were examined by live-cell microscopy.

The cells had not been exposed to UV-B. WT, wild-type. Scale bar, 10 µm.

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Supplementary Figure S4 | Interaction between mCh-NLS-COP1^C340 and H2B-GFP-UVR8(2x) or GFP-UVR8(2x)-H3 before and after exposure to a UV-B pulse. (a) Sustained interaction between mCh-NLS-COP1^C340 and H2B-GFP-UVR8(2x) after exposure to a UV-B pulse. U2OS cells expressing H2B-GFP-UVR8(2x) and mCh-NLS-COP1^C340 were exposed to a 25 J/m² UV-B pulse.

Live-cell images were acquired before exposure to UV-B (0h) and then immediately (0h, +UV), one (1h, +UV) and four (4h, +UV) hours after exposure to UV-B. Scale bar, 10 µm. (b) Interaction between mCh-NLS-COP1^C340 and GFP-UVR8(2x)-H3. Cells were examined before and immediately after exposure to a 25 J/m² UV-B pulse. Scale bar, 10 µm.

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Supplementary Figure S5 | Examples of mCh-NLS-COP1^C340 localization patterns. Representative examples of the three mCh-NLS- COP1^C340 localization patterns observed in U2OS cells expressing H2B- GFP-UVR8(2x) and mCh-NLS-COP1^C340 before or after exposure to UV- B: nucleoplasmic (mCh-NLS-COP1^C340 is present throughout the nucleus and the nucleoli are not visible); partially nucleoplasmic (mCh-NLS- COP1^C340 is present in the nucleoplasm, but the nucleoli are visible, suggesting some binding to chromatin) and chromatin-bound (mCh-NLS- COP1^C340 colocalizes well with the chromatin-bound H2B-GFP- UVR8(2x)). Scale bar, 10 µm.

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Supplementary Figure S6 | UV-B light-responsive gene expression in yeast. (a) Tight regulation of LexA^BD-UVR8 and GAL4^AD-COP1^C340 interaction by UV-B based on lacZ activity. Quantitative yeast two-hybrid assays were performed with either standard grown (- UV-B) or 16 h UV-B exposed (1.5 μmol m^-2 s^-1) yeast cells. EV: empty vector control. Mean values and standard deviations from three biological replicates are shown. (b) Interaction between UVR8 and COP1^C340 is specific to UV-B exposure. Yeast colonies expressing LexA^BD-UVR8 and GAL4^AD-COP1^C340 were exposed for 16 h with UV-B (1.5 μmol m^-2 s^-1), UV-A (20.1 μmol m^-2 s^-1), blue (28.7 μmol m^-2 s^-1), red (32.0 μmol m^-2 s^-1), and white light (3.6 μmol m^-2 s^-1) before quantitative yeast two-hybrid assay. Mean value and standard deviation from three biological replicates are shown.

(c) UV-B dose response of interaction between UVR8 and COP1^C340. Yeast colonies expressing LexA^BD-UVR8 and GAL4^AD-COP1^C340 were exposed 1 h with the indicated UV-B dose and then incubated in darkness for 2.5 h before quantification of the interaction. (d) Cell survival of yeast was not affected by the UV-B treatment in c. Aliquots of equal cell number from the yeast cells used for quantification of β-galactosidase activity in c were incubated for two days post-UV-B irradiation in standard growth conditions. Yeast colony numbers (representing a measure of viability of the initial, UV-B-irradiated cells) were counted and the number of the negative control (-UV-B) was set to 100%.

Mean value and standard deviation from three biological replicates are shown.

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Supplementary Figure S7 | UV-B induced γ-H2AX focus formation. U2OS cells grown on PMMA coverslips were exposed to UV-B using the setup shown in Fig. 4a. The UV-B LED was turned on for 0, 0.5, 1, 3, 10, and 30 sec, as indicated. U2OS cells grown on PMMA coverslips were also exposed to 25 J/m² UV-B using a transilluminator. Two hours after UV-B treatment, the cells were fixed and processed for immunofluorescence using antibodies specific for γ-H2AX (Abcam). The nuclei of the cells were counterstained with DAPI. Scale bar, 20 µm.

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