Light-induced nuclear export reveals rapid dynamics of epigenetic modifications

Abstract

We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.

Figure 1

Design of the Light Inducible Nuclear eXporter (LINX) and its use with the improved Light Inducible Dimer (iLID). (a) Design scheme for LINXa (cNES – conditional Nuclear Export Signal) illustrating its nuclear export upon binding to CRM1 after blue light illumination. Below the schematic the sequences for the WT Jα helix, the NES motif Super-PKI-2, and the engineered Jα helix in LINXa are shown. Residues important for nuclear export are shown in green. (b) Design scheme and sequences for LINXb. (c) Photoactivation of LINXa and LINXb fused to fluorescent proteins in mouse fibroblasts (IA32) (scale bar = 50 μm). Individual cells were activated in a field of cells, and nucleocytoplasmic ratios were measured as a function of time (Supplementary Movies 1 and 2 and Supplementary Fig. 3). (d) Quantification of nuclear/cytoplasmic fluorescence intensity change upon activation with blue light for LINXa3 and LINXb3. Mean values ± the standard error of the mean (s.e.m.) were calculated from images of multiple cells (LINXa3 n=5 and LINXb3 n=6). (e) LINX in combination with iLID enhances nuclear export and allows targeting to specific locations in the cytoplasm. One half of the light inducible dimer (nano) was fused to LINX and the other half (iLID) to a mitochondrial anchor. (f) Quantification of photoactivation in IA32 cells using LINXa3-nano and iLID-Mito (n=3, mean ± s.e.m.).

Figure 2

Control of gene transcription and histone modifications with LINX. (a, b) β-Galactosidase activity in the light (blue bars) and dark (black bars) induced with LINX variants fused to a LexA DNA binding domain and the Gal4 activation domain (n=3, mean ± s.e.m., two-tailed t-test). (c) Light-mediated control of the Bre1 E3 ligase in a BRE1 deletion strain as evidenced through immunoblotting with antibodies specific to various histone modifications (see Supplementary Fig. 12 for original film images). In all constructs the Bre1 NLS has been inactivated with mutations. Non-specific bands are indicated with asterisks. (d) Western blot quantification of histone modifications as a function of time after the transition from dark to blue light with the LINXa4-Bre1 switch (n=3, mean ± s.e.m. see Supplementary Fig. 11 and Supplementary Fig. 13 for original film images). (e) Western blot quantification of histone modifications as a function of time after the transition from blue light to dark with the LINXa4-Bre1 switch in (n=3, mean ± s.e.m. see Supplementary Fig. 11 and Supplementary Fig. 13 for original film images). Half-lives were determined from single exponential fits to the H2Bub1 and H3K4me3 relative abundance data using Prism 5.