Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Transcription

1 Supplementary Information Near-infrared optogenetic pair for protein regulation and spectral multiplexing Taras A. Redchuk 1, Evgeniya S. Omelina 1, Konstantin G. Chernov 1 and Vladislav V. Verkhusha 1,2 1 Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland, and 2 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA 1

2 Supplementary Results Supplementary Table 1. Functional characterization and structure prediction for tested mutants of a GAL4-Q-PAS1-VP16 chimeric transcription factor. Construct Coiled-coil probability, % Hydrophobic moment EGFP reporter expression level (a.u.) GAL4(68)-Q-PAS1-VP raw raw raw raw raw fine fine To utilize GAL4-Q-PAS1-VP16 heterodimerization with BphP1, a series of the GAL4-Q-PAS1- VP16 deletion mutants, which differ in a joining region between GAL4 and Q-linker, were constructed and tested in transcription activation assay. For all tested fusions the probability of forming a coiled-coil motif was predicted using a MARCOIL algorithm 24. An amphiphilicity of the helix joining regions was analyzed using a HeliQuest software 25. Generally, the constructs with higher coiled-coil probability and hydrophobic moment showed higher expression level of the EGFP reporter. 2

3 Supplementary Table 2. Comparison of transcription regulation systems that utilize engineered Q-PAS1. Transactivation construct Light controller Switch type Operation principle Reporter expression, fold DNA target Figure NLS-Q-PAS1- BphP1- VP16 mcherry-tetr BphP1- GAL4(148)-Q- mcherry- PAS1-VP16 CAAX ON OFF TF relocalization to nucleus 10 teto S6 7 TF caging at plasma GAL4 membrane 8 UAS 2b e GAL4(68)-Q- NLS-BphP1- GAL4 3a e, PAS1-VP16 mcherry OFF disruption of TF dimer 40 UAS S9 10 GAL4(148)-Q- PAS1-VP16 BphP1-SID OFF NLS- GAL4(148)-Q- BphP1- PAS1 mcherry-vp16 ON recruitment of histone deacetylase 5 TF recruitment to nucleus 42 GAL4 UAS S11 12 GAL4 UAS 4a-c TF, a transcription regulation factor. 3

4 Supplementary Table 3. Plasmids designed in this study. Plasmid Figure Vector backbone Promoter Insert ppps-dhth 1, S1 2 pet-22b T7 NdeI-Strep-tagII-NcoI- PpsR2dHTH -HindIII- 20aaLinker-AgeI-mRuby2-XhoI-His-tag ppps-dhth-pas2 1 pet-22b T7 NdeI-Strep-tagII-NcoI- PpsR2dHTH-PAS2- HindIII-20aaLinker-AgeI-mRuby2-XhoI- His-tag NdeI-Strep-tagII-NcoI- PpsR2dHTH-PAS1-2- ppps-dhth- 1 pet-22b T7 HindIII-20aaLinker-AgeI-mRuby2-XhoI- PAS1-2 His-tag ppps-q-pas1 1, S1 3 pet-22b T7 NdeI-Strep-tagII-NcoI- Q-PAS1-HindIII- 20aaLinker-AgeI-mRuby2-XhoI-His-tag ppps-q-linker 1, S1 2 pet-22b T7 NdeI-Strep-tagII-NcoI- Q-linker -HindIII- 20aaLinker-AgeI-mRuby2-XhoI-His-tag ppps-pas1 1, S1 pet-22b T7 NdeI-Strep-tag II-NcoI- PAS1-HindIII- 20aaLinker-AgeI-mRuby2-XhoI-His-tag pqp-207 S6 7 pires CMV NheI-NLS-AgeI- Q-PAS1-HindIII- 20aaLinker-NcoI-VP16-BglII-IRES-NcoI- BphP1-EcoRI-10aaLinker-mCherry-tetR-NotI pcmvd1-gv 2 pcmvd1 CMVd1 NcoI-GAL4-VP16-NotI pcmvd1- GAL4-68-VP16 2 pcmvd1 CMVd1 NcoI-GAL4(68)-VP16-NotI pqp-gal , S11 12 pcmvd1 CMVd1 NcoI-GAL4(148)-EcoRI-Q-PAS1-EcoRI- VP16-NotI pqp-gal NcoI-GAL4(148)-EcoRI-Q-PAS1-EcoRI- 2 pcmvd1 CMVd1 dnls VP16dNLS-NotI pqp-gal4-68 3, S9 10 pcmvd1 CMVd1 NcoI-GAL4(68)-Q-PAS1-EcoRI-VP16-NotI pqp-1raw 3, S9 10 pcmvd1 CMVd1 NcoI-GAL4(54)-Q-PAS1Δ7-EcoRI-VP16- NotI pqp-2raw 3, S9 10 pcmvd1 CMVd1 NcoI-GAL4(61)-Q-PAS1Δ14-EcoRI-VP16-4

5 pqp-3raw 3, S9 10 pcmvd1 CMVd1 pqp-4raw 3, S9 10 pcmvd1 CMVd1 pqp-5raw 3, S9 10 pcmvd1 CMVd1 pqp-i1-fine 3, S9 10 pcmvd1 CMVd1 pqp-d1-fine 3, S9 10 pcmvd1 CMVd1 pal-149-bphp1- SID S11-12 pcmv CMV pqp-nls-gal4-148-q-pas1 5 pcmvd1 CMVd1 pqp-t2a 4 psub- CMV CMV pqp-iris 5 pires2- EGFP CMV NotI NcoI-GAL4(68)-Q-PAS1Δ14-EcoRI-VP16- NotI NcoI-GAL4(68)-Q-PAS1Δ7-EcoRI-VP16- NotI NcoI-GAL4(68)-Q-PAS1Δ4-EcoRI-VP16- NotI NcoI-GAL4(68)-L-Q-PAS1-EcoRI-VP16- NotI NcoI-GAL4(68)-Q-PAS1Δ1-EcoRI-VP16- NotI KpnI-BphP1-EcoRI-10aaLinker-SpeImCherry-NheI-SID-NotI NcoI-NLS-5aaLinker-GAL4(148)-EcoRI-Q- PAS1-NotI XbaI-CMV-HindIII-BphP1-3 SAG- VP16dNLS-NotI-T2A-NLS-GAL4-Q-PAS1- XbaI NcoI-BphP1-mVenus-CAAX-IRES2-NESmCherry-Q-PAS1-AsLOV2cNLS-EcoRI 5

6 Supplementary Figure 1. Chemical cross-linking of purified PpsR2 mutants. Cross-linker MW kda Mild chemical cross-linking of PpsR2 mutants was performed using carbodiimide (EDC) and N- hydroxysuccinimide (NHS). It was shown that all constructs containing Q-linker constructs formed dimers. Dimers are indicated with arrows. The gel was stained with Coomassie blue. 6

7 Supplementary Figure 2. BphP1 interaction with the Q-PAS1, Q-linker and PpsR2dHTH fragments before background subtraction. Quantification of the densitometry data of pull-down experiments where the Q-PAS1, Q-linker and PpsR2dHTH interacted with BphP1 in the samples illuminated with 740 nm light during 2 h. The similar samples were also kept in darkness. All samples were resolved in the SDS-PAGE gel and visualized using the zinc-induced fluorescence assay. These results after the normalization and background subtraction are shown in Figure 1d. Error bars represent s.e.m., n=3 experiments. 7

8 Supplementary Figure 3. Chemical cross-linking of the purified BphP1 and Q-PAS1 proteins Crosslinker 162 kda 130 kda 100 kda Q-PAS1 Q-PAS1+ BphP1 BphP1 The purified samples of BphP1 and mruby2-tagged Q-PAS1 were analysed by the chemical cross-linking assay and visualized in the SDS-PAGE gel. A heterodimer band of ~130 kda, corresponding to the bound BphP1 Q-PAS1 pair, was clearly detectable in the BphP1 Q-PAS1 mixture (lane #3). The formed homodimers of Q-PAS1 (lane #1) and BphP1 (lane #5) and Q- PAS1 BphP1 heterodimer (lane #3) are indicated with arrows. Uncut gel images are shown in Supplementary Figure 4. 8

9 Supplementary Figure 4. Uncut gel images a b Coomassie Coomassie c Q-PAS1 Q-linker PpsR2 - + dhth 740 nm d Q-PAS1 - + Q-linkerr - + PpsR2 - + dhth H 740 nm e Zinc staining Q-PAS1 + - Q-PAS1+ BphP1 BphP Crosslinker f Coomassie Q-PAS1 + - Q-PAS1+ BphP1 Bph hp Coomassie Zinc staining Original gel images for Figures 1b (a, b), 1c (c, d) and Supplementary Figure 3 (e, f). 9

10 Supplementary Figure 5. Scheme of the BphP1 PpsR2 transcription activation based on the TetR-tetO system. cytoplasm nucleus No SEAP reporter expression darkness TATA nucleus 740 nm SEAP reporter expression TATA BphP1 (Pfr) BphP1 (Pr) mcherry TetR NLS signal PpsR2 VP16 Under 740 nm light, the NLS-PpsR2-VP16 fusion interacts with the BphP1-TetR protein, resulting in a relocalization of the whole protein complex to a nucleus. In the nucleus, VP16 activates transcription of a SEAP reporter after the recruitment of TetR to the tetracycline response elements (teto) upstream of a SEAP reporter sequence. 10

11 Supplementary Figure 6. Comparison of the TetR-tetO based transcription activation achieved with the original BphP1 PpsR2 system and the smaller BphP1 Q-PAS1 system. Illumination scheme: 12 h 24 h dark 740 nm 20 SEAP signal (a.u.) dark 740 nm dark 740 nm BphP1 Q-PAS BphP1 PpsR2 The SEAP reporter expression in HeLa cells co-transfected with constructs encoding either a BphP1 Q-PAS1 system (left bars) or a BphP1 PpsR2 system (right bars) and with a ptre- Tight-SEAP reporter plasmid, using the approach described in Supplementary Figure 5. The cells were illuminated using the 30 s 740 nm light / 180 s darkness pulsed mode. The observed light-to-dark contrast was 9.5-fold for BphP1 Q-PAS1 and 8.7-fold for BphP1 PpsR2 systems. Error bars represent s.e.m., n = 3 experiments. 11

12 Supplementary Figure 7. Kinetics of the TetR-tetO transcription activation using the original BphP1 PpsR2 system and the smaller BphP1 Q-PAS1 system. Illumination scheme: Time = 0 36 h 12 h dark 740 nm SEAP signal (a.u.) Time after illumination (h) The SEAP reporter expression in HeLa cells co-transfected with constructs encoding either a BphP1 Q-PAS1 system (red bars; pqpas207 plasmid) or a BphP1 PpsR2 system (black bars; pka207 plasmid) and with a ptre-tight-seap reporter plasmid, using the approach described in Supplementary Figure 5. The cells were illuminated using the 30 s 740 nm light / 180 s darkness pulsed mode. Error bars represent s.e.m., n = 3 experiments. 12

13 Supplementary Figure 8. Expression levels of EGFP reporter in HeLa cells expressing GAL4(148)-Q-PAS1-VP16 in darkness and under 740 nm light Fluorescence (a.u.) dark 740 nm HeLa cells were cotransfected with constructs expressing the GAL4(148)-Q-PAS1-VP16 and a reporter plasmid pg5-egfp without BphP1-containing construct. Data were normalized to the reporter expression level detected in darkness. Error bars represent s.e.m., n = 3 experiments. No difference between the dark and lit states was observed. 13

14 Supplementary Figure 9. Probability of coiled-coil formation calculated for various GAL4(DBD)-Q-PAS1-VP16 fusion mutants. 100 Coiled-coil probability (a.u.) Amino acid residue number The probability of the coiled-coil formation was calculated using the MARCOIL algorithm. Value of each probability was calculated for a certain position of amino acid residue in the amino acid sequence of the α-helices joining region. The GAL4(DBD)-Q-PAS1-VP16 fusion constructs with a high probability of the coiled-coil retention resulted in the higher reporter expression (see Supplementary Fig. 10). 14

15 Supplementary Figure 10. Expression levels of EGFP reporter in HeLa cells co-expressing different GAL4-Q-PAS1-VP16 mutants and NLS-BphP1-mCherry in darkness and under 740 nm light. HeLa cells were co-transfected with constructs expressing the GAL4-Q-PAS1-VP16 mutants having different positions of a fusion between the GAL4 and Q-linker helices (see Fig. 3b), an NLS-BphP1-mCherry and a pg5-egfp reporter in the 1:1:3 ratio and analysed using flow cytometry. The cells were either illuminated using the 10 s 740 nm light / 60 s darkness pulsed mode or kept in darkness. Error bars represent s.e.m., n = 3 experiments. 15

16 Supplementary Figure 11. Light control of chromatin epigenetic state. a darkness 740 nm Control of epigenetic state nucleus Luciferase expression TATA nucleus Histone deacetylation No luciferase expression b Bioluminescence TATA 10 BphP1 (Pfr) BphP1 (Pr) GAL4(148) Q-PAS1 mcherry VP16 SID 0 dark 740 nm (a) Schematic representation of light-controlled alteration of gene epigenetic state via histone deacetylation caused by SID domain. (b) Light-induced change in luciferase bioluminescence of HeLa cells expressing GAL4(148)-Q-PAS1-VP16, BphP1 4 SID constructs and pfr-luc reporter according to (a). Data were normalized to the bioluminescence signal of the 740 nm illuminated cells. Error bars represent s.e.m., n=3 experiments. 16

17 Supplementary Figure 12. Expression levels of Luc reporter in HeLa cells co-expressing GAL4(148)-Q-PAS1-VP16 and BphP1-SID, treated by trichostatin A Bioluminescence (a.u.) dark 740 nm HeLa cells were co-transfected with constructs expressing the GAL4(148)-Q-PAS1-VP16, BphP1-SID and pfr-luc, and treated by HDAC inhibitor trichostatin A during 12 h prior to measurement. Data were normalized to the reporter expression level detected in darkness. Error bars represent s.e.m., n = 3 experiments. 17

18 Supplementary Figure 13. Expression levels of EGFP reporter in HeLa cells co-expressing GAL4(148)-Q-PAS1-VP16 and BphP1-mCherry in darkness and under 740 nm light Fluorescence (a.u.) dark 740 nm HeLa cells were cotransfected with constructs expressing the GAL4(148)-Q-PAS1-VP16, BphP1-mCherry and a pg5-egfp (using the same experimental conditions as for Supplementary Fig. 11b). Data were normalized to the EGFP expression level detected in darkness. Error bars represent s.e.m., n=3 experiments. Data show inability of BphP1 itself to inhibit the transcription launched by GAL4(148)-Q-PAS1-VP16. 18

19 Supplementary Figure 14. Light-induced tri-directional subcellular targeting. a darkness 460 nm, 10 min b darkness 740 nm, 10 min (a) Relocalization of iris from a cytoplasm to the nucleus in HeLa cells was performed under 460 nm of 1 mw cm 2 illumination for 10 min. (b) Relocalization of the iris from a cytoplasm to the plasma membrane in HeLa cells was performed under 740 nm of 1 mw cm 2 illumination for 10 min. Bar, 10 µm. 19

20 Supplementary Figure 15. Cytoplasmic fluorescence in cells expressing iris. Cytoplasmic fluorescence (a.u.) nm darkness 740 nm darkness Time (min) mcherry fluorescence levels of iris successively measured in the cytoplasm of HeLa cells in darkness, after 10 min of 460 nm of 1 mw cm 2 illumination, after 60 min in darkness, after 10 min of 740 nm of 1 mw cm 2 illumination, and after 60 min in darkness again. Error bars represent s.e.m., n=10 cells for each bar. All images were acquired at 37 C using an epifluorescence microscope. 20

21 Supplementary Figure 16. Relocalization of iris from the cytoplasm to the plasma membrane and back to the cytoplasm. darkness 740 nm, 10 min darkness, 60 min HeLa cells were cotransfected with bicistronic vectors encoding iris and BphP1-mVenus- CAAX. iris exhibits translocation from the cytoplasm to the plasma membrane under 740 nm illumination and subsequent relocalization back to the cytoplasm in darkness. The plasma membrane is labeled with mvenus. Bar, 10 µm. NES-mCherry-Q-PAS1- AsLOV2cNLS (iris) BphP1-mVenus-CAAX Overlay 21

22 Supplementary Figure 17. Kinetics of iris dissociation from plasma membrane to the cytoplasm in darkness and under 620 nm light. 1.2 Cytoplasmic fluorescence (a.u.) t 1/2 (620 nm) t Time, min 1/2 (dark) mcherry fluorescence of iris measured in the cytoplasm of HeLa cells pre-illuminated with 740 nm light and subsequently kept in darkness (black line) or under 620 nm illumination (red line). The 1 min pulses of 620 nm light (1 mw cm 2 ) were alternated with 1 min of darkness. Epifluorescence images of cells were acquired every 4 min. The observed dissociation half-time was ~4.4 min in darkness and ~2.3 min under 620 nm illumination. Fitting by exponential curve was performed using OriginPro 8.6. Error bars represent s.e.m., n = 10 cells for each time point. 22

23 Supplementary Figure 18. Crystal structure of the RsPpsRdHTH fragment of Rhodobacter sphaeroides. a PAS1 Q-linker N-PAS PAS2 b Q-PAS1 RsPpsR from Rhodobacter sphaeroides is a typical representor of the PpsR-family of proteins and a close homolog of PpsR2. (a) RsPpsR2 is a multidomain protein consisting of the N-PAS, Q-linker, PAS1, PAS2 and HTH (not shown) domain. (b) RsPpsR, and likely PpsR2, form tetramers in solution and octamers when bound to DNA. The N-PAS and PAS2 domains responsible for the tetramer formation, as well as the HTH domain, were deleted from PpsR2, resulting in Q-PAS1. The used in (a) and (b) RsPpsR structure lacks the HTH domain (PDB ID: 4HH2). 23

24 Supplementary Video 1. Sequential light-induced relocalization of the iris tool from a cytoplasm to the nucleus and to the plasma membrane in a single cell after the illumination periods (either 5 min with 460 nm of 1 mw cm 2 or 5 min with 740 nm of 1 mw cm 2 light) followed by the dark relaxation periods. See Figure 5 for more details. 24