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Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions

Abstract

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin–microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin–transcription factor interaction. We retooled this coiled-coil-based GLI–KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

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Fig. 1: The DNA-binding domain of GLI2 directly binds the coiled-coil dimerization domain of KIF7.
Fig. 2: The coiled-coil dimerization domain of KIF7 is a DNA structural mimic for GLI binding.
Fig. 3: A second GLI interaction site on the KIF7 motor domain.
Fig. 4: GLI2 ZF increases the microtubule-binding affinity of KIF7, thereby increasing its own recruitment on microtubules.
Fig. 5: Synergistic regulation of GLI–KIF7 promotes KIF7 localization to cytosolic microtubules and the distal cilium tip.
Fig. 6: Re-engineering the DNA-mimicking coiled-coil domain of KIF7 as a tool for the sequestration of GLI away from the nucleus and cilia.

Data availability

The authors declare that all relevant data supporting the findings of this study are available within the paper and its supplementary information files. Structural data that support the findings of this study have been deposited in the publicly available repository PDB under accession number 7RX0. The previously published protein primary sequences that were re-analysed here are available at UniProtKB with the following accession codes: human GLI2 (P10070); the coiled-coil domain of human KIF7 (Q2M1P5); the coiled-coil domain of human KIF27 (Q86VH2); and the N-terminal motor domain of human KIF27 (Q86VH2). Previously published protein structures that were re-analysed here are available at the Research Collaboratory for Structural Bioinformatics Protein Data Bank with the following accessions codes: GLI1 zinc finger (PDB: 2GLI); ROCK1 coiled-coil (PDB: 3O0Z); and KIF7 motor domain (PDB: 6MLQ). The EM-derived structure of AMPPNP–KIF7 bound to microtubules is available at the PDB (6MLR) and the EMD (9141). Source data are provided with this paper.

Code availability

All custom codes used for analyses of the data are freely available on GitHub (https://github.com/Peii39/Gli-Subramanian-Lab).

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Acknowledgements

We thank R. E. Kingston (Massachusetts General Hospital), K. V. Anderson (Sloan Kettering Institute), R. Lipinski (University of Wisconsin), A. Salic (Harvard Medical School) and S. Angers (University of Toronto) for providing us with the HeLa and MEF (WT, Gli2–/–, Gli2–/–Gli3–/–, Kif7–/–) cell lines. We also thank M. He (University of Hong Kong) for valuable discussions. R.S. was supported through the Pew Biomedical Scholars program, the American Cancer Society (Ellison Foundation Research Scholar) and the Smith Family Award for Excellence in Biomedical Research. R.A.M. was supported through NIH R37GM052468.

Author information

Authors and Affiliations

Authors

Contributions

R.S. and F.H. designed the project. R.S., E.M.W.-K. and R.A.M. designed the cryo-EM experiments. F.H. performed the pull-down assays, homology modelling, in vitro reconstitution, TIRF microscopy, co-sedimentation and cell biological experiments. C.F. purified the recombinant proteins and performed the stoichiometry and BLI assays. Q.Y. performed initial pull-down assays. E.M.W.-K. performed cryo-EM experiments and obtained 3D reconstructions. N.M. did model building from the EM maps. P.-I.K. performed the cytoplasmic sequestration image analysis. F.H., C.F. and R.S. wrote the manuscript. All authors discussed the results and reviewed the manuscript.

Corresponding author

Correspondence to Radhika Subramanian.

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Competing interests

Massachusetts General Hospital has filed a provisional patent application (application number 63/089,770) covering the use of peptides inhibitors of glioma-associated oncogene derived from the coiled-coil dimerization domain of KIF7 that lists R.S., F.H., C.F. and Q.Y. as inventors. The remaining authors declare no competing interests.

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Nature Cell Biology thanks Jeremy Reiter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Purification, stoichiometry analysis and binding of recombinant Kif7-CC and Gli2-ZF proteins.

a, Pull-down of c-Myc-Gli2 (418-594aa) and FLAG-Kif7 (1-361aa) after co-transfection in Expi293F cells. Input (cell-lysate), FT (flow-through), wash and anti-FLAG immunoprecipitated(IP) beads samples were immunoblotted(IB) with anti-c-myc antibody to detect Gli2. Immunoblot with anti-FLAG antibody was used to detect the expression of Kif7. The blot is representative of three independent repeats. b, Chromatograms from size exclusion chromatography of GST-Kif7-CC-GFP, GST-Kif7-CC, GST-Kif7-SCC, Gli2-ZF-GFP and Gli2-ZF on Superdex 200 10/300 GL. Arrows indicate elution volumes of standards: (left to right) 1-ferritin(440kDa), 2-aldolase(158kDa), 3-ovalbumin(44kDa) and 4-carbonic anhydrase(29kDa). c, SDS-PAGE of purified GST-Kif7-CC-GFP, GST-Kif7-CC, GST-Kif7-SCC, Gli2-ZF-GFP and Gli2-ZF. MW – molecular weight markers. The gels are representative images of a minimum of three repeats. d, Single molecule fluorescence intensity histograms of Gli2-ZF-GFP (Intensity = 3.0 ×104 ± 1.4 ×104, N = 746) and GST-tagged Kif7-CC-GFP (Intensity = 4.2 ×104 ± 1.9 ×104, N = 536). Intensities are reported as mean ± standard deviation. e, Single molecule photobleaching traces for Gli2-ZF-GFP (1strow) and GST-Kif7-CC-GFP (2ndrow). Background subtracted integrated fluorescence intensity versus time plots used for step photobleaching analysis. Photobleaching steps are indicated by arrows. f, BLI assay to quantitatively examine the binding affinity of Kif7-CC-GFP to Gli2-ZF (black) & Gli2-ZF-GFP (green). Data represent mean and standard deviation from three independent repeats. The plots of binding response versus Gli2-ZF concentration were fit to a Hill equation to determine equilibrium dissociation constants (Kd). For Kif7-CC-GFP + Gli2-ZF: Kd = 65 ± 22  nM, for Kif7-CC-GFP  +  Gli2-ZF-GFP: Kd = 57 ± 9  nM. [Note: GFP-tagged versions of proteins show a consistent increase in Kd that is within error range]. g, Western blot of peak complex fraction from size exclusion chromatography of Kif7-CC-GFP and Gli2-ZF-GFP (see Fig.1d). Stoichiometry of components in complex is indicated in parenthesis. The blot is representative of three independent repeats.

Source data

Extended Data Fig. 2 Structural models of Kif7-CC-Gli2-ZF protein complex and DNA competition.

High confidence structural models of the Kif7-CC:Gli2-ZF protein complex from docking analysis (ClusPro 2.0). Gli2-ZF model is represented in blue and green colored ribbon diagrams and Kif7-CC models represented in salmon colored ribbon diagrams in the different models. a-b, Structural models that seem most probable based on our experiments. c, Overall electrostatic surface representation of the complex in a represented on a scale from -5 (red) to 0 (white) to +5 kT/e (blue) d-g, Structural models that are less probable from mutagenesis experiments (specifically Kif7-CC S2-mut, S3-mut1 and S3-mut2, see Extended Data Fig.3e,f,i). h-i, Structural models that appear less probable based on DNA competition experiments (See Fig. 2d). j, Competitive inhibition of Kif7-CC and Gli2-ZF (125 nM) binding response upon titration with Gli2 target DNA; TRE-2S (grey) and non-target DNA; TFIIIA-BS (red). Raw binding response from the BLI measurements is plotted against DNA concentration. Data represent mean and standard deviation from four independent repeats.

Source data

Extended Data Fig. 3 Site-directed mutagenesis to validate the structural model of the Kif7-CC:Gli2-ZF complex.

a, Summary of Gli2-ZF truncations binding to Kif7-CC (left). Data is representative of raw-trace from the association step in BLI assay (right) from a minimum of three repeats. b, SDS-PAGE of purified Gli2-ZF truncation proteins. Gels are representative images from three repeats. c, Chromatograms from size-exclusion chromatography of Gli2-ZF truncation proteins on Superdex 75 10/300GL. d, BLI binding response of Kif7-CC with Gli2 truncation constructs: ZF1-3 and ZF1-4. Data represent mean + /– SD from three independent repeats. The binding response plots were fit to a Hill equation to determine equilibrium dissociation constants(Kd). For Gli2-ZF1-4: Kd = 103 ± 15 nM and Gli2-ZF1-4: Kd = 160 ± 40  nM. e, Pull-down experiment with c-Myc-Gli2(418-594aa) and FLAG-Kif7-CC(460-600aa) mutant proteins in Expi293F cells. Input (cell lysate), FT (flow through), wash and anti-FLAG immunoprecipitated(IP) beads samples were immunoblotted(IB) with anti-c-Myc antibody to detect Gli2. The blots are representative images of a minimum of three repeats. Gli2-ZF mutants: ZF2-mut(H493A/R496A), ZF3-mut(H503A/R516A/K521A), ZF4-mut(R550A/K552A/R556A). Kif7-CC mutants: S1-mut(E500A/E501A/E502A/D505A), S2-mut(E511A/E515A), S3-mut1(E526A/E529A) and S3-mut2(E530A/R535A). f, Structural model of Kif7-SCC:Gli2-ZF protein-complex. In dark blue are Gli2-ZF residues when mutated to alanine abolish Kif7-CC binding; in red are Kif7-CC residues when mutated to alanine abolish Gli2-ZF binding; in yellow are residues when mutated to alanine did not abolish binding; in green are sections(S1,S2,S3) represent 3 patches of Kif7-CC residues predicted as potential Gli-binding sites by PDBsum analysis. g, SDS-PAGE of purified Kif7-CC-S1 point mutant proteins. Gels are representative from three repeats. h, Chromatograms from size-exclusion chromatography of Kif7-CC S1 point mutant proteins on Superdex 200 10/300GL. i, Raw traces from the association step in the BLI assay performed with Kif7-CC S1 point mutant proteins: D505A, E501A/E502A, E502A, E502A/D505A, with Gli2-ZF. Inset, Binding response in BLI assay for D505A Kif7-CC mutant to Gli2-ZF. Data represent mean + /– SD from three independent repeats.

Source data

Extended Data Fig. 4 Purification and stoichiometry analysis of recombinant Kif7-DM Kif7-MM and Gli2-ZF-SNAP proteins.

a, Chromatograms from size-exclusion chromatography of Kif7-DM(black), Kif7-DM-GFP(green) and Gli2-ZF-SNAP(magenta, *represents peak used for experiments) on Superdex 200 10/300GL. Dotted line indicates elution volume of Gli2-ZF. Arrows indicate elution volumes of the following standards: (left to right) 1-ferritin(440kDa), 2-aldolase(158kDa) and 3-ovalbumin(44kDa). b, SDS-PAGE of purified Kif7-DM, Kif7-DM-GFP and Gli2-ZF-SNAP. Gels are representative images from three repeats. c, Fluorescence intensity histograms of Kif7-MM-GFP (Intensity = 3.0 ×104 ± 1.7 ×104, N = 648) and Kif7-DM-GFP (Intensity = 4.0 ×104 ± 2.0 ×104, N = 1015). Intensities are reported as mean + /– SD. d, Single molecule photobleaching traces for Kif7-MM-GFP(top) and Kif7-DM-GFP(bottom). Photobleaching steps are indicated by arrows. e, BLI assay to quantitatively examine binding affinity of Kif7-DM(black) and Kif7-DM-GFP(green) to Gli2-ZF, & Kif7-DM to Gli2-ZF-SNAP(maroon). Data represent mean + /– SD from three independent repeats. The plots of binding response versus Gli2-ZF concentration were fit to a Hill equation to determine equilibrium dissociation constants (Kd). For Kif7-DM + Gli2-ZF: Kd = 99 ± 25  nM, for Kif7-DM  +  Gli2-ZF-SNAP: Kd = 99 ± 23  nM, for Kif7-DM-GFP + Gli2-ZF: Kd = 138  ± 41  nM, for [Note: GFP-tagged versions of proteins show a consistent increase in Kd that is within error range]. f, Scatter plot of Gli2-ZF-1-3-Alexa647 intensity per pixel on microtubules(MT) represents recruitment of Gli on MT by Kif7-MM and Kif7-DM. The no kinesin condition was a control for non-specific binding of Gli2-ZF1-3 to MT, and Kif27-DM was included as a negative control for Gli binding. Assay conditions: 100 nM Gli2-ZF-1-3-SNAP-Alexa647 with 100 nM of kinesin in each case. Data are presented as mean + /– SEM; N = 32 microtubules analysed over 3 independent experiments for each condition. One-way ANOVA (p < 0.0001) and post-hoc analysis (*indicates p < 0.0001 in Dunnett’s multiple comparisons test) show statistically significant differences in Gli intensity compared to no kinesin control; ns is not significant (p = 0.9425).

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Extended Data Fig. 5 Cryo-EM maps of Kif7 and Gli2-ZF on microtubules.

a, Fourier shell correlation (FSC) curve for Kif7-DM bound to GMPCPP microtubules in the presence of AMPPNP and Gli2-ZF-SNAP. b, Cryo-EM reconstruction of dimeric Kif7 motor domain (green) in complex with AMPPNP bound on the GMPCPP-microtubule lattice (grey) in the presence of Gli2-ZF-SNAP (pink), shown in two orientations, and when viewed from the –end and +end of the microtubule. Density of Gli2-ZF-SNAP is seen only attached to the Kif7 motor domain and no extra density is seen along the microtubule lattice. c, EM reconstructions of AMPPNP-bound Kif7-DM with Gli2-ZF-SNAP (black mesh) and AMPPNP-bound Kif7-MM with Gli2-ZF-SNAP (orange) superposed via the Kif7 motor domain. Structural model for AMPPNP-bound Kif7 motor (green) is shown. d, EM reconstructions of AMPPNP-bound Kif7-DM with Gli2-ZF-SNAP (black mesh) and ADP-bound Kif7-DM with Gli2-ZF (yellow) superposed via the Kif7 motor domain. Structural model for AMPPNP-bound Kif7 motor (green) is shown. Blue dotted region indicates density seen only with Gli2-ZF-SNAP and not with Gli2-ZF (thereby likely corresponding to part of SNAP peptide). e, Cryo-EM reconstructions of AMPPNP-bound Kif7-DM with Gli2-ZF-SNAP. Structural models for AMPPNP-bound Kif7 motor (green) and Gli2-ZF consisting of 2 complete and one partial zinc finger (magenta) are shown. Helix α-2 (dark green) and loop L2 (blue) of Kif7 make contacts with the density for Gli2-ZF-SNAP. See also Supplementary Video 1.

Extended Data Fig. 6 Single molecule residence time analysis and microtubule co-sedimentation assay with Kif7-DM-GFP in the presence of Gli2-ZF.

a, Representative kymographs from assays to visualize single molecules of Kif7-DM-GFP (1 nM) on X-rhodamine labeled microtubules with increasing Gli2-ZF concentrations (0, 25 & 50 nM). b, Representative kymographs from assays to visualize single molecules of Kif7-DM-GFP (0.1 nM) on X-rhodamine labeled microtubules with increasing Gli2-ZF concentrations (0, 25 & 50 nM). c, Cumulative frequency distribution plots of Kif7-DM-GFP residence time on microtubules from analysis of 0.1 nM Kif7-DM-GFP data set with increasing Gli2-ZF concentrations. Numbers of observed events at different Gli2-ZF concentrations: 0 nM, 1191; 25 nM, 576; 50 nM, 817. Inset shows expanded view of the same data. Dotted lines represent bi-phasic association functions fitted to the 25 nM and 50 nM Gli data sets. [Parameters from fit: (i) For 25 nM Gli; R2 = 0.99; TauFast = 0.11 s; TauSlow = 1.64 s; (ii) For 50 nM Gli; R2 = 0.99; TauFast = 0.13 s; TauSlow = 2.05 s]. See Methods for details. d, SDS-PAGE analysis of 1 μM Kif7DM-GFP co-sedimentation on GDP-taxol stabilized microtubules (0 – 10 μM) in the presence of 1 mM ATP, 2 mM MgCl2, with or without 5 μM Gli2-ZF, and the analysis of 5 μM Gli2-ZF non-specific co-sedimention on microtubules in the absence of Kif7 (S: Supernatant, unbound fraction. P: Pellet, bound fraction. BSA: Bovine Serum Albumin, gel loading control). The gels are representative images of a minimum of three repeats.

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Extended Data Fig. 7 Purification and binding of recombinant dimeric Kif7 mutant and chimera proteins to Gli2-ZF in solution and on microtubules.

a, SDS-PAGE of purified Kif7-DME502A-GFP, Kif7M-Kif27CC-GFP and Kif27M-Kif7CC-GFP. MW – molecular weight markers. The gels are representative images of a minimum of three repeats. b, Chromatograms from size exclusion chromatography of Kif7-DME502A-GFP (purple), Kif7M-Kif27CC-GFP (red) and Kif27M-Kif7CC-GFP (blue) on Superdex 200 10/300 GL. Dotted line indicates elution volume of Kif7-DM-GFP on the same column. c, Representative images of microtubule (MT) and Kinesin-DM-GFP (Kinesin) in the absence (left) or presence of Gli2-ZF (right, 100 nM). Fluorescence intensities were scaled similarly for images. Scale bars represent 2 μm. d, Recruitment of Gli to microtubules (MT) by Kif7-DM-GFP (grey), Kif7-DME502A-GFP (purple), Kif7M-Kif27CC-GFP (red) and Kif27M-Kif7CC-GFP (blue). Scatter plot shows the ratio of Gli2-ZF-Alexa647 intensity to Kinesin GFP intensity on microtubules. Assay conditions: 100 nM Gli2-ZF-SNAP-Alexa647 with 100 nM of kinesin in each case. Data are presented as mean + /– SD; N = 43 microtubules analysed over 3 independent experiments for each condition. One-way ANOVA (p < 0.0001) and post-hoc analysis (* indicates p < 0.0001 in Dunnett’s multiple comparisons test) show statistically significant differences in Gli intensity compared to Kif7-DM-GFP control. e, BLI assay to quantitatively examine the binding affinity of Kif7-DME502A-GFP (purple), Kif7M-Kif27CC-GFP (red) and Kif27M-Kif7CC-GFP (blue) to Gli2-ZF. Data represent mean and standard deviation from three independent repeats. The plots of binding response versus Gli2-ZF concentration were fit to a Hill equation to determine equilibrium dissociation constants (Kd). For Kif7-DME502A-GFP: Kd = 414 ± 103  nM, for Kif7M-Kif27CC-GFP: Kd = 397 ± 88  nM and for Kif27M-Kif7CC-GFP: Kd = 217  ± 70  nM.

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Extended Data Fig. 8 Immunofluorescence of microtubules and ER-marker calreticulin in cellular localization experiments.

a, Localization of co-transfected mRuby-tagged full-length Kif7 and mNeonGreen-tagged full-length Gli2 on microtubules. b, Localization of co-transfected mRuby-tagged Kif7-CC (460-600aa) with ER retention tag, mNeonGreen-tagged full-length Gli2 and ER-marker calreticulin in COS7 cells. DAPI staining was used to mark the nucleus. Kif7-CC alone shows localization at the endoplasmic reticulum in the cytoplasm. Gli2 is sequestered in the cytoplasm and shows co-localization with Kif7-CC both on the endoplasmic reticulum and in cytoplasmic puncta as seen in the zoomed insets (1 & 2). Scale bars represent 10 µm. The images are representative of three independent repeats.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Supplementary Fig. 1, Supplementary Video 1 legend.

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Supplementary Video 1

Cryo-EM reconstructions of AMPPNP-bound KIF7-DM with GLI2-ZF–SNAP. Structural models for AMPPNP-bound KIF7 motor (green) and GLI2-ZF consisting of two complete and one partial zinc finger (magenta).

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Haque, F., Freniere, C., Ye, Q. et al. Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions. Nat Cell Biol (2022). https://doi.org/10.1038/s41556-022-00935-7

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