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An efficient auxin-inducible degron system with low basal degradation in human cells

Nature Methods volume 16, pages 866–869 (2019)Cite this article

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An Author Correction to this article was published on 21 October 2019

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Abstract

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.

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Fig. 1: Identification of AtAFB2-miniIAA7 as an improved AID system.
Fig. 2: Depletion of endogenous transmembrane, cytoplasmic and nuclear proteins using the AtAFB2-miniIAA7 system.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

Custom software scripts (to be used with CellProfiler and MATLAB in Supplementary Fig. 5e,g) can be found at https://bitbucket.org/szkabel/lipidanalyser/get/master.zip.

Change history

  • 21 October 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank A. Uro for technical assistance and L. Kaipiainen for cholesterol measurements. HiLIFE Flow cytometry and Light microscopy platforms are acknowledged for access to research infrastructure. This study was supported by the Academy of Finland (Center of Excellence project grant no. 307415 to E.I. and I.V.; grant nos. 282192, 312491 to E.I.), the Sigrid Juselius Foundation (I.V. and E.I.), Finnish Medical Foundation (V.T.S.), Paulo Foundation (V.T.S.), Alfred Kordelin Foundation (V.T.S.), Maud Kuistila Foundation (V.T.S.), Biomedicum Helsinki Foundation (V.T.S.) and the Emil Aaltonen Foundation (V.T.S.).

Author information

Author notes

  1. These authors contributed equally: Xavier Prasanna, Veijo T. Salo.

Authors and Affiliations

  1. Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Shiqian Li, Veijo T. Salo & Elina Ikonen

  2. Minerva Foundation Institute for Medical Research, Helsinki, Finland

    Shiqian Li, Veijo T. Salo & Elina Ikonen

  3. Department of Physics, University of Helsinki, Helsinki, Finland

    Xavier Prasanna & Ilpo Vattulainen

  4. Computational Physics Laboratory, Tampere University, Tampere, Finland

    Ilpo Vattulainen

Authors
  1. Shiqian Li
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  2. Xavier Prasanna
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  3. Veijo T. Salo
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  5. Elina Ikonen
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Contributions

E.I. and S.L. conceptualized the project. S.L. designed and performed AID screen and test experiments. S.L. and V.T.S. designed and performed functional studies. X.P. and I.V. provided theoretical and mechanistic insights. S.L. and E.I. wrote the manuscript with input from all authors. E.I. supervised the study.

Corresponding author

Correspondence to Elina Ikonen.

Ethics declarations

Competing interests

A patent application covering the use of AtAFB2-miniIAA7 for rapid protein depletion (application no. FI 20195239) has been filed in which the University of Helsinki is the applicant, and E.I. and S.L. are the inventors.

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Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Depletion of endogenous seipin in the absence of IAA using the OsTIR1-miniAID system.

(a) Scheme showing establishment of A431 cell line with OsTIR1-miniAID system for depletion of endogenous seipin. (b) Western blot analysis of endogenous seipin tagged with miniAID-mEGFP. The tagged protein was depleted in OsTIR1 expressing cells before IAA addition. The immunoblot is representative of two independent experiments; the uncropped scan of the immunoblot is shown in Supplementary Fig. 10. (c) Widefield images of lipid droplets (LDs) stained with LD540, showing numerous tiny and few giant LDs characteristic of seipin knockout phenotype in OsTIR1 expressing cells before IAA addition. Images are representative of >100 cells in 8 fields of view. This experiment was repeated once independently with similar results.

Supplementary Figure 2 Screen of different auxin receptor F-box proteins using miniAID degron in human cells.

(a) Scheme for screening different auxin receptor F-box proteins using miniAID-mEGFP tagged seipin as the target. (b) Relative mCherry intensities analyzed by FACS in cells expressing different mCherry-tagged auxin receptor F-box proteins. Bars represent mean; n=2 independent experiments. At: Arabidopsis thaliana; Gh: Gossypium hirsutum; Mn: Morus notabilis; Nc: Noccaea caerulescens; Os: Oryza sativa. (c) Live cell Airyscan images showing subcellular localization of mCherry-tagged auxin receptor F-box proteins. This experiment was repeated once independently with similar results. (d) FACS plots showing overlays of GFP histograms at 0 h (blue line), 1 h (green line) and 16 h (red line) IAA in cells expressing the nontagged (left) or mCherry-tagged (right) constructs indicated. Figures exemplifying the gating strategy with SSC, FSC for cells, and trigger pulse width for singlets are shown above. Ctrl: no auxin receptor F-box protein, WT: wildtype A431 cells without GFP, blue dotted line and red dotted line: drawings for comparison of GFP peaks. This experiment was repeated once independently with similar results. (e) Mean GFP intensities in (d). Bars represent mean ± s.d.; n=4 biologically independent samples, except for ctrl (n=2). Individual data points of different auxin receptor F-box proteins are combinations of nontagged and mCherry-tagged constructs.

Supplementary Figure 3 Screen of different degrons in combination with AtAFB2 or OsTIR1 in human cells.

(a) Scheme for screening different degrons using mEGFP tagged seipin and AtAFB2 or OsTIR1. (b) FACS plots showing overlays of GFP histograms at 0 h (blue line), 1 h (green line) and 16 h (red line) IAA in cells expressing the indicated constructs. Ctrl: seipin-mEGFP without degron, *: KR dipeptide in domain II, blue dotted line and red dotted line: drawings for comparison of GFP peaks. MiniIAA7 and KR-miniIAA7 showed the best reduction of GFP peak intensity at 1 h IAA. KR dipeptide as part of a potential nuclear localization signal is unfavorable in the tag. This experiment was repeated once independently with similar results. (c) Mean GFP intensities in (b). Bars represent mean ± s.d.; for OsTIR1 combinations, n=2 biologically independent samples, except for IAA3(37–86) (n=3), IAA17(33-104) (n=4), N.D.=not determined; for AtAFB2 combinations, n=2 except for IAA17(31-104) (n=3), IAA17(33-104) (n=4), IAA17(31-132) (n=3), IAA7(37-104) (n=4), IAA14(32-98) (n=4), IAA3(37-86) (n=4), and IAA3(39-86) (n=4).

Supplementary Figure 4 Supplements to depletion of endogenous targets with the AtAFB2-miniIAA7 system.

(a) Western blot analysis of endogenous EGFR tagged with miniIAA7-mEGFP in A549 cells using anti-EGFR and anti-GFP antibodies. Experiments were repeated once independently with similar results; the uncropped scans of immunoblots are shown in Supplementary Fig. 10. (b) Scheme showing establishment of A431 cell lines using miniIAA7-mEGFP (1.) or mEGFP-miniIAA7 (2.) to tag endogenous SEC61B N-terminally. (c) Relative GFP intensities analyzed by FACS in ctrl cells at 0 h IAA. Bars represent mean ± s.d.; n=3 biologically independent samples. (d) Live cell Airyscan images showing similar endoplasmic reticulum localization of tagged proteins. Representative images from 2 independent experiments. (e) Mean GFP intensities analyzed by FACS in cells with indicated endogenously miniIAA7-tagged Sec61beta and auxin receptor F-box proteins or ctrl. Bars represent mean ± s.d.; n=3 biologically independent samples. (f) Time-lapse widefield images of A431-LBR cells with different AtAFB2-mCherry constructs. Representative images from 2 independent experiments. (g) Mean GFP intensity analyzed by FACS in cells with endogenously miniIAA7-tagged targets treated with or without IAA for 0 to 48 h. Bars represent mean ± s.d.; n=3 independent experiments.

Supplementary Figure 5 Loss-of-function studies using the AtAFB2-miniIAA7 system.

(a) Live cell Airyscan images of homozygously tagged cell lines. N or C in parentheses indicate N- or C-terminal tagging. DHC1 and EGFR refer to Fig. 1. Representative images from 2 independent experiments. (b) Auxin-inducible reduction in glucose uptake in cells with tagged Glut1. Bars represent mean ± s.d.; paired two-tailed t-test; n=3 biologically independent samples. (c) Phalloidin staining showing auxin-inducible changes in F-actin structures in cells with tagged NMIIA. Maximum intensity projections of deconvolved widefield images are shown. (d) Widefield images of filipin stained cells showing auxin-inducible perinuclear cholesterol accumulation in cells with tagged NPC1. (e) LD540 staining of lipid droplets and DAPI staining of nuclei showing auxin-inducible changes in LDs in cells with tagged seipin. Right: quantification of fraction of tiny LDs (<0.05 um2)/ cell. Bars represent mean ± s.e.m.; unpaired two-tailed t-test; n=238, 266, 217, 201 cells for ctrl no IAA, ctrl IAA, AtAFB2 no IAA, AtAFB2 IAA. (f) Auxin-inducible reduction in cellular cholesterol content in cells with tagged LBR. Bars represent mean ± s.d.; paired two-tailed t-test; n=4 biologically independent samples. (g) Auxin-inducible reduction in peroxisomal membrane proteins in cells with tagged PEX3. Left: western blot analysis of endogenous PMP70 levels, right: widefield maximum intensity projections and quantification of overexpressed PMP22-mCardinal fluorescence. Bars represent mean ± s.e.m.; unpaired two-tailed t-test; n=107, 184 cells for no IAA, IAA. Experiments in c-e, and g were repeated once independently with similar results; uncropped scans of immunoblots in g are shown in Supplementary Fig. 10.

Supplementary Figure 6 Testing of different miniIAA7 tags.

(a) Scheme for testing different miniIAA7 tags. *: results estimated from panel b. (b) Western blot analysis of overexpressed seipin tagged with different miniIAA7 tags. Note that seipin is an oligomeric transmembrane protein partly resolved as higher MW bands on SDS-PAGE. All immunoblots are representative of two independent experiments; uncropped scans of immunoblots are shown in Supplementary Fig. 10.

Supplementary Figure 7 Testing of the dTAG system for BRD4 degradation.

(a) Principle of protein depletion with the dTAG system. CRBN is an endogenous component in this system. (b) Scheme for tagging endogenous BRD4 in A431 and HEK293A cells using CRISPR/Cas9-mediated Precise Integration into Target Chromosome (PITCh). (c-d) Western blot analysis of dTAG-13 induced depletion of endogenously tagged BRD4 (c) and dTAG-13 washout (d) in the indicated cell pools. All immunoblots are representative of two independent experiments; uncropped scans of immunoblots are shown in Supplementary Fig. 10.

Supplementary Figure 8 Comparison of the dTAG system and the improved AID system for depleting endogenous LMNA.

(a) Scheme for tagging endogenous LMNA in A431 and HEK293A cells using CRISPR/Cas9-mediated HDR. A coselection strategy was employed for one-step generation of cells with both miniIAA7-mEGFP tagged LMNA and AtAFB2-mCherry-weak NLS. (b) Western blot analysis of endogenously tagged LMNA depleted with the dTAG or the improved AID system in the indicated cell pools. All immunoblots are representative of two independent experiments; uncropped scans of immunoblots are shown in Supplementary Fig. 10.

Supplementary Figure 9 Reversibility in the improved AID system compared to the OsTIR1-miniIAA7 system or the dTAG system.

(a-b) Left: FACS plots showing GFP histograms before and after IAA washout with the indicated miniIAA7-mEGFP tagged proteins in A549 or A431 cells. Right: mean GFP intensities analyzed by FACS. Bars represent mean ± s.d; n=3 independent experiments. (c) Western blot analysis of endogenously tagged LMNA before and after washout with the dTAG system or the improved AID system in HEK293A cell pools. All immunoblots are representative of two independent experiments; uncropped scans of immunoblots are shown in Supplementary Fig. 10. (d) Schematic illustration of dTAG-13 bifunctional binding and IAA coreceptor binding.

Supplementary Figure 10 Uncropped scans of immunoblots.

Uncropped scans of immunoblots are shown with the cropped area in frame and the corresponding figures indicated.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Supplementary Note 1.

Reporting Summary

Supplementary Table 1

Sequence information: proteins, cDNAs, sgRNAs and primers sequences.

Supplementary Video 1

Time-lapse imaging of cell division in DHC1 cells. Representative videos recording cell division events in DHC1 control or degron A431 cells, treated with or without IAA during 16 h. IAA was added to the cells at 0 h; n = 12 fields each in the experiment. The experiment was repeated once independently with similar results.

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Li, S., Prasanna, X., Salo, V.T. et al. An efficient auxin-inducible degron system with low basal degradation in human cells. Nat Methods 16, 866–869 (2019). https://doi.org/10.1038/s41592-019-0512-x

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