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LIN28 phosphorylation by MAPK/ERK couples signalling to the post-transcriptional control of pluripotency

Abstract

Signalling and post-transcriptional gene control are both critical for the regulation of pluripotency1,2, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein, has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA biogenesis and direct modulation of mRNA translation3. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells, which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced the effect of LIN28 on its direct mRNA targets, revealing a mechanism that uncouples LIN28’s let-7-dependent and -independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naive to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signalling, post-transcriptional gene control, and cell fate regulation.

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Figure 1: MAPK/ERK phosphorylates LIN28A on Ser200.
Figure 2: LIN28A phosphorylation increases its protein stability.
Figure 3: LIN28A phosphorylation can uncouple its let-7-dependent and -independent activities.
Figure 4: LIN28A phosphorylation contributes to the regulation of pluripotency transitions.

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Acknowledgements

We thank P. Sharp, L. Cantley, G. Ruvkun, and members of the Daley laboratory for invaluable discussions, A. D. L. Angeles for critical feedback on the manuscript, X. Wu/Yi Zhang’s laboratory and R. Rubio/DFCI CCCB for assistance with RNA-seq, and R. Tomaino at the Taplin Biological Mass Spectrometry Core for assistance with mass spectrometry. Bioanalyser analysis was performed in the BCH IDDRC Molecular Genetics Core, which is supported by NIH (NIH-P30-HD 18655). Sequencing analysis was conducted on the Orchestra High Performance Computing Cluster at Harvard Medical School. K.M.T. was an HHMI International Student Research Fellow and a Herchel Smith Graduate Fellow. D.S.P. was supported by a grant from NIGMS (T32GM007753). R.I.G. was supported by a grant from NIGMS (R01GM086386). G.Q.D. is an investigator of the Howard Hughes Medical Institute and the Manton Center for Orphan Disease Research, and was supported by a grant from NIGMS (R01GM107536).

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Authors

Contributions

K.M.T. designed and performed the experiments, and wrote the manuscript. D.S.P. helped with RNA-seq, HeLa clonal series generation and expression analysis. Z.W. performed reprogramming experiments. A.H. performed RNA-seq bioinformatics analysis. R.T. and R.I.G. shared unpublished results and generated the isogenic HeLa cells. M.T.S. performed expression analysis. J.T.P. and J.K.O. helped with experimental design. S.K. generated the human pLIN28A antibody. S.P.G. supervised the proteomics experiments. G.Q.D. designed and supervised experiments, and wrote the manuscript.

Corresponding author

Correspondence to George Q. Daley.

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Competing interests

G.Q.D. and R.I.G. hold options and intellectual property related to 28/7 Therapeutics, a company seeking to develop inhibitors of the LIN28/let-7 pathway. S.K. is an employee of Cell Signaling Technology.

Integrated supplementary information

Supplementary Figure 1 Phosphoproteomic analysis of LIN28A.

(a) Schematic of the phosphoproteomic strategy. (b) Coomassie-stained SDS-PAGE gel showing FLAG-LIN28A purified from mESCs, without or with Calyculin A (100 nM) treatment. (c) Digestion patterns of proteases used for generation of LIN28A peptides. The amino acid sequence of mouse LIN28A is shown. Trypsin and chymotrypsin sites are highlighted in blue and red, respectively. (d) LIN28A phosphosites and kinases predicted to phosphorylate them, as determined by ScanSite 3 (http://scansite3.mit.edu). Site numbers refer to human LIN28A. (e) Western blot analysis of LIN28A (S200) phosphorylation in a panel of human PSCs. A representative image of two independent experiments is shown. Unprocessed scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 2 LIN28A and let-7 levels after MEK/ERK inhibitor treatment.

(a) Western blot (left) and qRT-PCR (right) analysis of transgenic FLAG-LIN28A in HeLa-LIN28A cells after 48-hour treatment with DMSO or PD0325901 (1 μM). n = 3 independent experiments. Error bars represent s.e.m. n.s. = non-significant; P = 0.19 (two-tailed Student’s t-test). (b) qRT-PCR analysis of pri/pre- (left) and mature (right) let-7s in PA1 cells treated with DMSO or PD0325901 (1 μM) for 48 h. n = 3 independent experiments. Error bars represent s.e.m. P > 0.05 (two-tailed Student’s t-test, PD versus DMSO). Statistics source data are shown in Supplementary Table 5. Unprocessed scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 3 Effects of mild LIN28A depletion on let-7 and LIN28’s direct mRNA targets.

qRT-PCR analysis of mature let-7s (a) and LIN28A’s mRNA targets (b), and Western blot analysis of LIN28A’s mRNA targets (c) in PA1 cells after mild LIN28A knockdown. siNC = negative control siRNA. n = 3 independent experiments. Error bars represent s.e.m. P > 0.05 (two-tailed Student’s t-test, siLIN28A versus siNC). Statistics source data are shown in Supplementary Table 4. Unprocessed scans of blots are shown in Supplementary Fig. 5.

Supplementary Figure 4 Effects of LIN28A level and S200 phosphorylation on let-7 and LIN28A’s direct mRNA targets.

(a) Corresponding levels of mature let-7s (top) and transgenic FLAG-LIN28A protein (middle and bottom) in a clonal series of HeLa-LIN28A cells. PA1 cells are included as a reference for native levels of LIN28A in hPSCs. Data are representative of two independent experiments. (b) Western blot analysis of transgenic FLAG-LIN28A in HeLa Flp-In cells stably expressing Dox-inducible wild-type (WT) or phospho-null (S200A) FLAG-LIN28A, without or with treatment with Dox. Dox = doxycycline (100 ng ml−1). A representative image of three independent experiments is shown. (c) qRT-PCR analysis of mature let-7 species in HeLa Flp-In cells stably expressing Dox-inducible wild-type (WT) or phospho-null (S200A) LIN28A, without or with treatment with Dox. Dox = doxycycline (100 ng ml−1).n = 3 independent experiments. Error bars represent s.e.m. P > 0.05 (two-tailed Student’s t-test). (d) Quantification of immunoprecipitated RNA (>200 nt) from HeLa Flp-In cells stably expressing wild-type (WT) or phospho-null (S200A) LIN28A. Western blot validation of the immunoprecipitation is shown on the bottom. n = 3 independent experiments. Error bars represent s.e.m. P < 0.05 (two-tailed Student’s t-test). (e) RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-null (S200A) FLAG-LIN28A in HeLa Flp-In cells. Each dot represents an average enrichment value for transcripts from a given gene. n = 3 independent experiments. Data were normalized to the amount of immunoprecipitated LIN28A prior to sequencing. Detailed description of the analysis is provided in the Methods section and the complete data set is available in Supplementary Table 3. (f) Western blot analysis of RNA immunoprecipitation in HeLa Flp-In cells overexpressing wild-type (WT) or phospho-mimetic (S200E) FLAG-LIN28A. Cells were treated with doxycycline (100 ng ml−1) to induce FLAG-LIN28A 48 h prior to analysis. A representative image of three independent experiments is shown. (g) qRT-PCR analysis of representative mRNA targets immunoprecipitated by wild-type (WT) or phospho-mimetic (S200E) FLAG-LIN28A in HeLa Flp-In cells. n = 3 independent experiments. Data were normalized to cell number prior to RT. Error bars represent s.e.m. P < 0.05; P < 0.01 (two-tailed Student’s t-test, S200E versus WT). Statistics source data are shown in Supplementary Table 4. Unprocessed scans of blots are shown in Supplementary Fig. 5.

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Tsanov, K., Pearson, D., Wu, Z. et al. LIN28 phosphorylation by MAPK/ERK couples signalling to the post-transcriptional control of pluripotency. Nat Cell Biol 19, 60–67 (2017). https://doi.org/10.1038/ncb3453

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