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Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells

Nature Neuroscience volume 19, pages 807–815 (2016)Cite this article

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Abstract

Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB–REST–miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons.

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Figure 1: Quantitative neuronal induction by shPTB in combination with small molecule inhibitors on HAFs.
Figure 2: Generation of functional neurons from HAFs by sequential knockdown of PTB and nPTB.
Figure 3: Neuronal maturation induced by BRN2 in combination with PTB knockdown.
Figure 4: BRN2 is essential to neuronal maturation in hNPCs and activation of mature-neuron-specific miRNAs.
Figure 5: Specific miRNAs repress nPTB during neuronal differentiation.

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Acknowledgements

We thank Z.-N. Zhang from A.R. Muotri's laboratory (University of California, San Diego) for sharing hNPCs and culture protocols and K. Zhang (University of California, San Diego) for sharing various antibodies and GFP transgenic rats. We are grateful to S. Dowdy for help on FACS analysis, to J. Ravits (University of California, San Diego) for human adult fibroblasts, and to Y. Yu from G. Hannon's laboratory (Cold Spring Harbor Laboratory) for the pTRIPZ lentiviral plasmid. This work was supported by a grant from the National Natural Science Foundation of China (91440101) to Y.X., by key programs of Chinese Academy of Sciences to X.-D.F. and Y.X. (KJZD-EW-L12) and by NIH grants (GM049369 and HG004659) to X.-D.F.

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  1. Yuanchao Xue and Hao Qian: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,

    Yuanchao Xue & Xihao Hu

  2. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA.,

    Yuanchao Xue, Hao Qian, Jing Hu, Bing Zhou, Yu Zhou & Xiang-Dong Fu

  3. Child Health Institutes of New Jersey, Rutgers-Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA

    Aziz Karakhanyan & Zhiping Pang

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Contributions

Y.X. and X.-D.F. conceived the project. Y.X. designed and performed most biochemical experiments. H.Q. was responsible for all electrophysiology analysis and hNPC differentiation with input from A.K. and Z.P. B.Z. performed bioinformatics analysis and Y.Z. and X.H. contributed to RNA-seq analysis. J.H. performed EdU labeling, ChIP-qPCR, and FACS analysis. Y.X. and X.-D.F. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 PTB knockdown in human adult fibroblasts dampens cell proliferation and cell fate switch to the neuronal lineage.

(a) Characterization of HAFs by immunostaining with various cell type markers. HAFs show homogeneous staining of the fibroblast markers Fibronectin, FSP1 and Vimentin, but not markers for keratinocytes (K5, P63), melanocytes (MITF, P75), neural crest derivatives (SOX10, BRN3A) and neurons (indicated in the lower panel). n=2 biological repeats.

(b) FAC analysis of EdU-pulse labeled HAFs with or without PTB knockdown. Nuclei were stained by DAPI and EdU positive cells were stained with Alexa fluor 647. n=3 biological repeats.

(c) Example of Tuj1 (Green) positive cells that lack EdU (Red) staining. n=3 biological repeats.

(d) Example of induced neuronal-like cells by PTB knockdown only or in combination with specific small molecules. Red: Tuj1 expression, Blue: DAPI-stained nuclei. Scale bar: 20 µm. n=2 biological repeats.

(e) Phase contrast pictures of control and PTB shRNA-treated HAFs with small molecules treatment at different time points. The 3 small molecules were either present through the assay periods or washed away from the first treatment for 3 hrs. n=2 biological repeats.

(f) Immunostaining of the neural crest markers Nestin and Sox2 during PTB knockdown induced neuronal conversion. Scale bar: 100 µm. n=3 biological repeats.

Supplementary Figure 2 Validation of nPTB knockdown efficiency by RT-qPCR and Western blotting.

(a) Immunostaining of NeuN and NF (neurofilament) expression after different days of PTB knockdown. Scale bar: 50 µm. n=3 independent experiments.

(b) Relative expression of PTB and nPTB examined by RNA-seq analysis during neuronal conversion from HAFs. n=2 biological repeats.

(c, d) The mRNA and protein levels of nPTB assessed on shPTB-treated HAFs by RT-qPCR after the addition of doxycycline for 4 days to induce shnPTB expression. Beta-Actin (ACTB) served as loading control. Data are presented as mean± SD. Two-tailed unpaired Student’s t test was applied to calculate significance. n=3 independent experiments. NS: υ=4, t=0.628, P=0.564; 1#: υ=4, t=16.752, P=0.0000744; 2#: υ=4, t=5.872, P= 0.00420); 3#: υ=4, t=27.311, P= 0.0000107; 4#: υ=4, t=39.992, P=0.00000234. **P<0.01, ***P<0.001. Uncropped versions of Western blots are shown in Supplementary Figure 7.

Supplementary Figure 3 ChIP-qPCR analysis of REST targets and characterization of calcium influx in response to membrane depolarization in induced neurons with shPTB plus various neuronal lineage-specific transcription factors.

(a) ChIP-qPCR analysis of REST binding at the promoters of indicated neuronal transcription factors and miRNA loci in HAFs. Two-tailed unpaired Student’s t-test was applied to calculate significance. Data are presented as mean± SD. n=3 independent experiments. Ascl1: υ=4, t=5.336, P=0.00594; Myt1l: υ=4, t=11.605, P=0.000315); NeuroD1: υ=4, t=34.602, P=0.000004; Olig2: υ=4, t=12.332, P=0.000248; Brn2: υ=4, t=3.737, P=0.020; miR-124: υ=4, t= 5.812, P=0.00436. *P<0.05, **P<0.01, ***P<0.001.

(b) Screening for transcription factors capable of promoting MAP2 expression (Green) when combined with PTB knockdown in HAFs.

(c) Calcium influx in neuronal cells induced by co-expression of shPTB and BRN2 in HAFs. Scale bar: 30 µm.

Supplementary Figure 4 BRN2 regulation of a diverse repertoire of targets to control neuronal maturation.

(a) Characterization of hNPCs by immunostaining Nestin (Green), BRN2 (Red) and SOX2 (Red).

(b) Immunofluorescence analysis of neurons derived from hNPCs after BRN2 knockdown.

(c) The impact of BRN2 knockdown on the expression of MAP2 (Green) and SOX2 (Red) in neurons derived from hNPCs. n= 3 independent experiments for (a), (b) and (c). Scale bar: 20 µm.

(d) Immunostaining of NeuN after knockdown of BRN2 in PTB/nPTB sequentially depleted HAFs. n=3 random 20x fields.

(e) Impaired Na+ (Red) and K+ (Blue) currents in BRN2 knockdown neurons from hNPCs.

(f) Western blotting analysis of MAP2, Tuj1 and BRN2 in differentiated hNPCs. GAPDH served as loading control. n= 2 biological repeats. Uncropped versions of Western blots are shown in Supplementary Figure 7.

(g) Test of 4 commercially available anti-BRN2 antibodies by immunoprecipitation. C-20 and B-2 were chosen for ChIP-seq library construction. GAPDH served as input control. Uncropped versions of Western blots are shown in Supplementary Figure 7.

(h, i) GO term analysis of BRN2 target genes. Both GO terms of biological processes (g) and biological components (h) indicate BRN2 is critical for neuronal maturation. Background was based on all expressed genes in hNPCs.

Supplementary Figure 5 Regulation of nPTB by BRN2.

(a) Western blot analysis of PTB and nPTB expression in hNPC derived neurons upon depletion of BRN2. Beta-Actin and GAPDH served as loading controls. n=3 independent experiments. Two-tailed unpaired Student’s t test was applied to calculate significance. Data are presented as mean± SD. υ=4, t=13.208, P=0.000190. ***P<0.001.

(b) Western blot analysis of nPTB expression upon the ectopic expression of BRN2. Beta-Actin served as loading control. n=3 independent experiments. Uncropped versions of Western blots are shown in Supplementary Figure 7.

Supplementary Figure 6 Two consecutive RNA loops to control neuronal conversion and maturation in HAFs.

The previously deduced PTB-miR-124-REST loop for neuronal conversion28 and the newly elucidated nPTB-miR-9-BRN2 loop for neuronal maturation. Each component of the loops likely regulates a set of downstream targets to generate specific neuronal phenotypes.

Supplementary Figure 7 Uncropped versions of western blots.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 2148 kb)

Supplementary Methods Checklist

(PDF 453 kb)

Supplementary Table 2

BRN2 ChIP-seq targets in the human genome (XLSX 257 kb)

Supplementary Table 3

PCR primers used in this study (XLSX 14 kb)

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Xue, Y., Qian, H., Hu, J. et al. Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells. Nat Neurosci 19, 807–815 (2016). https://doi.org/10.1038/nn.4297

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