SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) harbour the ability to undergo lineage-specific differentiation into clinically relevant cell types. Transcription factors and epigenetic modifiers are known to play important roles in the maintenance of pluripotency of hESCs. However, little is known about regulation of pluripotency through splicing. In this study, we identify the spliceosome-associated factor SON as a factor essential for the maintenance of hESCs. Depletion of SON in hESCs results in the loss of pluripotency and cell death. Using genome-wide RNA profiling, we identified transcripts that are regulated by SON. Importantly, we confirmed that SON regulates the proper splicing of transcripts encoding for pluripotency regulators such as OCT4, PRDM14, E4F1 and MED24. Furthermore, we show that SON is bound to these transcripts in vivo. In summary, we connect a splicing-regulatory network for accurate transcript production to the maintenance of pluripotency and self-renewal of hESCs.

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Figure 1: The effects of SON depletion in hESCs.
Figure 2: Rescue of SON depletion phenotype in hESCs.
Figure 3: RNA-seq analysis of transcripts regulated by SON.
Figure 4: SON regulates splicing of small introns with weak splice sites.
Figure 5: SON targets specific introns of transcripts that encode pluripotency regulators.
Figure 6: Unspliced transcripts induced by SON depletion are potentially targeted by the NMD pathway.
Figure 7: SON regulates alternative splicing in hESCs.
Figure 8: SON knockdown induces an isoform switch of PRDM14.

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Acknowledgements

We are grateful to the Agency for Science, Technology and Research (A*STAR) for their generous funding, and also for NIH R15 GM084407 to P.A.B. We acknowledge T. C. Peow, L-P. Yaw, T. Tng and G. K. Jee for their technical assistance. We acknowledge the Genome Technology Biology (GTB) group from the Genome Institute of Singapore (GIS) for their help and expertise in performing the RNA sequencing. We thank T-L. Huber for critical comments on the manuscript. J. Göke is supported by a fellowship within the Postdoc-Programme of the German Academic Exchange Service, DAAD.

Author information

X.L.: conception, design, collection of data, data analysis and manuscript writing; J.G.: design, collection of data, data analysis and manuscript writing; F.S., H.L. and B.F.: collection and data analysis; P-É.J. and G.B.: collection and data analysis; P.A.B: design, and provision of study material; H-H.N.: conception, design, supervision, data interpretation and manuscript writing.

Correspondence to Huck-Hui Ng.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression pattern of SON in different cell types.

(a) Expression profile of SON according to probe 201086 and probe 214988 (Chip U133P2) of the AmaZonia human whole genome expression resource Supplementary Ref. S56. (bSON expression changes during reprogramming, PRDM14 depletion and NFRKB depletion according to published microarray data5. OSKC: the 4 Yamanaka reprogramming factors: OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC(C). (cSON expression in MRC5 cells and hESCs according to real-time PCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (dSON expression during hESC differentiation induced by FGF removal. Mean±s.e.m. of biological triplicate data are shown in all graphs.

Supplementary Figure 2 Characterisation of the hESC differentiation phenotype after SON depletion.

(a) Western blot of SON protein in total cell extract from H1 hESCs. (b) Western blot of SON, pluripotency markers (OCT4 and NANOG) and differentiation markers (GATA6 and MSX1) after transfecting cells with constructs expressing control shRNA or SON shRNAs. β-ACTIN and GAPDH were used as loading control. (c) Immunostaining of pluripotency cell surface marker (SSEA4) and differentiation markers (GATA6 and MSX1) in hESCs. DAPI positively stains the nucleus. Scale bar, 20 μm. (d) Flow cytometry analysis of percentage of cells positive for pluripotency makers (OCT4, TRA-1-81 and TRA-1-60) and differentiation markers (GATA6 and MSX1) after SON knockdown. Isotype control is from secondary antibody staining alone. (e) Expression of housekeeping genes after SON depletion in hESCs according to real-time PCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (f) Number of TRA-1-60 positive colonies after reprogramming in the presence of SON shRNA. Mean±s.e.m. of biological triplicate data are shown in all graphs. OSKC: the 4 Yamanaka reprogramming factors: OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC(C). (g) Number of alkaline phosphatase colonies after SON depletion and rescue SON expression in hESCs. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 3 Influences of SON depletion on hESC survival and cell cycle progression.

(a) TUNEL assay for cell apoptosis after treating hESCs with control shRNA or SON shRNAs. Scale bar, 20 μm. (b) Expression of SON in MRC5 cells after SON knockdown for 4 days according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (c) Expression of SON at protein level after SON depletion in MRC5 cells according to Western blot. Mean±s.e.m. of biological triplicate data are shown in all graphs. (d) TUNEL assay after SON depletion in MRC5 cells. Scale bar, 20 μm. (e) Mitotic spindle staining after SON depletion. α-tubulin was immunostained for imaging. Representative examples of cells in mitosis are shown. After SON knockdown, cells with monopolar spindle pole or incomplete separation of bipolar spindle poles are shown. Scale bar, 2 μm. (f) Percentage of cells in different mitotic stages. α-Tubulin/Aurora A immunostaining was done in hESCs with or without SON knockdown. The number of mitotic cells in different phases and spindle pole status were counted. Monopolar and multipolar spindles are abnormal spindle structure. n = 100 cells. (g) Percentage of cells in mitosis calculated based on DAPI staining. Mean±s.e.m. of biological triplicate data are shown in all graphs. n>400 cells per experiment. (h) Morphology of hESCs after depleting SON and TUBA1C expression. Scale bar, 20 μm. shRNA1 was used for SON depletion. (i) TUNEL assay for cell apoptosis after treating cells with control shRNA or CYCLIN A2shRNAs. (j) Expression of pluripotency markers after TUBA1C depletion according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (k) CYCLIN A2depletion (right panel) according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs.

Supplementary Figure 4 Relationship between SON-knockdown induced differentiation and cell death.

(a) Time-course morphology of hESCs after depletion of SON. Scale bar, 20 μm. (b) Time-course expression of pluripotency markers after SON knockdown according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (c) Time-course expression of differentiation markers after SON knockdown according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (d) Time-course analysis of cell cycle profile after SON depletion. Mean±s.e.m. from biological triplicate data are shown. (e) Cell cycle profile of hESCs after depletion of SON to various levels. Mean±s.e.m. from biological triplicate data are shown. (f) Flow cytometry analysis of percentage of cells positive for pluripotency makers (OCT4, TRA-1-81 and TRA-1-60) and differentiation markers (GATA6 and MSX1) after SON depletion in the presence of ROCK inhibitor. Isotype control is from secondary antibody staining alone. (g) Western blot of pluripotency marker OCT4 and differentiation marker GATA6 after SON depletion in the presence of ROCK inhibitor. (h) Flow cytometry analysis of TUNEL assay staining of cells treated with SON shRNA (shRNA1), TUBA1C shRNA or control shRNA and SON shRNA in the presence of ROCK inhibitor. (i) Flow cytometry analysis of pluripotency marker (SOX2) expression in γ-H2AX +/- populations. Isotype control is from secondary antibody staining alone. -, negatively stained population. +, positively stained population. (j) Flow cytometry analysis of pluripotency marker (TRA-1-60) expression by immunostaining in hESC population at different cell cycle stages after SON depletion. Isotype control is from secondary antibody staining alone. Cell cycle stages were determined by PI staining.

Supplementary Figure 5 Analysis and validation of RNA-seq results.

(a) Density plot showing the RNA-Seq data at the SON and ACTIN loci before and after SON depletion. RPKM values were calculated by normalising the number of reads in these loci by the length of these loci and the number of reads mapped to exons. (b) Gene ontology analysis of genes with decreased expression after SON depletion. (c) Histogram of the t-test statistic for intron inclusion events. The black line indicates the cutoff used in this study (-8) to select a stringent set of intron inclusion events. (d) Illustration to PCR primer locations in RT-PCR to detect inclusion of introns (left) or isoform switch (right) after SON depletion. (e) Analysis of intron inclusion events or isoform switches after SON depletion. Percentage of unspliced transcripts was measured according to band intensity. Control introns that are not regulated by SON are shown at the bottom of the figure. (f) Boxplot showing the normalised intron expression for SON-targeted introns as described in the main text. Intron expression was estimated by normalising the number of reads that mapped to the intron by intron length, exon read count and exon length. Significance was estimated using the Wilcoxon test.

Supplementary Figure 6 Characterisation of SON-targeted introns.

(a) Intron length for SON-targeted introns (estimated using intron expression) and unaffected introns from the same set of genes to account for gene to gene variation. (b) Intron length for SON-targeted introns (estimated using the ratio of spliced over unspliced reads) and non-targeted introns of SON. SONtargeted introns were identified by 3’ junction reads of the last exon only. (c) Splice strength for the 5’ splice site, estimated using MaxEntScan. (d) Splice strength for the 3’ splice site, estimated using MaxEntScan. (e) Nucleotide content for all introns in the genome (black), SON-targeted introns (red), and non-targeted introns from genes which had at least one affected intron (green). Left: 5’ end, right: 3’ end. (f) Nucleotide Frequencies for the 5’ and 3’ splice sites of introns unaffected introns from the same genes that had at least one intron targeted by SON to account for gene to gene variation (See Supplementary Fig. S6h). (g) Nucleotide Frequencies for the 5’ and 3’ splice sites of introns affected by SON knockdown, only genes with at least one affected and one unaffected intron were investigated to account for gene to gene variation (see Supplementary Fig. S6g). (h) Examples of noncandidate genes with weak splicing sites. Top panel: structure of genes Solid box: exon. Line: intron. Bottom panel: real-time PCR analysis of intron expression after SON depletion. Mean±s.e.m. from biological triplicate data are shown. (i) Morphology of hESCs after depletion of another weak splicing regulator TIA1. Scale bar, 20 μm. (j) Expression of pluripotency markers (left panel) and differentiation markers (right panel) after TIA1 depletion according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (k) Expression of SON regulated introns after depletion of TIA1 by qPCR. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 7 SON regulates the splicing of specific introns of pluripotency genes.

(a) Venn diagram showing the overlap of hESC specific genes (orange) with genes regulated by SON. The p-value was calculated using Fisher’s test. (b) Intron length of SON-targeted hESC specific introns. Significance was estimated using the Wilcoxon test. (c) Intron length of SON-targeted hESC-state-maintaining genes. Significance was estimated using the Wilcoxon test. (d) Intron length of SON-targeted cell survival genes. Significance was estimated using the Wilcoxon test. (e) Time-course expression fold change of candidate introns after SON depletion. The fold changes of intron expression levels were normalised to gene expression fold change (measured by primers designed for constitutive exonic sequence) fold change (set as 1). TUBA1B intron 2 as negative control. Mean±s.e.m. from biological triplicate data are shown. (f) Expression of introns and exons in the OCT4 gene after SON depletion. Solid box: exon. Line: introns. Arrows: locations of primers. Mean±s.e.m. from biological triplicate data are shown. (g) Gene structure of NANOG and position of primers and qPCR analysis of expression change of NANOG introns after SON depletion for 3 days. Solid box: exon.Line: introns. Arrows: locations of primer pairs. SJ: splice junction. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 8 Analysis of SON regulated alternative splicing.

(a) Venn diagram showing the overlap of pluripotency genes (green) with genes that show alternative splicing after depletion of SON. The p-value was calculated using Fisher’s test. (b) Venn diagram showing the overlap of self-renewal genes (red) with genes that show alternative splicing after depletion of SON. Genes essential for hESC survival were defined as genes with average cell survival Nscore<−2 (Supplementary Table S2 from genome-wide RNAi screening in hESCs5). The P-value was calculated using Fisher’s test.

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Lu, X., Göke, J., Sachs, F. et al. SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells. Nat Cell Biol 15, 1141–1152 (2013) doi:10.1038/ncb2839

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