C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4

Abstract

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPα (Bα′ cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPα also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Bα′ cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPα.

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Figure 1: An improved ultrafast reprogramming system of B cells to pluripotency.
Figure 2: C/EBPα- and OSKM-induced proteome changes during reprogramming.
Figure 3: C/EBPα-induced Lsd1 and Hdac1 upregulation and requirement of Lsd1 for silencing of the B cell program.
Figure 4: C/EBPα-induced Brd4, Med1 and Cdk9 upregulation and requirement of Brd4 for activation of the pluripotency program.
Figure 5: Analysis of chromatin accessibility changes and transcription factor binding in C/EBPα-pulsed B cells.
Figure 6: Comparison between molecular properties of Bα′ cells and GMPs.
Figure 7: Summary diagrams.

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Acknowledgements

We thank J. E. Bradner (Dana Farber Cancer Institute, Harvard Medical School, USA) for the JQ1 compound, J. Zuber (Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Austria) for the Brd4 shRNA construct, R. Levine (Memorial Sloan Kettering Cancer Center, USA) for the Tet2 shRNA construct, L. de Andres for help with GMP isolations, L. Batlle for help with the chimaeric mice, the Genomics Facility and the Biomolecular Screening & Protein Technologies Facility of the CRG for technical assistance and members of the Graf laboratory for discussions. This work was supported by Ministerio de Educacion y Ciencia, SAF.2012-37167, Fundacio La Marató TV3 120410, AGAUR SGR 1136 and European Research Council Synergy Grant (4D-Genome). R.S. was supported by an EMBO Long-term Fellowship (ALTF 1201-2014) and a Marie Curie Individual Fellowship (H2020-MSCA-IF-2014). J.L.S. was supported by MINECO (IJCI-2014-21872). Work in the Porse laboratory was supported through a Centre grant from the NovoNordisk Foundation (Section for Stem Cell Biology in Human Disease and R179-A1513).

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Contributions

B.D.S. and T.G. conceived the study and wrote the manuscript. B.D.S. performed the cell culture, animal and molecular biology experiments and analysed the data. S.C., D.T. and M.F. performed the bioinformatics analysis, J.S.J. and B.P. the ChIP-seq experiments, J.L.S. and C.S.-M. the western blots and the protein immunoprecipitations, and A.L. the ATAC-seq experiments, M.W. and M.M. collected and analysed the proteomics data, R.S. performed and analysed the 4C-seq experiments; F.L. performed cell culture experiments.

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Correspondence to Bruno Di Stefano or Thomas Graf.

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Integrated supplementary information

Supplementary Figure 3 Characterization of Bα′ cell reprogramming into iPS cells.

(A) Representative chimeric mouse obtained after blastocyst injection of αiPS clone. (B) Heatmap of RNA-seq data showing genes changing >2fold during reprogramming (FDR < 1%, LRT test). (C) Gene Ontology (GO) analysis of protein clusters shown in panel A. The size of each circle represents the proportion of GO sets found in each cluster; the intensity of the color represents the P-value, determined by a hypergeometric test. (D) Gene expression (qRT-PCR) of selected pluripotency genes. Values were normalized against Pgk expression. Error bars indicate s.d. (n = 3 biologically independent samples). (E) Representative western blots for selected pluripotency transcription factors. See Suppl. Fig. 8 for uncut gel images.

Supplementary Figure 4 Protein dynamics during reprogramming.

(A) PANTHER classification for all the proteins identified by mass spectrometry in the samples tested. (B) Correlation between biological duplicates of RNA-seq and proteomic data. (C) C-means clustering of proteins changing >2 fold at any time points during reprogramming.

Supplementary Figure 5 Gene silencing induced by C/EBPα, protein interactions and B cell specific gene enhancer activities during reprogramming.

(A) Representative western blots of Brd4, Lsd1, Klf4 and Hdac1 in B and Bα′ cells. See Suppl. Fig. 8 for uncut gel images. (B) RNA-seq expression values for selected B cell specific genes. The data represent the average from two biologically independent samples. (C) Western blots of Cdk9 after induction of C/EBPα in B cells. See Suppl. Fig. 8 for uncut gel images. (D) Bα′ cell extracts were fractionated on Superose 6 10/300 GL column and Hdac1, Lsd1 and C/EBPα were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (E) Peptide counts, P-value and enrichment over IgG of C/EBPα, Hdac1 and Lsd1, for the IP-mass spectrometry shown in Fig. 3B. (F) C/EBPα co-immunoprecipitation experiment. Lsd1 or C/EBPα were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (G) Co-immunoprecipitation of C/EBPα, Lsd1 and Hdac1. Parp1 and Pcna (negative controls) were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (H) Screenshots of H3K27ac histone decoration and Brd4 binding by ChIP-seq at enhancers of selected B cell transcription factors. (I) Gene expression of selected B cell genes as measured by qRT-PCR in B cells (data from Fig. 3F), B cells treated for 18 h with E2 (Bα′ cells) and B cells treated for 18 h with both E2 and the Hdac1 inhibitor VPA. Error bars indicate s.d. (n = 3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P < 0.05, P < 0.01).

Supplementary Figure 6 Effect of Lsd1 and Brd4 inhibitions on iPS reprogramming.

(A) Representative flow cytometry analysis of B cells treated with JQ1 or S2101 for 24 hours using Pacific Blue Annexin V/SYTOX AADvanced Apoptosis Kit. (B) Representative BrdU (6 h pulse) FACS staining of B cells treated with JQ1 or S2101 or DMSO as a control. (C) shRNA sorting strategy. (D) Gene expression by qRT-PCR of Lsd1 and Brd4 after specific knockdown in B cells. Error bars indicate s.d. (n = 3 biologically independent samples). (E) Representative alkaline phosphatase positive iPS colonies obtained from reprogramming of B cells after Lsd1 and Brd4 knockdown. (F) Oct4-GFP and alkaline phosphatase positive iPS colonies obtained from reprogramming of B cells (OSKM alone without C/EBPα pulse) treated with S2101 or DMSO as a control. Error bars indicate s.d. (n = 3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (n.s. P > 0.05). (G) Genome browser screenshots of Rarg and Egln3 loci showing C/EBPα, Brd4 and H3K27ac ChIP-seq data. (H) Representative alkaline phosphatase positive iPS colonies obtained from reprogramming of Bα′ cells induced with OSKM and treated with JQ1 during C/EBPα (E2) or OSKM (Doxy) induction.

Supplementary Figure 7 ATAC-seq cluster analysis.

(A) Gene ontology enrichment for genes associated with ATAC-seq peaks in each cluster shown in Figure 5A (nearest gene relative to the peak). P-values were determined by a hypergeometric test. (B) Genome browser screenshots of Id1 and Ifitm6 loci showing C/EBPα and H3K27ac ChIP-seq, as well as ATAC-seq data. (C) Selected over-represented DNA motifs shown in Figure 5B discovered (de novo) in ATAC-seq peaks, and similar motifs found in the JASPAR or HOCOMOCO database. (D) Genome browser screenshot of the Klf4 locus showing C/EBPα and PU.1 ChIP-seq data, and 4C data using the newly discovered −90 kb enhancer as view point (black triangle at the bottom). The second highlighted region (right) correspond to the second −280 kb enhancer, as shown in Figure 5E. (E) Comparison of our ATAC-seq data (Fig. 5A), with Brd4 (GSE36561) and Klf4 (ref. 56) ChIP-seq data in ES cells. (F) Venn diagram showing the overlap between C/EBPα ChIP-seq peaks in Bα′ cells and Klf4 ChIP-seq peaks in ES cells. (G) Average plots of C/EBPα ChIP-seq (top) and MNAse-seq signal (bottom) in the C10 pre-B cell line at different timepoints after induction of C/EBPα, for each ATAC-seq cluster (Fig. 5A). Profiles were normalized to B cells and centered on the median.

Supplementary Figure 8 C/EBPα induced changes in chromatin accessibility at myeloid and ES cell loci.

(A) Genome browser screenshots of the Rarg and Lefty2 loci showing ChIP-seq data for Oct4, Nanog, Klf4 and Brd4 in ESCs (ref. 56, 57 and GSE36561). (B) Gene expression profile by RNA-seq for Rarg and Lefty1 during iPS reprogramming. The data represent the average from two biologically independent samples. (C) Comparison of GMPs and Bα′ cells for the number of upregulated and downregulated genes (>2fold) between B and Bα′ cells as well as between B cells and GMP, indicating the number of genes that overlap. (D) Canonical component analysis (CCA) of RNA-seq from B cells and Bα′ cells, together with RNA-seq from different hematopoietic cell populations (ref. 58). (E) Heatmaps of ATAC-seq data from clusters I to IV of B cells, GMPs, Bα′ cells, ESCs. (F) Average peak intensities of ATAC-seq data from clusters I to IV of GMPs, Bα′ cells, ESCs and MEFs (ref. 44). (G) Genome browser screenshots of selected genomic loci displaying ATAC-seq data. (H) Average plot of C/EBPα ChIP-seq signal in GMPs for each ATAC-seq cluster.

Supplementary Figure 9 Comparison of fast and slow cycling GMPs.

(A) FACS plots showing sorting strategy to obtain GMPs and their separation into fast and slow cycling fractions after CSFE treatment. (B) Klf4 expression as determined by qRT-PCR in fast and slow cycling GMPs. Error bars indicate s.d. (n = 3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P < 0.01). (C) Array expression values for selected genes in fast and slow cycling GMPs. The data represent the average from two biologically independent samples. (D) Tet2 knockdown efficiency tested by qRT-PCR. Error bars indicate s.d. (n = 3 biologically independent samples). (E) Representative Oct4-GFP FACS analysis of OSKM-induced MEFs overexpressing TFIID and treated with JQ1 or S2101.

Supplementary Figure 10 Uncut gels.

Supplementary Table 1 List of proteins annotated with the GO term “protein degradation”.
Supplementary Table 2 List of primers used.

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Di Stefano, B., Collombet, S., Jakobsen, J. et al. C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4. Nat Cell Biol 18, 371–381 (2016). https://doi.org/10.1038/ncb3326

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