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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007).
Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007).
Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70 (2007).
Mikkelsen, T. S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008).
Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008).
Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008).
Li, R. et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51–63 (2010).
Samavarchi-Tehrani, P. et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010).
Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).
Di Micco, R. et al. Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes. Cell Rep. 9, 234–247 (2014).
Liu, L. et al. Transcriptional pause release is a rate-limiting step for somatic cell reprogramming. Cell Stem Cell 15, 574–588 (2014).
Buganim, Y., Faddah, D. A. & Jaenisch, R. Mechanisms and models of somatic cell reprogramming. Nat. Rev. Genet. 14, 427–439 (2013).
Wang, Y. et al. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478–1483 (2008).
Krizhanovsky, V. & Lowe, S. W. Stem cells: the promises and perils of p53. Nature 460, 1085–1086 (2009).
Chen, J. et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat. Genet. 45, 34–42 (2013).
Liang, G., He, J. & Zhang, Y. Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming. Nat. Cell Biol. 14, 457–466 (2012).
Onder, T. T. et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598–602 (2012).
Maherali, N. & Hochedlinger, K. Tgfβ signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr. Biol. 19, 1718–1723 (2009).
Korpal, M., Lee, E. S., Hu, G. & Kang, Y. The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J. Biol. Chem. 283, 14910–14914 (2008).
Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49–52 (2009).
Di Stefano, B. et al. C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells. Nature 506, 235–239 (2014).
Rais, Y. et al. Deterministic direct reprogramming of somatic cells to pluripotency. Nature 502, 65–70 (2013).
Bar-Nur, O. et al. Small molecules facilitate rapid and synchronous iPSC generation. Nat. Methods 11, 1170–1176 (2014).
Vidal, S. E., Amlani, B., Chen, T., Tsirigos, A. & Stadtfeld, M. Combinatorial modulation of signaling pathways reveals cell-type-specific requirements for highly efficient and synchronous iPSC reprogramming. Stem Cell Rep. 3, 574–584 (2014).
Eminli, S. et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nat. Genet. 41, 968–976 (2009).
Guo, S. et al. Nonstochastic reprogramming from a privileged somatic cell state. Cell 156, 649–662 (2014).
Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).
Shi, Y. et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941–953 (2004).
Whyte, W. A. et al. Enhancer decommissioning by LSD1 during embryonic stem cell differentiation. Nature 482, 221–225 (2012).
Wang, G. L. et al. HDAC1 cooperates with C/EBPα in the inhibition of liver proliferation in old mice. J. Biol. Chem. 283, 26169–26178 (2008).
Grebien, F. et al. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia. Nat. Chem. Biol. 11, 571–578 (2015).
Adam, R. C. et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521, 366–370 (2015).
Northrup, D. L. & Allman, D. Transcriptional regulation of early B cell development. Immunol. Res. 42, 106–117 (2008).
Laurent, B. et al. A specific LSD1/KDM1A isoform regulates neuronal differentiation through H3K9 demethylation. Mol. Cell 57, 957–970 (2015).
Loven, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).
Roe, J. S., Mercan, F., Rivera, K., Pappin, D. J. & Vakoc, C. R. BET bromodomain inhibition suppresses the function of hematopoietic transcription factors in acute myeloid leukemia. Mol. Cell 58, 1028–1039 (2015).
Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
van Oevelen, C. et al. C/EBPa activates pre-existing and de novo macrophage enhancers during induced pre-B cell transdifferentiation and myelopoiesis. Stem Cell Rep. 5, 232–247 (2015).
Zhang, D. E. et al. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein α-deficient mice. Proc. Natl Acad. Sci. USA 94, 569–574 (1997).
Hasemann, M. S. et al. C/EBPα is required for long-term self-renewal and lineage priming of hematopoietic stem cells and for the maintenance of epigenetic configurations in multipotent progenitors. PLoS Genet. 10, e1004079 (2014).
Maza, I. et al. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nat. Biotechnol. 33, 769–774 (2015).
Chen, J. et al. Vitamin C modulates TET1 function during somatic cell reprogramming. Nat. Genet. 45, 1504–1509 (2013).
Canettieri, G. et al. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation. Nat. Cell Biol. 12, 132–142 (2010).
Wu, Y. et al. The deubiquitinase USP28 stabilizes LSD1 and confers stem-cell-like traits to breast cancer cells. Cell Rep. 5, 224–236 (2013).
Pijnappel, W. W. et al. A central role for TFIID in the pluripotent transcription circuitry. Nature 495, 516–519 (2013).
Di Tullio, A. et al. CCAAT/enhancer binding protein α (C/EBP(α))-induced transdifferentiation of pre-B cells into macrophages involves no overt retrodifferentiation. Proc. Natl Acad. Sci. USA 108, 17016–17021 (2011).
Soufi, A. et al. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell 161, 555–568 (2015).
Ye, M. et al. Hematopoietic differentiation is required for initiation of acute myeloid leukemia. Cell Stem Cell 17, 611–623 (2015).
Ohlsson, E. et al. Initiation of MLL-rearranged AML is dependent on C/EBPα. J. Exp. Med. 211, 5–13 (2014).
Krivtsov, A. V. et al. Cell of origin determines clinically relevant subtypes of MLL-rearranged AML. Leukemia 27, 852–860 (2013).
Harris, W. J. et al. The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells. Cancer Cell 21, 473–487 (2012).
Bueno, C. et al. Reprogramming human B-cells into induced pluripotent stem cells and its enhancement by C/EBPα. Leukemia http://dx.doi.org/10.1038/leu.2015.294 (2015).
Carey, B. W., Markoulaki, S., Beard, C., Hanna, J. & Jaenisch, R. Single-gene transgenic mouse strains for reprogramming adult somatic cells. Nat. Methods 7, 56–59 (2010).
Boiani, M., Eckardt, S., Scholer, H. R. & McLaughlin, K. J. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev. 16, 1209–1219 (2002).
Di Stefano, B. & Graf, T. Rapid generation of induced pluripotent stem cells from mouse pre-B cells. Nat. Protoc. Exch. http://dx.doi.org/10.1038/protex.2016.001 (2016).
Bussmann, L. H. et al. A robust and highly efficient immune cell reprogramming system. Cell Stem Cell 5, 554–566 (2009).
Zuber, J. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011).
Figueroa, M. E. et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18, 553–567 (2010).
Di Stefano, B. & Graf, T. Very rapid and efficient generation of induced pluripotent stem cells from mouse pre-B cells. Methods Mol. Biol. 1357, 45–56 (2014).
Stadhouders, R. et al. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions. Nat. Protoc. 8, 509–524 (2013).
Jakobsen, J. S. et al. Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq. BMC Genomics 16, 46 (2015).
Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014).
Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).
Ji, X. et al. Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions. Proc. Natl Acad. Sci. USA 112, 3841–3846 (2015).
Chen, X., Xu, H., Yuan, P. & Fang, F. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).
Lara-Astiaso, D. et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014).
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).
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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).
(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.
(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.
(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.
About this article
Cite this article
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
EMBO reports (2021)
The corepressor NCOR1 and OCT4 facilitate early reprogramming by suppressing fibroblast gene expression
[1,2,3]Triazolo[4,5-d]pyrimidine derivatives incorporating (thio)urea moiety as a novel scaffold for LSD1 inhibitors
European Journal of Medicinal Chemistry (2020)
The FEBS Journal (2020)
Perspectives on somatic reprogramming: spotlighting epigenetic regulation and cellular heterogeneity
Current Opinion in Genetics & Development (2020)