G9a and Jhdm2a regulate embryonic stem cell fusion-induced reprogramming of adult neural stem cells - PubMed (original) (raw)

G9a and Jhdm2a regulate embryonic stem cell fusion-induced reprogramming of adult neural stem cells

Dengke K Ma et al. Stem Cells. 2008 Aug.

Abstract

Somatic nuclei can be reprogrammed to pluripotency through fusion with embryonic stem cells (ESCs). The underlying mechanism is largely unknown, primarily because of a lack of effective approaches to monitor and quantitatively analyze transient, early reprogramming events. The transcription factor Oct4 is expressed specifically in pluripotent stem cells, and its reactivation from somatic cell genome constitutes a hallmark for effective reprogramming. Here we developed a double fluorescent reporter system using engineered ESCs and adult neural stem cells/progenitors (NSCs) to simultaneously and independently monitor cell fusion and reprogramming-induced reactivation of transgenic Oct4-enhanced green fluorescent protein (EGFP) expression. We demonstrate that knockdown of a histone methyltransferase, G9a, or overexpression of a histone demethylase, Jhdm2a, promotes ESC fusion-induced Oct4-EGFP reactivation from adult NSCs. In addition, coexpression of Nanog and Jhdm2a further enhances the ESC-induced Oct4-EGFP reactivation. Interestingly, knockdown of G9a alone in adult NSCs leads to demethylation of the Oct4 promoter and partial reactivation of the endogenous Oct4 expression from adult NSCs. Our results suggest that ESC-induced reprogramming of somatic cells occurs with coordinated actions between erasure of somatic epigenome and transcriptional resetting to restore pluripotency. These mechanistic findings may guide more efficient reprogramming for future therapeutic applications of stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

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Figures

Figure 1

Figure 1

Cre-loxP-based, enhanced green fluorescent protein-inducible assay for reprogramming (CLEAR). (A): A diagrammatic illustration of CLEAR analysis. CIPOE NSC lines were established by infection of adult NSCs derived from transgenic mice harboring Oct4-enhanced green fluorescent protein reporter with retroviruses to coexpress the Cre recombinase and puromycin resistance gene. Z-Red ESCs carry an inducible DsRed expression cassette upon Cre-mediated excision. PEG-induced fusion of Z-Red ESCs and CIPOE NSCs leads to GFP expression as an indicator of Oct4 reactivation and DsRed expression as a reporter for fusion events. The dual-color reporter system can be monitored by both live fluorescence microscopy and quantitative flow cytometry to probe reprogramming processes. (B, C): Live images of fused ES-like colonies. Shown are sample images of fused ES-like colonies at 48 hours (B) and 96 hours (C) after PEG-induced fusion between CIPOE NSCs and Z-Red ESCs. Arrows point to DsRed+GFP− cells that were successfully fused but incompletely reprogrammed. Scale bar = 20 _μ_m. Abbreviations: ES, embryonic stem; GFP, green fluorescent protein; ires, internal ribosome entry site; NSC, neural stem cell/progenitor; O4E, Oct4-enhanced green fluorescent protein; PEG, polyethylene glycol.

Figure 2

Figure 2

Flow cytometry analysis of Oct4-EGFP reactivation using Cre-loxP-based, enhanced green fluorescent protein-inducible assay for reprogramming. (A): Representative dot plots. Shown are sample plots from a control cell population including CIPOE NSCs only; Z-Red ESCs only; Z-Red ESCs transfected with a constitutive Cre expression plasmid; CIPOE NSCs transfected with a Cre reporter plasmid (pCAGT-bGeo-LoxP) and a mix of Z-Red ESCs without polyethylene glycol (PEG); and from PEG-induced fusion cell population at 2, 4, and 6 DIV. (B): Analysis of Rf. Rf was calculated from multiple independent experiments and quantified for fusion population over 2, 4, 6, and 8 days after PEG treatment. Values represent mean ± SEM (n = 5; *, p < .01, one-way analysis of variance). (C): Analysis of reprogramming efficacy. Shown is the summary of cumulative distribution plot of EGFP intensity for grouped DsRed+ cells over 2, 4, 6, and 8 days after PEG treatment. Values represent mean ± SEM (n = 5; *, p < .01, Kolmogorov-Smirnov test). Abbreviations: DIV, days in vitro; EGFP, enhanced green fluorescent protein; mix, mixture; NSC, neural stem cell/progenitor; Rf, reprogramming frequency.

Figure 3

Figure 3

Dioxygenase inhibitor DMOG impedes reprogramming. (A): Inhibition of Jhdm2a-induced histone demethylation by DMOG. Blocking effects of DMOG on Jhdm2a were examined in 293T cells transfected with a plasmid expressing the Jhdm2a-EGFP fusion protein. In control cells treated with DMSO, H3K9diM immunostaining signal was lost in Jhdm2a-EGFP transfected cells (arrows). With the treatment of 10 _μ_M DMOG, H3K9diM signals were present in all cells regardless of Jhdm2a-EGFP expression. Scale bar = 20 _μ_m. (B–D): DMOG attenuates ESC-induced reactivation of Oct4 expression from adult neural stem cells/progenitors. Polyethylene glycol-mediated cell fusion population treated with DMSO or DMOG (10 _μ_M) was analyzed at day 4 and is displayed in dot plots. Representative plots from multiple experiments are shown (B). Reprogramming frequencies from multiple experiments were quantified (C) for fusion population with 48-hour treatment with DMSO or DMOG. Data represent mean ± SEM (n = 3; **, p < .01, Student’s t test). Shown in (D) is the cumulative distribution plot of DsRed+ population with graded EGFP fluorescence intensities for comparison of the reprogramming efficacy in the presence of DMSO or DMOG (n = 3 experiments; *, p < .01, Kolmogorov-Smirnov test). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DMOG, dimethyloxalylglycine; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; H3K9diM, histone H3 lysine 9 dimethylation.

Figure 4

Figure 4

Histone methyltransferase G9a restricts Oct4-EGFP reactivation during ESC-induced reprogramming. (A): Expression of G9a in ESCs and adult neural stem cells/progenitors (NSCs). Shown is a summary of quantitative real-time polymerase chain reaction analysis of the expression level of G9a in Z-Red ESCs, CIPOE NSCs, CIPOE NSCs with shCon, and shG9a. The mRNA abundance was normalized to the levels in CIPOE cells. Values represent mean ± SEM (n = 3; *, p < .01, Student’s t test). (B): Summary of reprogramming frequencies. Shown are results from cell fusion experiments between Z-Red and CIPOE, CIPOE-shCon, or CIPOE-shG9a cells. Values represent mean ± SEM (n = 3; *, p < .01, Student’s t test). (C): Summary of reprogramming efficacy. Cumulative distribution plots of DsRed+ population with graded EGFP fluorescence intensities are shown for comparison of the reprogramming efficacy of Z-Red ESCs with CIPOE-shCon, or CIPOE-shG9a cells at days 2 and 4 after cell fusion. Values represent mean ± SEM (n = 3; *, p < .01, Kolmogorov-Smirnov test). Abbreviations: EGFP, enhanced green fluorescent protein; shCon, control short hairpin RNA; shG9a, G9a-targeted short hairpin RNA.

Figure 5

Figure 5

Histone demethylase Jhdm2a facilitates Oct4 reactivation during ESC-induced reprogramming. (A): EST profiles of histone demethylases. EST counts (transcripts per million) were collected from National Center for Biotechnology Information Unigene expression resources (

http://www.ncbi.nlm.nih.gov/sites/entrez

) and plotted against a panel of currently identified histone demethylases. Distributions of EST for individual demethylases across different developmental stages are shown. (B): Expression of Jhdm2a in ESCs and adult neural stem cells/progenitors (NSCs). Shown are quantitative real-time polymerase chain reaction analyses of the expression levels of Jhdm2a in Z-Red ESCs, CIPOE NSCs, and CIPOE NSCs with virally transduced Jhdm2a WT or H1120Y enzymatically inactive mutant. mRNA abundance is normalized to the levels of Z-Red cells, and values represent mean ± SEM (n = 3; *, p < .01, Student’s t test). (C): Ectopic expression of Jhdm2a, but not the enzyme-inactive mutant, induces global loss of H3K9 dimethylation in CIPOE NSCs, as indicated by arrows. Scale bar = 20 _μ_m. (D): Summary of reprogramming frequencies. Shown are results from cell fusion experiments between Z-Red and CIPOE, CIPOE-J2WT, or CIPOE-J2HY cells. Values represent mean ± SEM (n = 3; *, p < .01, Student’s t test). (E): Summary of reprogramming efficacy. Cumulative distribution plots of DsRed+ population with graded EGFP fluorescence intensities are shown for comparison of the reprogramming efficacy of Z-Red ESCs with CIPOE-J2 or CIPOE-J2HY cells at day 4 after cell fusion. Values represent mean ± SEM (n = 3; *, p < .01, Kolmogorov-Smirnov test). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag; H3K9diM, histone H3 lysine 9 dimethylation; TPM, transcripts per million; WT, wild-type.

Figure 6

Figure 6

Synergistic enhancement of reprogramming by a combination of Jhdm2a with the pluripotency-specific transcription factor Nanog. (A): A schematic drawing of the model on mechanisms underlying ESC fusion-induced Oct4-EGFP reactivation during reprogramming of somatic neural stem cells/progenitors. (B): Summary of reprogramming frequencies. Shown are results from cell fusion experiments between Z-Red and CIPOE, CIPOE-Nanog, or CIPOE-Nanog plus Jhdm2a cells. Values represent mean ± SEM (n = 3; *, p < .01, Student’s t test). (C): Summary of reprogramming efficacy. Cumulative distribution plots of DsRed+ population with graded EGFP fluorescence intensities are shown for comparison of the reprogramming efficacy of Z-Red ESCs with CIPOE, CIPOE-Jhdm2a, or CIPOE-Nanog plus Jhdm2a cells at day 4 after cell fusion. Values represent mean ± SEM (n = 3; *, p < .01, Kolmogorov-Smirnov test). Abbreviations: EGFP, enhanced green fluorescent protein; H3K9diM, histone H3 lysine 9 dimethylation.

Figure 7

Figure 7

Oct4 reactivation and promoter demethylation in adult neural stem cells/progenitors (NSCs) after G9a knockdown. (A): A schematic drawing of the Oct4 promoter with 16 CpG sites located between the proximal enhancer and exon 1. Bisulfite sequencing analysis was performed using genomic DNA extracted from CIPOE, Z-Red ESCs, fusion clone B3, CIPOE-shCon, and CIPOE-shG9a NSCs. (B, C): Conventional real-time polymerase chain reaction (PCR) (B) and quantitative real-time PCR (C) were used to compare the mRNA abundance of Oct4 in CIPOE, Z-Red ESCs, fusion clone B3, CIPOE-shCon, and CIPOE-shG9a NSCs. Data represent mean ± SEM (n = 3; *, p < .01, Student’s t test). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shCon, control short hairpin RNA; shG9a, G9a-targeted short hairpin RNA.

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