Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells

Abstract

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.

Keywords: Cited2; Nanog; Pluripotency; Self-renewal; Transcriptional regulation.

Figure 1

Mouse embryonic stem cells (ESC) require Cited2 for proliferation, survival, and pluripotency. (A): AP activity in C2^fl/fl ESC cultured on gelatine in the presence (top panel) or 4 days after removal (bottom panel) of LIF. (B): Expression of Cited2 transcripts determined by quantitative real-time PCR in C2^fl/fl, and C2^fl/fl ESC stably transfected with a Cre-ERt expressing plasmid, the ESC lines C2^fl/fl[Cre]A, C2^fl/fl[Cre]B, and C2^fl/fl[Cre]C, treated with 1 μM 4HT or ethanol (vehicle for 4HT) for 48 hours. Expression level is normalized for Gapdh and reported as relative to the expression in C2^fl/fl ESC treated with ethanol which is set at 1. Results are presented as the mean ± SEM of three independent biological replicates (each performed in technical duplicate). (C): Proliferation of C2^fl/fl[Cre]A, C2^fl/fl[Cre]B, and C2^fl/fl[Cre]C ESC lines plated at 500 cells per well of gelatinized wells (12-well plate) in the presence of ethanol or 0.5 μM 4HT at day 0. Cells were maintained in culture with the conditions applied at day 0 until counted at the indicated time points. Results are presented as the mean ± SEM of three biological replicates, each performed in technical duplicate. (D): Percentage of AP-positive colonies over the total number of colonies at day 6 after ethanol or 4HT treatment in the presence of LIF. Results are the mean ± SEM of three independent biological replicates (each performed in technical duplicate). (E): Representative morphology of control ESC (C2^fl/fl[Cre]) and cells 3 days after Cited2 depletion by 4HT (C2^Δ/Δ[Cre]). (F): Percentage of C2^fl/fl[Cre] and C2^Δ/Δ[Cre] adherent ESC positive for Annexin V (early apopotosis) or propidium iodide (PI, late apoptosis) determined by flow sorting anlysis (FACS) 20 or 48 hours after ethanol or 4HT treatment. (G): Total number of C2^fl/fl[Cre] and C2^Δ/Δ[Cre] ESC in the culture supernatants stained with PI, 20 and 48 hours after treatment with ethanol or 4HT, determined by FACS. Results in (F) and (G) are shown as the mean ± SEM of three technical replicates performed with two biological replicates (i.e., C2^fl/fl[Cre]A and C2^fl/fl[Cre]B ESC). Abbreviations: AP, alkaline phosphatase; CPD, cumulative population doublings; LIF, leukemia inhibitory factor.

Figure 2

Cited2 knockout defects in embryonic stem cells (ESC) are rescued by human CITED2 expression. (A): C2^fl/fl[Cre]A and C2^fl/fl[Cre]B ESC were transduced with a lentiviral vector constitutively expressing the human CITED2 or a control vector to generate C2^fl/fl[Cre]/CITED2 and C2^fl/fl[Cre]/Control, respectively. Cited2 protein levels were detected by Western blotting in extracts from C2^fl/fl[Cre]/Control, C2^fl/fl[Cre]/CITED2, C2^Δ/Δ[Cre]/Control, and C2^Δ/Δ[Cre]/CITED2 ESC 48 hours after incubation with ethanol or 4HT. Loading in each lane was monitored by detection of β-tubulin. (B): Relative expression of Cited2 and pluripotency markers in cells described in (A). Gene expression in C2^fl/fl[Cre]/Control ESC was set to 1. Results are presented as the mean ± SEM of technical triplicates performed using two independent biological replicates (i.e., C2^fl/fl[Cre]A and C2^fl/fl[Cre]B ESC). (C): CPD of cells as described in A (LIF) or further supplemented with 1 μM PD0325901 and 3 μM CHIR99021 (LIF+2i), after treatment with ethanol or 4HT for 24 hours. Cells were plated at 10,000 cells per gelatinized well of a six-well plate at day 0. Results are presented as the mean ± SEM of three independent experiments performed with C2^fl/fl[Cre]B ESC. (D): Percentage of AP-positive colonies over the total number of colonies in C2^fl/fl[Cre]/Control, C2^Δ/Δ[Cre]/Control, C2^fl/fl[Cre]/CITED2, and C2^Δ/Δ[Cre]/CITED2 ESC cultures 6 days after ethanol or 4HT treatment. Results are presented as the mean ± SEM of technical triplicates performed in two independent biological replicates. (E): FACS analysis of the indicated ESC cell cycle profile by propidium iodide staining. Data are the mean ± SEM of three independent experiments performed with C2^fl/fl[Cre]/Control, C2^Δ/Δ[Cre]/Control, C2^fl/fl[Cre]/CITED2, and C2^Δ/Δ[Cre]/CITED2 ESC. Abbreviations: AP, alkaline phosphatase; CPD, cumulative population doublings; LIF, leukemia inhibitory factor.

Figure 3

Cited2 knockdown results in spontaneous differentiation in embryonic stem cells. (A): Relative Cited2 gene expression in E14TG2A cells detected by quantitative real-time PCR (qPCR), 6 days post-transfection of a vector expressing a double stranded RNA targeting Cited2 (KD-Cited2), a nontargeting double stranded RNA (KD-control), and the empty (KD-empty) vectors. Cited2 expression in E14TG2A cells with the empty vector is set to 1. Results are presented as the mean ± SEM of three independent experiments performed in technical triplicates. (B): Cited2 protein levels detected in E14TG2A extracts with anti-Cited2 antibody by Western blotting. Equal loading in each lane was monitored by β-tubulin detection. (C): Proliferation of E14TG2A cells transfected with KD-empty, KD-control, or KD-Cited2 vectors. Cells were plated at 500 cells per gelatinized wells (12-well plate) the day after transfection. Cells were maintained in culture under the conditions applied at day 0 until counted at the indicated time points. Results are presented as the mean ± SEM of three independent experiments, each performed in technical duplicate. (D): Percentage of AP-positive colonies over the total number of colonies, 5 days post-transfection of E14TG2A cells with KD-empty, KD-control, or KD-Cited2 vectors. Results are presented as the mean ± SEM of three biological experiments. (E): KD-control or KD-Cited2 transfected E14TG2A cells coimmunostained with anti-Cited2 (green), anti-H3-triMeK9 (red) antibodies, and DAPI (blue). Arrow heads indicate cells with undetectable Cited2 protein, with enlarged nuclei and increased number of H3-triMeK9 foci. (F): Distribution of H3-triMeK9 foci number in KD-control or KD-Cited2 transfected cells described in (E). The results are presented as cluster of cells displaying up to 10 (0–10), between 11 and 20 (11–20), and more than 21 H3-triMeK9 foci in KD-control (black bars) and all KD-Cited2 (white bars) transfected cells. The number of H3-triMeK9 in KD-Cited2 transfected cells expressing high levels of Cited2 (red bars) and low levels or no detected Cited2 (gray bars) is also presented. (G): Transcript levels of pluripotency (black bars), mesoderm (Brachyury, Cdx2), endoderm (Foxa2, Gata6, Sox17), ectoderm (Fgf5, Sox1) markers, and Cited2 (gray bar) detected by qPCR using the primer set Cited2#2 (Supporting Information Table S1) in E14TG2A cells 6 days post-transfection of KD-empty or KD-Cited2. Gene expression presented as fold of expression relative to KD-empty treated cells. Data are the mean ± SEM of three independent experiments, each performed in technical triplicate. Abbreviation: AP, alkaline phosphatase.

Figure 4

Cited2 controls Nanog expression. (A): Immunocytochemistry using anti-Cited2 (green), anti-Nanog (red) antibodies, and DAPI (blue) in E14TG2A. (B): Cited2, Nanog, and Oct4 protein levels detected in E14TG2A extracts prepared 48 hours post-transfection with KD-control or KD-Cited2. Loading in each lane was monitored by detection of β-tubulin. (C): Top: diagram of the mouse Nanog genomic contig showing the transcriptional start site (arrow), exon 1 (gray box), and the positions of PCR primers (arrow heads) used in chromatin immunoprecipitation (ChIP) assays. Bottom: enrichment analyzed by quantitative real-time PCR (qPCR) of the “Stat3” element, the distal enhancer and the proximal promoter of Nanog, as well as the CR1 proximal promoter and the CR4 distal enhancer of Oct4, and c-Myc proximal promoter in ChIP assays using anti-Cited2 and control anti-flag antibodies. Results are presented as the mean ± SEM of three independent experiments. (D): Enrichment of Nanog proximal promoter from E14/T cells expressing flag-CITED2 or control vector in ChIP assays using anti-flag or control antibodies. Results are presented as the mean ± SEM of three biological experiments. (E): Endogenous Nanog, Oct4, Sox2, Klf4, Tbx3, c-Myc, and ectopic CITED2 transcript levels detected by qPCR in E14/T cells transfected with pPyCAGIP or pPyCAGIP-flagCITED2. Expression is presented as fold relative to pPyCAGIP. Results are shown as the mean ± SEM of three independent experiments. (F): Ectopic flag-CITED2 and endogenous Nanog protein levels in E14/T embryonic stem cells (ESC) transfected with pPyCAGIP or pPyCAGIP-flagCITED2 detected by Western blotting. Loading in each lane was monitored by detection of β-tubulin. (G): Left panel: pNanog-luc or pGL3basic activity in E14TG2A cells cotransfected with KD-Cited2 (gray bars) or KD-empty (black bars). RLU are presented relative to RLU of pGL3basic transfected with the control vector KD-empty set at 1. Right panel: pNanog-luc or pGL3basic activity in E14TG2A cells cotransfected with pPyCAGIP-flagCITED2 (gray bars) or pPyCAGIP (black bars). Results are presented as the mean ± SEM of three biological experiments performed in duplicate. (H): pOct4-luc or pGL3basic activity in E14TG2A ESC cotransfected as in (G). Results are presented as the mean ± SEM of three independent experiments (each performed in technical duplicate). Abbreviation: RLU, relative luminescence units.

Figure 5

Cited2 is present at the Klf4 and Tbx3 promoters in embryonic stem cells (ESC). (A): Diagram of the mouse Klf4 and Tbx3 genomic contigs showing the transcriptional start site (arrow), exon 1 (gray box), and the positions of PCR primers (arrow heads) used in chromatin immunoprecipitation (ChIP) assays. The region of mouse Klf4 promoter responsive to Cited2 overexpression in epithelial cells determined elsewhere is indicated by brackets. (B): Enrichment of the promoter region of Klf4 (Klf4 A and B), the junction of intron 1 and exon 2 of Klf4 (Klf4 C), the distal (Tbx3 A), proximal promoter (Tbx3 B), and the junction of exon 1 and intron 1 of Tbx3 (Tbx3 C) in ChIP assays with anti-Cited2 compared to control antibodies. (C): ChIP assays using anti-flag or control antibodies as described above with Klf4 primers in E14/T expressing flag-CITED2 or control vector. (D): LTBX3-luc or pGL3basic activity in E14TG2A ESC cotransfected as in Figure 4G. Data are the mean ± SEM of three independent experiments (each performed in technical duplicate). Results in (B) and (C) are presented as the mean ± SEM of three independent biological experiments. Abbreviation: RLU, relative luminescence units.

Figure 6

Nanog overexpression rescues defects caused by knockdown of Cited2. (A): pCITED2-luc or pGL3basic activity in E14TG2A embryonic stem cells (ESC) cotransfected with pPyCAGIP (black bars) or pPyCAGIP-Nanog (gray bars). RLU are presented relative to RLU of pGL3basic transfected with the control vector pPyCAGIP set at 1. Results are shown as the mean ± SEM of three independent experiments. (B): Percentage of AP-positive colonies over the total number of colonies in E14/T-vector and E14/T-Nanog cells transfected with KD-control or KD-Cited2 vectors. Results are presented as the mean ± SEM of three biological experiments. (C): Proliferation of E14/T-vector and E14/T-Nanog treated as in (B). Results are presented as the mean ± SEM of three independent experiments. (D): Nanog and Cited2 transcript levels detected by quantitative real-time PCR (qPCR) in E14/T-vector or E14/T-Nanog cells transfected for 48 hours KD-control or KD-Cited2. Gene expression is presented relative to expression in E14/T-vector (which is set at 1). Results are presented as the mean ± SEM of three independent experiments. (E): Cited2 and Nanog protein levels in E14/T-vector or E14/T-Nanog cell extracts. Equal loading in each lane was monitored by β-tubulin detection. (F): Proliferation of C2^fl/fl/Control or C2^fl/fl/Nanog ESC as well as C2^Δ/Δ/Control and C2^Δ/Δ/Nanog ESC isolated 48 hours post-transfection of a Cre and green fluorescent protein (GFP) expressing plasmid. Sorted GFP⁺ cells were plated at 10,000 cells per gelatinized wells (six-well plate) at day 0. Results are presented as the mean ± SEM of three independent experiments. (G): Cited2, Nanog, Oct4, Sox2, Rex1, Tbx3, Klf4, and c-Myc transcript levels detected by qPCR from C2^fl/fl/Control, C2^fl/fl/Nanog, C2^Δ/Δ/Control, and C2^Δ/Δ/Nanog GFP-positive cells purified by FACS, 4 days post-transfection of a Cre and GFP coexpressing plasmid. Data are the mean ± SEM of three independent experiments. Abbreviations: AP, alkaline phosphatase; CPD, cumulative population doublings; RLU, relative luminescence units.

Figure 7

Cited2 is essential for the generation of induced pluripotent stem cells and enhances the efficiency of reprogramming. (A): Relative expression of Cited2 mRNA in control (MEF-C2^fl/fl[Cre]) and Cited2^Δ/Δ (MEF-C2^Δ/Δ[Cre]) MEFs measured by quantitative real-time PCR using the Cited2#3 primer set and normalized to Tbp expression (Supporting Information Table S1). (B): mOrange expression in control and Cited2^Δ/Δ MEFs 2 days after transfection of PB transposon harboring MKOS-IRES-mOrange cassette, a constitutively active CAG-rtTA transactivator construct and a transposase expression vector. (C): AP staining following 15 days in culture in the presence of doxycycline. (D): Nanog expression in primary colonies 15 days after doxycycline treatment. (E): MEFs harboring a doxycycline-inducible MKOS-IRES-mOrange cassette and Nanog-eGFP reporter (MKOS-MEFs) were transduced with MSCV retroviruses expressing Cited2 or empty control retroviruses and treated with doxycycline. MKOS-MEFs transduced with Cited2 and control retroviruses are referred to as MKOS-MEFs-Cited2 and MKOS-MEFs-Control, respectively. (F): AP staining in MKOS-MEFs-Cited2 or MKOS-MEFs-Control cultures 20 days after the initiation of doxycycline treatment. (G): Model of direct (red) and indirect (gray) interactions between Cited2 and core pluripotency. Results in (A), (C), and (F) are presented as the mean ± SEM of three independent experiments with at least two different biological replicates. Abbreviations: AP, alkaline phosphatase; MEF, mouse embryonic fibroblast.