Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May 2;24(5):785-801.e7.
doi: 10.1016/j.stem.2019.03.017. Epub 2019 Apr 25.

Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency

Tüzer Kalkan  1 , Susanne Bornelöv  2 , Carla Mulas  3 , Evangelia Diamanti  2 , Tim Lohoff  4 , Meryem Ralser  2 , Sjors Middelkamp  5 , Patrick Lombard  2 , Jennifer Nichols  4 , Austin Smith  6
Affiliations

Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency

Tüzer Kalkan et al. Cell Stem Cell. .

Abstract

The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.

Keywords: ETS factors; RBPJ; commitment; differentiation; embryonic stem cell; epiblast; gene regulatory network; pluripotency; self-renewal.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
ETV4/5 Expression and Function (A and B) RNA-seq expression values (FPKM or RPKM) for naive and transitioning ESCs (A) and early embryo lineages (B). Error bars represent SD from (A) 2 and (B) 3 independent biological replicates. (C and D) qRT-PCR expression in 2i and after 2i withdrawal (C) and in 2i/L components for 3 passages (D). Data are means ± SD from (C) 2 wells of cells differentiated in parallel and (D) 3 independent biological replicates. CH, CHIR99021; L, LIF; P, PD0325901. (E) Western blot with anti-FLAG antibody on Etv5-3×FLAG knockin ESCs. (F) Schematic for (G)–(I). (G) Quantitation of colony assays on WT (wild type), Etv4/5-dKO ESCs, and Etv4/5-dKO ESCs expressing monomeric Kusabira Orange (mKO), ΔN-Etv5, or canonical Etv5 transgenes. Error bars show SD from 2 technical replicates. (H) Rex1-GFP profiles of RGd2 and independently generated clonal lines (c) of Etv4-KO; Etv5-KO and Etv4/5-dKO at 25 and 42 h post-2i withdrawal. RGd2-2 is a clonal line derived from parental RGd2-1. (I) Colony assay. (J) GFP profiles for parental RGd2 and mutant ESC lines at 72 h post-CH/LIF withdrawal (N72h) and end of passage 2 (p2). Red cross indicates failure of replating after passage. ET, Etv5/Tcf3. See also Figures S1 and S2.
Figure 2
Figure 2
RBPJ Expression and Function (A) RBPJ western blot: C, cytoplasmic fraction; N, nuclear fraction; T, total cell lysate. Oct4 and GAPDH were used as loading controls for nuclear and cytoplasmic fractions, respectively. (B) GFP profiles of RGd2 and three clonal Rbpj mutant lines in N2B27 at 25 h (N25h) and 42 h (N42h) post-2i withdrawal. (C) Colony assay. (D) MA plot showing mean expression against fold change per gene in Rbpj-KO ESCs at 16 h post-2i withdrawal (N16h). Gene symbols and colored tags are shown for selected genes listed. (E) RNA-seq expression values for naive pluripotency factors in RGd2 and Rbpj-KO ESC in 2i and at N16h. Error bars show SD from biological replicates plated in parallel; 3 independent clonal lines for Rbpj-KO and 2 different lines for RGd2 (one parental and one clonal). (F) The University of California, Santa Cruz (UCSC) genome browser tracks for Nanog and Tbx3 loci showing normalized RNA-seq read coverage for parental and Rbpj-KO ESCs at N16h. RBPJ binding sites are indicated with red arrowheads. The RBPJ-binding motif within the Nanog locus is highlighted. (G) ChIP-qPCR for binding sites shown in Figures 2F and S4E. Two primer sets were used for the Tbx3 locus. y axis shows absolute enrichment normalized to input DNA for each sample. Error bars indicate SD from two ChIP replicates. (H) GFP profiles at 40 h after 2i withdrawal following a 7-h period of siRNA transfection. (I) Colony assay at 40 h after 2i withdrawal. See also Figures S2, S3, and S4 and Tables S1 and S2.
Figure 3
Figure 3
Dual- and Triple-Knockout Phenotypes (A) GFP profiles of RbpJ-KO (R-KO), Etv5/Rbpj-dKO (ER-dKO), and Rbpj/Tcf3-dKO (RT-dKO) ESCs at 72 h post-CH/LIF withdrawal (N72h) or at the end of passage 2 (p2). Red crosses indicate failure of replating upon passage. (B) Profiles of RGd2 and ER-dKO ESCs cultured in CHIRON (CH) only. (C) Clonogenicity in 2i/LIF or N2B27. Error bars show SD from 2 technical replicates. (D) Whole well images of colony formation in 2i/LIF or N2B27. (E) GFP profiles of ETR-tKO and RGd2 ESCs cultured in CH/LIF. (F) Phase contrast images. Scale bar represents 75 μM. (G) GFP profiles of ETR-tKO ESCs cultured in N2B27 only. (H) Immunofluorescent staining (IF) of RGd2 ESCs cultured in 2i, ER-dKO in CH, and ETR-tKO in N2B27 after 6 passages. (I) IF after 8 days of neural differentiation. (H and I) Images were taken using 20× (H) and 10× (I) objective. See also Figures S4 and S5.
Figure 4
Figure 4
Transition Failure of Etv5 and Triple-Knockout ESCs (A) Phase contrast images of RGd2 and Etv5-KO ESCs during first three passages (p1–p3) in AFX taken using a 10× objective. Scale bars represent 75 μM. (B) GFP profiles at the end of p1. (C) Alkaline phosphatase staining at the end of p3. (D and E) qRT-PCR (D) and GFP (E) profiles after 10 passages in AFX. (F) qRT-PCR on embryoid bodies on days 3–9. Day 0 is starting ESCs in CH/LIF. Error bars in (D) and (F) show SD from 2 technical replicates for qPCR.
Figure 5
Figure 5
Transcriptome Analysis of Single and Combined Mutants (A and B) Hierarchical clustering (A) and PCA plot (B) based on normalized gene expression for all genes. ∗ denotes cells expanded in CH/LIF and switched to 2i for 48 h prior to sample conditions. (C and D) Heatmaps showing relative expression for pluripotency genes (C) and lineage markers (D). Values are shown as Log2 fold change of RNA-seq read counts relative to RGd2 2i-p5. Only the genes with a mean expression value of FPKM ≥ 1 in either RGd2 2i-p5 or ETR-tKO N-p5 samples were included. Genes were sorted by mean expression within each group. (E) KEGG pathway enrichment for differentially expressed genes. (F) MA plot showing mean expression against fold change per gene in ETR-KO cells cultured in N2B27 for 5 passages (ETR-tKO N-p5) versus RGd2 2i-p5 sample. Gene symbols are shown for selected genes listed below. See also Tables S2 and S3.
Figure 6
Figure 6
Chimera Contribution and Perturbation by Triple-Knockout ESCs Reporter fluorescence and whole-mount immunofluorescence staining on chimeric embryos obtained from RGd2 or ETR-tKO ESCs labeled with H2B-tdTomato (red). (A) In vitro matured blastocysts at E4.5 stained for Gata4 and Sox2. Scale bars represent 50 μm. (B) E6.5 embryos with T (Brachyury) staining and Rex1-GFP fluorescence. Arrowheads in the lower DAPI panel point to separate ETR-tKO chimeras. Scale bars represent 100 μm. (C) E7.5 embryos with Pou3f1 (Oct6) and Nanog staining. Scale bars represent 200 μm. (D) E7.5 T and Nanog staining. Scale bars represent 200 μm. Note the different magnifications for RGd2 and ETR-tKO chimeras in (C) and (D). See also Table S4.
Figure 7
Figure 7
Etv5 Association with Transcriptionally Dynamic Genes (A) Numbers of ETV5 ChIP-seq peaks in 2i or at 16 h post-2i withdrawal (N16h). (B) Heatmap showing relative expression of downregulated genes in Etv5 mutants (fold ≤ 0.66) at 16 h post-2i withdrawal (N16h) with proximal ETV5 binding. (C–E) UCSC Genome browser tracks of Lef1 (C), Fgf5 (D), and Pou3fl (E) loci showing normalized ChIP-seq read coverage for Etv5 and H3K4me3 (this study), p300, H3K27Ac, and H3K4me1 (Buecker et al., 2014). (F) GFP profiles of EpiLCs (48 h in Activin/Fgf2/KSR) generated from RGd2 ESCs or Etv5-KO ESCs (2 clonal lines). (G) qRT-PCR on time course samples during EpiLC formation. Error bars show SD from 2 wells of cells differentiated in parallel. (H) ChIP-qPCR showing H3K27Ac levels on upstream and downstream loci adjacent to the Etv5 peaks shown in (C)–(E). y axis shows absolute enrichment normalized to input DNA from each sample. ChIP was performed in duplicate (1 and 2) for each sample. Error bars show SD from 2 qPCR replicates. (I) Mean read coverage for p300, H3K27Ac, and H3Kme1 (ChIP-seq from Buecker et al., 2014) on “non-promoter”-associated ETV5-bound loci. Read depth is scaled to 1×. See also Figures S6 and S7 and Tables S5 and S6.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akagi T., Kuure S., Uranishi K., Koide H., Costantini F., Yokota T. ETS-related transcription factors ETV4 and ETV5 are involved in proliferation and induction of differentiation-associated genes in embryonic stem (ES) cells. J. Biol. Chem. 2015;290:22460–22473. - PMC - PubMed
    1. Alexandrova S., Kalkan T., Humphreys P., Riddell A., Scognamiglio R., Trumpp A., Nichols J. Selection and dynamics of embryonic stem cell integration into early mouse embryos. Development. 2016;143:24–34. - PMC - PubMed
    1. Arber S., Ladle D.R., Lin J.H., Frank E., Jessell T.M. ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell. 2000;101:485–498. - PubMed
    1. Betschinger J., Nichols J., Dietmann S., Corrin P.D., Paddison P.J., Smith A. Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell. 2013;153:335–347. - PMC - PubMed
    1. Boroviak T., Loos R., Bertone P., Smith A., Nichols J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat. Cell Biol. 2014;16:516–528. - PMC - PubMed

Publication types

MeSH terms

Substances