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PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK–ERK signaling

Abstract

The transcriptional network acting downstream of LIF, WNT and MAPK–ERK to stabilize mouse embryonic stem cells (ESCs) in their naive state has been extensively characterized. However, the upstream factors regulating these three signaling pathways remain largely uncharted. PR-domain-containing proteins (PRDMs) are zinc-finger sequence-specific chromatin factors that have essential roles in embryonic development and cell fate decisions. Here we characterize the transcriptional regulator PRDM15, which acts independently of PRDM14 to regulate the naive state of mouse ESCs. Mechanistically, PRDM15 modulates WNT and MAPK–ERK signaling by directly promoting the expression of Rspo1 (R-spondin1) and Spry1 (Sprouty1). Consistent with these findings, CRISPR–Cas9-mediated disruption of PRDM15-binding sites in the Rspo1 and Spry1 promoters recapitulates PRDM15 depletion, both in terms of local chromatin organization and the transcriptional modulation of these genes. Collectively, our findings uncover an essential role for PRDM15 as a chromatin factor that modulates the transcription of upstream regulators of WNT and MAPK–ERK signaling to safeguard naive pluripotency.

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Figure 1: PRDM15 is essential for naive pluripotency and self-renewal maintenance of ESCs.
Figure 2: PRDM15 zinc fingers are essential to maintain the naive state of ESCs, independently of PRDM14.
Figure 3: PRDM15 is essential in both mouse and human iPSC reprogramming.
Figure 4: PRDM15 acts as a positive modulator of WNT–β-catenin signaling and a negative regulator of MAPK–ERK signaling.
Figure 5: Genome-wide profile of PRDM15 binding to chromatin.
Figure 6: PRDM15 directly binds the Rspo1 and Spry1 promoters to modulate their transcription.
Figure 7: Proposed model for PRDM15 activity in ESCs.

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Acknowledgements

We thank F. Aguilo, M. Walsh and W.W. Tee for helpful discussions and critically reading the manuscript, H.-H. Ng (GIS/Singapore) for the kind gift of antibodies, and T. Teng and BRC Shared facilities for technical support. We acknowledge the technical expertise provided by the Advanced Molecular Pathology Laboratory (AMPL) at IMCB, and in particular we thank C.B. Ong. This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. We acknowledge support from NMRC/OFIRG/0032/2017, an AGA-SINGA (Singapore Graduate Award) fellowship to S.M. and M.B., the French College of Gynecology and Obstetrics to D.H. and an NRF fellowship to D.M.M.

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Contributions

Overall design of the project: S.M. and E.G. Acquisition of experimental data: S.M., D.H., S.X.T., H.F., Q.R.X., E.M.S. Detailed molecular and genomics approaches: S.M., J.Z., H.W., E.G. Generation of reagents and scientific inputs: E.S.M.W., M.B., M.K.Y.S., S.L.M.O., C.L.S., V.T., Y.-H.L., N.R.D., D.M.M. Bioinformatic analysis: J.Z. Organization of experiments and figures: S.M., D.M.M., E.G. Histopathology: M.A.-H. S.M. and E.G. wrote the manuscript with comments from all authors.

Corresponding author

Correspondence to Ernesto Guccione.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Addition of 2i rescues Prdm15–/– phenotype.

(a) qPCR analysis of Prdm15 mRNA levels in Prdm15(+/+;+/–;–/–) ESCs. (b) Western blot analysis of PRDM15 protein levels in Prdm15(+/+;+/–;–/–) ESCs. TUBA is a loading control. (c) Analysis of PRDM15 protein (top) and RNA (bottom) levels in ESCs cultured in 2i versus SL. TBX3 is a naive pluripotency marker; uncropped gels are in Supplementary Data. In a and c, data are from three experiments; three independent ESC clones are shown per genotype. (d) Short-term growth analysis of Prdm15(+/+; +/–; –/–) ESCs in 2i versus SL conditions. Cells (three independent cultures) were cultured in 2i for either 5 d (left) or switched to SL at day 3 (right); three independent clones are shown for each genotype. (e) Analysis of Prdm15 mRNA (top) and protein (bottom) levels in Prdm15fl/fl versus Prdm15Δ/Δ ESCs 3 d after OHT induction. (f) Colony formation assay in Prdm15fl/fl versus Prdm15Δ/Δ ESCs after 6 d of culture in SL. Data from two experiments with a total of n = 6 independent cell cultures are shown (g) Cell cycle analysis by flow cytometry; Prdm15fl/fl versus Prdm15Δ/Δ ESCs were pulsed with BrdU for 30 min and then stained with anti-BrdU/propidium iodide. (h) AP staining on Prdm15fl/fl versus Prdm15Δ/Δ ESCs cultured either in SL or 2i; data are presented as percent of the total number of colonies from three independent cultures. (h) Box plot of mean volumes for the teratomas formed by Prdm15fl/fl versus Prdm15Δ/Δ ESCs (n = 5 mice). Representative photos are shown to the right. Center values, mean; error bars, s.d. Student’s t test (two-sided) was used.

Supplementary Figure 2 Prdm15 overexpression does not affect ESC properties.

(a) Prdm15 overexpression (PR15-OE) levels measured by qPCR. (b) Cell cycle analysis by flow cytometry in ESCs upon exogenous expression of Prdm15 or empty vector (EV) control. (c) Colony formation assay for control versus PR15-OE ESCs after 6 d of culture in SL. (d) AP staining on control (EV) versus PR15-OE ESCs; two independent clones are shown. (e) Histological analysis of teratomas formed by control (EV) and PR15-OE ESCs. In both conditions, teratomas contained derivatives of all three germ layers (ectoderm/neural ectoderm in the second panel; mesoderm and endoderm). Representative images are shown (n = 4). One representative experiment with three independent cultures is shown in ad (n = 2). Center values, mean; error bars, s.d. Student’s t test (two-sided) was used.

Supplementary Figure 3 Genetic redundancy/interaction between Prdm14 and Prdm15 in ESCs.

(a) mRNA levels of Prdm14/Ubiquitin in Prdm15fl/fl and Prdm15Δ/Δ ESCs in SL versus 2i conditions. (b) AP staining of wild-type (Prdm15fl/fl; Prdm14+/+), Prdm15-null (Prdm15Δ/Δ ; Prdm14+/+), Prdm14-null (Prdm15fl/fl; Prdm14–/–) and Prdm15/Prdm14 double-knockout (Prdm15Δ/Δ; Prdm14–/–) ESCs. 1–3 depicts the number of independent clones for each genotype. (c) mRNA levels of Dnmt3b, Cdx2, Klf5 and Tcl1 in the same cells as in b. In a–c, one representative experiment is shown (n = 2); data are from three independent cultures. (d) mRNA levels of Prdm15 (N-terminus- versus C-terminus-specific primers), Tbx3 and Klf4 in Prdm15fl/fl and Prdm15Δ/Δ ESCs upon exogenous expression of EV, full-length PRDM15 (PR15-FL) or mutant PRDM15 (ΔZNFs). Data are from three independent experiments; center values, mean; error bars, s.d. Student’s t test (two-sided) was used.

Supplementary Figure 4 Analysis and comparison of Prdm15Δ/Δ and Prdm14–/– ESC transcriptomes.

(a) Scatterplot of RNA-seq expression analysis in Prdm15fl/fl versus Prdm15Δ/Δ ESCs cultured either in SL (top) or 2i (bottom). The numbers of upregulated (red) and downregulated (blue) genes are indicated on the graphs. Highlighted are Spry1 and Rspo1 (which are downregulated upon PRDM15 depletion in both conditions) and Wif1 whose expression is upregulated in Prdm15Δ/Δ ESCs under SL conditions but rescued by 2i. (b) Venn diagram depicting the overlap among differentially expressed genes in Prdm15-knockout versus Prdm14-knockout (top) and Prdm15-knockout versus Mtgr1-knockout (bottom) ESCs in SL and 2i conditions. (c) Differentially expressed genes between Prdm15fl/fl and Prdm15Δ/Δ ESCs in 2i versus SL conditions, identified by RNA-seq (3 d after deletion (n = 3)). Four main clusters have been identified. Cluster 1 and clusters 2/3 comprise genes up- and downregulated, respectively in Prdm15Δ/Δ cells, yet rescued by 2i. Cluster 4 contains genes unaffected by PRDM15 deletion in both conditions (2i and SL). The most dominant categories enriched in each cluster are indicated.

Supplementary Figure 5 PRDM15-mediated regulation of the WNT/β-catenin pathway.

(a) Box plot of β-catenin (left) and CDH1 (right) quantification in Prdm15fl/fl, Prdm15Δ/Δ and Prdm15Δ/Δ ESCs upon rescue with recombinant WNT3a or CHIR. Integrated density was measured for each channel, using ImageJ, and normalized to integrated density for DAPI (n = 6). (b) Immunofluorescence staining for β-catenin (β-CAT) and TUBULIN (TUBA) in Prdm15fl/fl, Prdm15Δ/Δ ESCs. (c) Western blot analysis of total levels of β-CAT, plakoglobin and CDH1 in Prdm15fl/fl and Prdm15Δ/Δ ESCs; uncropped images are in Supplementary Data. (d) Immunofluorescence staining for β-catenin (β-CAT) and plakoglobin (JUP) in Prdm15fl/fl, Prdm15Δ/Δ ESCs. (e) Immunofluorescence staining for TBX3 (green) and NANOG (red) in Prdm15fl/fl and Prdm15Δ/Δ ESCs; note the restoration of TBX3+NANOG+ in Prdm15Δ/Δ cells upon addition of recombinant WNT3a or CHIR. (f) Immunofluorescence staining for β-CAT and PRDM15 in Prdm15fl/fl, Prdm15Δ/Δ and Prdm15Δ/Δ ESCs expressing exogenous Prdm15. (g) qPCR analysis of canonical WNT target activation in Prdm15fl/fl and Prdm15Δ/Δ ESCs, 8 h after stimulation with recombinant WNT3a (10 ng). Data from three experiments are shown; center values, mean; error bars, s.d. Student’s t test (two-sided) was used. Representative images are shown in bf (n = 10).

Supplementary Figure 6 Exogenous expression of Prdm15 partially substitutes for 2i and supports naive pluripotency.

(a) Differentially expressed genes in wild-type (EV) versus PRDM15-overexpressing ESCs, associated with primed or naive pluripotency. (b) mRNA levels of naive (Tbx3 and Prdm14) and primed (Otx2, Fgf5 and Krt18) markers in wild-type (EV) versus PRDM15-overexpressing ESCs. Cells were cultured in medium containing two (2i), one (Meki or CHIR) or zero (N2B27) inhibitors. Expression levels are normalized to those of Ubiquitin and presented as fold change compared to wild-type cells under the 2i condition. One representative experiment is shown (n = 2); data are from three independent cultures; center values, mean; error bars, s.d. Student’s t test (two-sided) was used.

Supplementary Figure 7 Analysis and comparison of Prdm15Δ/Δ and Prdm14–/– ESC cistromes.

(a) Immunofluorescence staining for PRDM15 (red) in wild-type and PRDM15-overexpressing ESCs. (b) Pie chart showing the distribution of PRDM15-specific ChIP–seq peaks across the ESC genome under 2i conditions. (c) Venn diagram depicting the overlap between PRDM15-bound gene promoters in SL versus 2i. (d) Venn diagram showing the overlap between PRDM15 peaks in Prdm15fl/fl and Prdm15Δ/Δ in SL (left and middle) and 2i (right). (e) Venn diagram depicting the overlap between PRDM15 versus PRDM14 peaks (top) and PRDM15 versus MTGR1 peaks (bottom) in SL (left) and 2i (right). (f) PRDM15 binding motif in 2i identified by de novo motif discovery analysis. (g) The in silico prediction tool (http://zf.princeton.edu/) (bottom) is compared to the PRDM15 consensus motif derived from the ChIP–seq analysis (top). (h) Sequence comparison of probe 1 (containing the PRDM15-binding motif) and probe 2 (carrying random mutations of the same motif) used in EMSA. (i) EMSA performed using nuclear lysate from Prdm15fl/fl versus Prdm15Δ/Δ ESCs with probes described in h; an asterisk indicates unknown protein–DNA binding. (j) EMSA using wild-type probe 1 (left) or mutated probe 2 (right) with increasing amounts of nuclear lysate from Prdm15fl/fl ESCs. (k) Competition assay performed with non-labeled (cold) Probe#1 in excess. (l) EMSA performed using nuclear lysates from Prdm15fl/fl vs. Prdm15Δ/Δ ESCs with AP1-binding motif (AP1 was used as a reference/control transcription factor). (m) Supershift assay performed with PRDM15 antibody and matching IgG control. PRDM15–DNA–antibody complex is indicated with an arrow. In im, representative images of uncropped gels are shown (n = 3).

Supplementary Figure 8 Identification of potential PRDM15 direct transcriptional targets.

(a,b) Venn diagram depicting the overlap among differentially expressed genes in Prdm15fl/fl versus Prdm15Δ/Δ ESCs and genes bound by PRDM15 on their prompter region (a) or on distal enhancers associated with their respective promoters (b), as identified by ChiA-PET (Nature 504, 306–310, 2013). (c) Snapshots from PRDM15 peak alignment along with those from transcriptional regulators described in Figure 5c.

Supplementary Figure 9 Transcriptional regulation of Rspo1 and Spry1 in PRDM15-binding-site-mutated clones.

(a) qPCR analysis of Rspo1 expression in WT versus mutant clone R15 (left) and in Prdm15fl/fl versus Prdm15Δ/Δ ESCs; expression was normalized to Ubiquitin and WT cells were set as the reference. The sequences of the PRDM15-binding site in WT and R15 clones are shown to the right. (b) Similar to a, Spry1 expression levels are analyzed by qPCR in three single clones targeted with CRISPR–CAS9 (using three different gRNAs). Genotypes and sequences of the CRISPR–CAS9-targeted region in WT clone and CRISPR clones S16, S19 and S24 are shown to the right; note the disruption within the consensus motif sequence in clones S19 and S24 and upstream of it in clone S16, which is used as the control for further experiments. In a and b, data are from four independent experiments; center values, mean; error bars, s.d. (c) ChIP–qPCR analysis of PRDM15 and RNAPII binding along with H3k27ac, H3k27me3 and total histone H3 enrichment on the promoter region of Spry1 in WT versus S16 clones. Data are from one representative experiment (n = 2) and are averaged across two technical replicates ± s.d. Student’s t test (two-sided) was used.

Supplementary Figure 10 Restoration of naive pluripotency in Prdm15Δ/Δ ESCs by rescue of target gene expression.

(a) Immunofluorescence staining of Prdm15fl/fl versus Prdm15Δ/Δ ESCs in SL using antibodies against β-CAT (green) and CDH1 (red). DNA was stained with DAPI (blue); cells were cultured either in untreated (n.t.) or RSPO1 conditioned medium (CM). (b) Immunofluorescence staining of TBX3 (green) and NANOG (red) in Prdm15fl/fl, Prdm15Δ/Δ, Prdm15Δ/Δ; SPRY1-OE and Prdm15Δ/Δ; RSPO1-OE ESCs. (c) Immunofluorescence staining of TBX3 (green) and NANOG (red) in Prdm15fl/fl, Prdm15Δ/Δ and Prdm15Δ/Δ; Wif1–/–; SPRY1-OE + RSPO1-CM ESCs. Representative images are shown (n = 10).

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Mzoughi, S., Zhang, J., Hequet, D. et al. PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK–ERK signaling. Nat Genet 49, 1354–1363 (2017). https://doi.org/10.1038/ng.3922

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