The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells

Abstract

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.

Figure 1.

SNON expression in hESCs is controlled by SMAD2/3 and OCT4, SOX2, and NANOG. (A) hESCs were differentiated into DE for 5 d according to the schematic. Activin A and BMP4 (both at 50 ng/mL) were added on day 0. Only Activin A was replenished on day 3. (B) SNON expression levels during DE differentiation by qPCR. Data were normalized against GUSB and are shown relative to undifferentiated hESCs (= 1.0). (C) Western blot analysis for SNON protein levels during DE differentiation. (D) OCT4 and SNON immunofluorescence on undifferentiated HES3 (hESCs). Bar, 100 μm. (E) Luciferase reporter assays of SNON promoter activity in HES3. Black shading indicates specific mutation of the OCT4/SOX2/NANOG-binding sites in the 5′ distal enhancer or of the four SBEs proximal to the SNON transcriptional start site.

Figure 2.

SNON knockdown induces differentiation in hESCs. (A) Schematic of the experimental design using two stable DOX-inducible S4TR5 hESC lines—shSNON(954) and shSNON(1307)—where DOX was replenished daily for 5 d. (B) SNON, FOXA2, and SOX17 expression analysis by qPCR. RNA was collected on days 0–5 according to A. Data were normalized against GUSB and are depicted relative to S4TR5 (DOX−) at day 0 (= 1.0). P-values were calculated according to the Student's t-test. (*) P < 0.05. (C) Western blot analysis for SNON, FOXA2, and SOX17.

Figure 3.

SNON overexpression inhibits DE formation. (A) Western blot analysis detects abundant C-terminally truncated, constitutively active SNON(1–366) in two stable, clonally selected lines (#1 and #9). (B) Expression analysis of the indicated genes by qPCR. hESCs were differentiated according to the schematic in Figure 1A. Data were normalized against GUSB and are shown relative to parental wild-type undifferentiated hESCs (= 1.0). The “vector control” HES3 line contains only the pCAG-IRES-Puro construct. P-values were calculated according to the Student's t-test. (*) P < 0.05; (**) P < 0.01.

Figure 4.

SNON highly and selectively occupies the promoters of PS and mesendodermal genes in hESCs. (A) ChIP-qPCR analysis of SNON-binding (αSn), SMAD2-binding (αS2), and SMAD3-binding (αS3) sites at the indicated gene regions in HES3 cultured in mTeSR1 medium. Relative occupancy values are shown as the apparent immunoprecipitation efficiency (percentage) (ratio = immunoprecipitated DNA/input DNA). (IgG) Normal rabbit IgG as negative control. P-values were calculated according to the Student's t-test. (*) P < 0.05; (**) P < 0.01. (B) Proposed model for SNON function in hESCs. In pluripotent hESCs, extrinsic signals (e.g., TGFβ/Activin/Nodal) regulate stem cell factor gene expression through activated SMAD2/3/4 complexes. OCT4/SOX2/NANOG positively regulate their own promoters and activate SNON transcription. SMAD2/3 also regulate SNON. SNON is selectively recruited to SMAD2-bound mesendodermal genes and suppresses their transcription. During early differentiation, stem cell factor gene expression and SMAD2/3/4 occupancy at the SNON promoter/enhancer decline. Consequently, SNON expression decreases. Existing SNON protein is rapidly degraded in a ligand-dependent manner by E3 ubiquitin ligases (e.g., SMURF2, ARKADIA, or APC). This leads to the derepression of early PS and DE target genes (collectively referred to as mesendoderm). (X) Tissue-specific transcriptional coactivators such as FOXH1.