Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency

Abstract

To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.

Fig. 1

CRISPR-Cas9 screen reveals FLCN as an essential gene during the exit from the naïve pluripotent state. a H3K27me3 profile 5KB around transcription start site in developmental genes in naïve, primed and naive NNMT CRISPR KO samples were plotted from ChIPseq analysis. b Only 27% of the genes with H3K27me3 marks enriched in primed vs. naïve hESC are increased in NNMT KO naïve hESC. c Flowchart of genome wide CRISPR screen for exit of naïve state using GeCKO v2 library. d Schematic model of action of methotrexate/acetaldehyde selection. e Methotrexate/acetaldehyde selectively kills primed hESC and cells exiting naïve pluripotent state (4D TeSR). S.e.m.; *p < 0.05, ***p < 0.001; two-tailed t-test, n = 3 or 4 biological replicates. f CRISPR candidate hits selected by integrating CRISPR screen and RNA-seq data in primed hESC. Genes that are significant CRISPR hits (FDR < 0.05, at least 2.5-fold higher in day 17 compared to day 14) and expressed in primed hESC (Elf1 AF, >10 normalized read counts) are colored in red. Apoptotic genes were removed (Supplementary Data 2). g Secondary screen. Naïve hESC were infected with lentiCRISPR virus targeting single genes and induced to exit naïve state in TeSR media. The number of surviving cells after 3 days of methotrexate/acethaldehyde (MT/AC) was counted. S.e.m.; *p < 0.05, **p < 0.005; two-tailed t-test, n = 3 biological replicates

Fig. 2

FLCN KO hESC express naïve pluripotency markers. a Schematic representation of FLCN protein and location of CRISPR guide RNAs used. Sanger sequencing analysis of Elf1 2iL-I-F FLCN KO hESC reveals introduction of STOP codons in exons 3. b, c Generation of WIBR3 5iLA FLCN KO and Elf1 2iL-I-F FLCN KO line, and overexpression of FLCN-GFP fusion protein in wild type and FLCN KO background. d The pluripotency marker Oct4 is expressed in FLCN KO 2iL-I-F and 5iLA hESC. Scale bars represent 50 μm. e Expression pattern of genes up-regulated by FLCN KO compared to genes up-regulated in monkey pre- vs. post-implantation stage. Red: genes up-regulated in FLCN KO and pre-implantation, known ground state pluripotency markers are labeled. f tSNE analysis of naïve markers DNMT3L, TFCP2L1, and ESRRB in in vivo blastocysts from non-human primate, cynomolgus monkey (Macaca fascicularis). g, h RT-qPCR analysis of naïve markers DNMT3L and TFCP2L1 in naïve 2iL-I-F wild type (WT), FLCN KO, and rescue line (FLCN KO + OE FLCN-GFP) (g) and in naïve 5iLA WT and FLCN KO (h). S.e.m.; **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–10 biological replicates

Fig. 3

FLCN is essential for the exit from the naïve pluripotency. a Schematic representation of experimental procedures used to exit naïve pluripotent state. b Principal component analysis after RNA-seq analysis reveals that FLCN KO hESC do not fully exit naïve state. RNA-seq from various pre-implantation in vivo human embryo, naïve, and primed in vitro hESC were plotted, and separated in PC1 axis^,,,,,,. c Expression pattern of genes regulated by FLCN KO during exit of naïve state compared to cynomolgus monkey (Macaca fascicularis) pre- vs. post-implantation stage. Red: genes up-regulated in 7D TeSR FLCN KO and pre-implantation; green: genes up-regulated in 7D TeSR WT and post-implantation. d, e RT-qPCR analysis of naïve (DNMT3L, d) and primed (IDO1, e) markers after exit of 2iL-I-F naïve state (7 day TeSR) in WT, FLCN KO, and rescue line (FLCN KO + OE FLCN-GFP). S.e.m.; *p < 0.05, **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–6 biological replicates. Presented are the fold changes compared to naïve 2iL-I-F. f Western blot analysis of primed markers JARID2, LDHA, and H3K27me3 marks in naïve 2iL-I-F hESC and 10D TeSR hESC. g RT-qPCR analysis of NNMT expression. S.e.m.; **p < 0.005; two-tailed t-test, n = 3–6 biological replicates. h Model of doxycycline inducible- NNMT-GFP fusion construct inserted into AAVS1 locus of 2iL-I-F FLCN KO hESC. i Western blot analysis of NNMT, GFP, H3K27me3, JARID2, and OCT4 after NNMT OE in FLCN KO 7D TeSR hESC. j, k FLCN KO cells retain naïve 5iLA morphology (j) and naïve markers DNMT3L, TFCP2L1, and KLF4 (k) when pushed to exit the naïve state (Exit assay: FGF 4 or 7 days; relative mRNA expression: fold changes of expression in FLCN KO vs. control (WT FGF: 4D or 6D, red bar). Complete data presented in Supplementary Fig. 3B. S.e.m.; *p < 0.05, **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–6 biological replicates. Scale bars represent 100 μm

Fig. 4

FLCN regulates TFE3 subcellular localization in hESC. a Enrichment of transcription factor targets in genes that are up-regulated in FLCN KO 7D TeSR compared to WT 7D TeSR. b Immunofluorescence staining of TFE3 in naïve (WIBR3 5iLA) and primed (H1) hESC observed with confocal microscopy. c, d WT and FLCN KO lines were stained for TFE3 in naïve conditions (2iL-I-F, C; 5iLA, D) and 4 days after culture in TeSR (c) or FGF (d) media. TFE3 was detected in the nucleus in WT naive hESC and in FLCN KO grown in all conditions. Scale bars represent 10 μm. e Western blot analysis of TFE3 in WT and FLCN KO in naïve (5iLA) and 10D TeSR samples. f TFE3 targets are upregulated in naïve hESC (2iL-I-F) FLCN KO compared to WT, including TFCP2L1, ESRRB, and Wnt pathway components. g Generation of FLCN/ESRRB double KO in naïve hESC. Western blot analysis of CRISPR-Cas9-generated ESRRB mutant clones in WIBR3 5iLA FLCN KO. h FLCN/ESRRB double mutant does not rescue FLCN KO phenotype during the exit of naïve pluripotency. RT-qPCR analysis of naive hESC markers (TFCP2L1, KLF4, and DNMT3L) in WIBR3 6D FGF (WT), WIBR3 FLCN KO 6D FGF, and WIBR3 FLCN KO/ESRRB mutants 6D FGF. S.e.m.; **p < 0.005, ***p < 0.001; two-tailed t-test. i TFE3 targets are upregulated in 7D TeSR FLCN KO hESC compared to 7D TeSR WT hESC, including several Wnt pathway components. j TFE3-target Wnt ligands and Wnt pathway targets are up-regulated in naïve (2iL-I-F) hESC FLCN KO compared to WT (log2 fold change from RNAseq data is presented and p-value indicated). k, l Wnt-pathway targets are upregulated in 7D TeSR FLCN KO hESC compared to 7D TeSR WT hESC, as analyzed by RT-qPCR (k, s.e.m.; *p < 0.05,**p < 0.005, ***p < 0.001; two-tailed t-test, n = 3 biological replicates) and western blot (l). m Principal component analysis after RNA-seq analysis reveals that inhibition of Wnt by IWP2 during the exit of naïve state (Elf1 7D TeSR) rescues FLCN KO phenotype. RNA-seq from various pre-implantation in vivo human embryo, naïve, and primed in vitro hESC were plotted, and separated in PC1 axis^,,,,,,

Fig. 5

FLCN differentially regulates mTOR pathway in various pluripotency states. a Proteins associated with FLCN were co-immunoprecipitated and identified using mass spectrometry. Protein abundance (label free quantification (LFQ) intensity) differences were quantified between plus and minus dox-treated cell lines. Black line represents the 5% false discovery rate calculated in Perseus 1.5.6. N = 3–4 technical measurements. b Western blot analysis of p-mTor, mTOR, p-AKT(Ser 473), AKT, p-S6, and S6 in WT or FLCN KO naïve hESC (5iLA, 2iL-I-F) and hESC growing in FGF (7D) or TeSR (7D). c Effect of Rapamycin and INK-128 on naïve hESC and hESC exiting the naïve pluripotent state. Naïve 5iLA and 7D TeSR hESC were treated with either DMSO (control), INK-128 (100 nM), or Rapamycin (100 nM) for 24 h (at day 6 for 7D TeSR cells). d Western blot analysis of SIN1 expression in naïve (2iL-I-F) hESC WT and SIN1 mutant lines. e, f SIN1 mutant hESC exhibit a reduction of p(S473)AKT (e) and pS6 (f) in cells exiting the naïve state (TeSR 7D). g RICTOR mutant hESC exhibit a reduction of p(S473)AKT and pS6 in cells exiting the naïve state (TeSR 7D). h TFE3 is observed in the cytoplasm of hESC exiting the naïve state (Elf1 2iL-I-F → 4D TeSR, WT, SIN1 mutant clone #18, RICTOR KD mutant) and in the nucleus when treated for 24 h with mTORC1 inhibitor Rapamycin (100 nM) or mTORC1/2 inhibitor INK-128 (100 nM), using confocal microscopy. Scale bars represent 50 μm. i Hypothetical model of FLCN-regulation of mTORC1, mTORC2, and TFE3 localization in hESC