Interestingly, we also observed that LEFTY1, a known TGF-beta direct target, was also co-bound by ZNF398 (Fig.?7a). from your GEO database for the following datasets: “type”:”entrez-geo”,”attrs”:”text”:”GSE54471″,”term_id”:”54471″GSE54471 (H3K27ac and H3K4me1), “type”:”entrez-geo”,”attrs”:”text”:”GSE76084″,”term_id”:”76084″GSE76084 (H3K27me3, H3K36me3, H3K4me3, H3K9ac, SOX2), “type”:”entrez-geo”,”attrs”:”text”:”GSE118325″,”term_id”:”118325″GSE118325 (H3K9me3), “type”:”entrez-geo”,”attrs”:”text”:”GSE73725″,”term_id”:”73725″GSE73725 (NANOG). Data for POU5F1 (R)-MG-132 and EP300 was instead obtained from the ENCODE database (https://www.encodeproject.org/). All plasmids, materials and data supporting the findings of this study are available from corresponding authors upon affordable request. The source data underlying Figs.?1b, 2a, b, 3a, c, 4a, 5d, e, 6c, 7a, b, 8c, e and Supplementary Figs.?1b, f, g, 2d, 3a, c, d, 4a, b, 5aCc, 6b, 7a-d, 8a, cCe are provided as a Source Data file. Abstract Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for growth of standard hPSCs as smooth epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have obvious implications for the generation of bona fide hPSCs for regenerative medicine. test. Source data are provided as a Source Data file. c Approach used to identify potential SMAD3 direct targets. See also Supplementary Fig.?1e. d Top: Transcriptome analysis of hESCs treated with SB43 for 48?h (microarray data from ref. 10). Dark grey dots show differentially expressed genes (DEGs) for ?1? ?Log2 fold-change? ?1 and and test relative to Empty SB43 samples. Right: Representative images of clonal assay performed in KiPS. Observe (R)-MG-132 also Supplementary Fig.?3a for results obtained in H9 hESCs. Level bars 500?m. Source data are provided as a Source Data file. b Morphology of HES2 colonies stably expressing an empty vector (Empty) in presence of DMSO or SB43 and HES2 stably expressing the eight SMAD3 targets in presence of SB43. Representative images of three impartial experiments are shown. (R)-MG-132 Observe also Supplementary Fig.?3b PLAT for results obtained in H9. Level bars 200?m. c Gene expression analysis by qPCR of HES2 (light green bars) and KiPS (dark green bars) stably expressing an Empty vector or the eight SMAD3 targets and treated with or without SB43 for 5 days. Bars show mean??SEM of indie experiments, shown as dots (test. Source data are provided as a Source Data file. Open in a separate windows Fig. 4 A quartet of transcriptional regulators maintain pluripotency.a Left: immunostaining for the pluripotency markers NANOG and POU5F1/OCT4 of KiPS stably expressing an empty vector control (Empty) in presence of DMSO or SB43 and KiPS stably expressing NANOG, KLF7, MYC or ZNF398 in presence of SB43 for 5 days. Representative images of three impartial experiments are shown. Right: Violin plots showing fluorescence intensity quantification of NANOG and OCT4. For each condition, at least 1200 nuclei from five randomly selected fields were analysed. Box plot indicates 25th, 50th and 75th percentile; whiskers show minimum and maximum. Scale bars 20?m. Observe also Supplementary Fig.?3c for results obtained in H9. Source data are provided as a Source Data file. b Diagrams showing an extended set of pluripotency regulators. Gene expression analysis by RNA-seq of KiPS stably expressing an empty vector, NANOG, KLF7, MYC or ZNF398 and treated with SB43 for 5 days. Colours show the fold-change relative to Empty DMSO sample, thus yellow indicates the endogenous expression of a given gene in undifferentiated hPSCs. c Box plot showing complete expression levels (normalised counts, TPM) of 538 genes DOWN-regulated by SB43 treatment (5 days) in KiPS stably expressing an empty vector (observe Fig.?5a, blue dots). Shown data refers to KiPS transfected with the vacant vector in the presence of DMSO or SB43 (test. Source data are provided as a Source Data file. Murine epiblast stem cells (EpiSCs) are primed pluripotent cells derived from (R)-MG-132 the post-implantation epiblast35,36. EpiSCs share several molecular features with primed hPSCs37, including the requirement of TGF-beta for.