Fig 2: Exploring molecular determinants of transcriptional bursting.(A to C) Kinetic properties of transcriptional bursting of genes either with or without a TATA box. (D) Schematic representation of calculating reads per million (RPM) at the promoter and gene body from ChIP-seq data. In addition, similar calculations were also performed for enhancers (see Materials and Methods). (E) Heat maps of Spearman’s rank correlation between promoter-, gene body–, or enhancer-associated factors and either normalized intrinsic noise (N. int. noise), burst size, or burst frequency (burst freq.). (F) Effect of the Pol II pause release inhibitor, DRB, and flavopiridol treatment on the kinetic properties of transcriptional bursting. ?normalized intrinsic noise, ?burst size, and ?burst frequency are residuals of normalized intrinsic noise, burst size, and frequency of inhibitor-treated cells from that of control cells, respectively. Error bars indicate 95% confidence interval. (G) Effect of Suz12 K/O on normalized intrinsic noise. Suz12 K/O cell lines derived from Dnmt3l, Dnmt3b, Peg3, and Ctcf KI cell lines were established. Upper panel represents the result of Western blotting. In the lower part of the panel, the ?normalized intrinsic noise, ?burst size, and ?burst frequency compared with the control (cont1) are shown. Error bars indicate 95% confidence interval. Asterisks indicate significance at P < 0.05.

Fig 3: CRISPR library screening of genes involved in intrinsic noise regulation.(A) Schematic diagram of CRISPR lentivirus library screening. Screening was performed independently for each of the three (Nanog, Trim28, and Dnmt3l) KI cell lines. (B) Ranked differentially expressed (DE) score plots obtained by performing CRISPR screening on three cell lines. The higher the DE score, the more the effect of enhancing intrinsic noise. (C) KEGG pathway enrichment analysis. KEGG pathway enrichment analysis was performed using clusterProfiler (see Materials and Methods), with the upper or lower 100 genes of DE score obtained from the CRISPR screening (referred as posi and nega, respectively). The pathways shown in red indicate hits in multiple groups of genes. Genes corresponding to these pathways are labeled in (B). (D) Simplified diagram of MAPK, Akt, and mTOR signaling pathways. These pathways are included in the pathways highlighted in red in (C) and cross-talk with each other. (E) Western blot of cells treated with signal pathway inhibitors. (F) ?normalized intrinsic noise of cells treated with signal pathway inhibitors against control [dimethyl sulfoxide (DMSO)–treated] cells. Error bars indicate 95% confidence interval. (G) Twenty-four KI cell lines were conditioned to 2i or PD-MK conditions and subjected to flow cytometry analysis. ?normalized intrinsic noise, ?burst size, and ?burst frequency against control (DMSO-treated) cells are shown. Error bars indicate 95% confidence interval.

Fig 4: Chemical reversion of conventional primed hPSC with a tankyrase/PARP inhibitor promotes stable rewiring to a human preimplantation epiblast-like state with intact epigenomic imprints.The TIRN method is a two-step culture system109,111 comprised of one brief adaptation step of conventional hPSC with LIF and five small molecules (LIF-5i) that includes XAV939 (tankyrase/PARP inhibition), CHiR99021 (GSK3ß inhibition), PD0325901 (MEK inhibition), forskolin (adenylate cyclase activation), and purmorphamine (Hedgehog signaling activation); followed by continuous, stable culture in only LIF/XAV939/CHIR99021/PD0325901 (LIF-3i) for at least 30 passages. This tankyrase/PARP inhibitor-mediated modification of the classical LIF-2i method has been validated to stably revert over 30 hPSC lines, and is independent of genetic donor background109,111. a (Left panels) Three representative hPSC lines109,110 (RUES1 hESC, cord blood (CB)-hiPSC 6.2, and fibroblast (fib)-hiPSC C.2 are shown in their starting primed (i.e., E8 medium; PRIMED) conditions, and 6–10 passages post culture in continuous TIRN-reverted (NAÏVE) conditions109,111. Monolayer bFGF-dependent hPSC colonies in primed conditions became tolerant to bulk single-cell passaging and acquired a typical dome-shape morphology following TIRN reversion. (Middle panels) TIRN-hPSC retained strong expression of TRA-1–81 and SSEA4 surface antigens by flow cytometry. (Right panels) Western blot analyses in these three primed (P) and TIRN (N) lines demonstrated that TIRN-hPSC acquired de novo expression of phosphorylated STAT3 and reduced ERK1/2 phosphorylation. b TIRN-hPSC uniformly shifted their transcriptomes toward a pre-implantation naïve epiblast identity. Mean expression ratios of naïve epiblast-specific genes from Illumina gene arrays from isogenic primed-to-naïve-reverted pairs of genetically independent hPSC (n = 9 lines)109. c Western blot analysis of primed (P) and TIRN (N) CB-hiPSC (E5C3). TIRN-CB-hiPSC markedly upregulated naïve epiblast-specific DNMT3L whilst maintaining DNMT1 protein expressions following TIRN reversion109,110. d Infinium CpG methylation heatmaps of genomic Imprinted promoter regions of a panel of primed and TIRN-reverted isogenic hPSC samples revealed that TIRN-hPSC retains normal somatic epigenomic imprint configurations, similar to their isogenic primed states. Heatmaps compare previously published Zambidis lab109 methylation data alongside published Court et al.121 data of identical imprinted genomic regulatory regions as controls. Zambidis lab methylation beta values were subset to exact imprinted regions provided by Court et al.121 (methylation beta values: 0—completely hypomethylated probe; 1—completely methylated probe). Matching Infinium probes are sorted by chromosomal location and arranged into their adjacent primed (-) and naïve (+) hPSC isogenic pairs. The heatmaps of the same imprinted regions from Court et al.121 include abnormal androgenetic hydatidiform mole, normal human tissues (e.g., brain, muscle, placenta, and sperm), and primed hESC lines.

Fig 5: RHOX10 drives ProSG differentiation in vitro and in vivo(A) Luciferase analysis of constructs harboring promoters from the indicated genes ligated upstream of the Firefly luciferase gene. All promoter constructs contain between 1 to 2 kb of sequence upstream of the TSS (see Table S3). These reporter constructs were transiently co-transfected with an Rhox10-expression vector into GC1 cells, which only express trace levels of endogenous Rhox10. Data are represented as mean ± SD (n = 4). Staistical significance was determined using the two-tailed unpaired Student’s t test. *p < 0.05.(B) Left, IF analysis of testis sections from P3 Rhox10-null (KO) or control (WT) mice co-stained with antisera against DNMT3L and GCNA1, which mark T1-ProSG and all germ cells, respectively. Cell nuclei were stained with DAPI (blue). Scale bar, 30 µm. Right, quantification of the percentage of all germ cells (GCNA1+ cells) that express either high (Bright), low (Dim), or no detectable (Neg) DNMT3L. The analysis was done blind to genotype. Statistical significance was determined using the chi-square test. Data are represented as mean ± SD (n = 3). *p < 0.05.(C) Left, IF analysis of testis sections from P3 Rhox10-null (KO) or control (WT) mice co-stained with antisera against ETV4 and GCNA1, which stain emergent SSCs and all germ cells, respectively. Cell nuclei were stained with DAPI (blue). Scale bar, 30 µm. Right, quantification of ETV4-positive germ cells, determined as in (B). Data representation and statistical significance (n = 3) as in (B).(D) Quantification of germ cells with the indicated levels of DNMT3L-signal intensity in primary Rhox10-null and control testicular cell cultures, analyzed as in (B), at the indicated time points after initiation of culture. Data representation and statistical significance (n = 3) as in (B).See also Figure S4.