Fig 2: Sth-independent Thpok repression by Bcl11b in pre-selection DP thymocytes. a Quantitative RT-PCR showing amount of promoter-specific Thpok transcripts in splenic CD4+ T cells and splenic B220+ B lymphocytes. Summary of three measurements. b Quantitative RT-PCR for Thpok-gfp expression in CD4+ T cells from mutant reporter alleles, Thpok gfp:?TE and Thpok gfp:?PE, lacking the thymic enhancer (TE) or proximal enhancer (PE), respectively. Levels of P1- and P2 promoter-specific transcripts relative to those from the control Thpok gfp allele harboring intact enhancers is shown. One representative of two independent experiments. c Promoter-specific Thpok transcripts in Bcl11b m/m pre-selection DP thymocytes and CD4+ T cells relative to those in control CD4+ T cells. Summary of three measurements. d Histograms showing Thpok-GFP expression from the Thpok gfp:?TESPE allele in the indicated cell subsets differentiated in Rag1-deficient recipients from control (dotted line) and Bcl11b m/m (solid line) fetal liver cells. Expression pattern of Thpok gfp is shown as reference (shaded). Schematic structure of the Thpok gfp:?TESPE allele is included (upper panel). One representative of two independent experiments. e ATAC-seq tracks at the Thpok locus in CD24hiTCRßloCD4+CD8+ pre-selection thymocytes of 6-week-old Bcl11b +/+ mice, newborn Bcl11b +/fl :Lck-cre, newborn Bcl11b HM/fl :Lck-cre and newborn Bcl11b HM/HM :Lck-cre mice. ATAC-seq results in immature B cells (GSM2123187) is shown as reference. DP thymocyte- and B lymphocyte-specific peaks are marked with blue and red arrowheads, respectively. Position of the P1 promoter is indicated with black arrowhead

Fig 3: Transcriptional profile of Bcl11b deletion is highly correlated with HD mouse models and postmortem human HD tissue. (A) Gene perturbation enrichment analysis. (B) Disease perturbation enrichment analysis. (C) Drug perturbation enrichment analysis.

Fig 4: Large overlap between Bcl11b- and Foxp3-occupied sites in Treg cells.ChIP-seq was performed for Bcl11b on sorted CD4+Foxp3+ Treg (Tr) and CD4+Foxp3-CD62lhi T naïve (Tn) cells. (A) Overview of the percentage of Bcl11b-dependent Up or Down genes bound by Bcl11b within ±20 kb of their transcription start sites (TSSs). (B) Genome-wide occupancy of Bcl11b in Tr and Tn cells. Each column depicts Bcl11b binding in Tr or Tn cells within a window ±3 kb centered around Bcl11b-bound sites (indicated as a yellow triangle). Normalized enrichments of indicated Bcl11b-bound peak sets are shown on the right as histogram plots. (C) Representative tracks, as viewed in Integrative Genome Viewer (IGV), of Bcl11b ChIP-seq reads aligned to the corresponding gene loci. Y scale is normalized tag intensity (reads per million mapped reads). Red arrows point toward peaks in each category that are otherwise not obvious. (D) Plots depicting enrichment of Tn/Tr-common (top) or Tr-specific (bottom) sites within a given distance to promoter of randomly selected genes, or genes that are down-regulated or up-regulated in Bcl11b-deficient Treg cells. Wilcoxon rank-sum test P values are shown. (E) CDF analysis of gene expression changes between Bcl11b-deficient versus Bcl11b-sufficient Treg cells for gene subsets that “exclusively” contain Tr-specific Bcl11b-specific sites. Of note, there was a subcategory of genes prebound by Bcl11b in Tn and gained new Bcl11b peaks at different site(s) in Tr. While these genes are also categorized as Tr-specific site–containing genes, they are not included in this analysis. Two-tailed Kolmogorov-Smirnov test P value is shown. (F) Genome-wide occupancy of Bcl11b and Foxp3 in Treg cells. Each column depicts Bcl11b or Foxp3 binding within a window ±3 kb centered on peaks identified by ChIP-seq analyses (indicated as a yellow triangle). Normalized enrichments of indicated Bcl11b- and Foxp3-bound peak sets are shown on the right as histogram plots. (G) Examples of Bcl11b- and Foxp3-bound peaks representing common sites (Bcl11b-Foxp3-Overlapping) or sites where Bcl11b binds in the absence of Foxp3 (Bcl11b-specific). (H) Plots depicting enrichment of Bcl11b-specific (top) or Bcl11b-Foxp3-Overlapping (bottom) sites within a given distance to promoter of randomly selected genes, or genes that are down-regulated or up-regulated in Treg cells in the absence of Bcl11b. (I) Normalized enrichment of indicated chromatin marks around Bcl11b binding sites within ±20 kb of TSS for the gene subsets analyzed. The blue line represents genes with Bcl11b-Foxp3-Overlapping sites that are Tr-specific for Bcl11b. The red line represents genes that are only bound by Bcl11b in Treg cells. (J) CDF analysis of gene expression changes between Bcl11b-deficient versus Bcl11b-sufficient Treg cells for indicated gene subsets. Genes that are bound by both Bcl11b and Foxp3, but at two different sites (nonoverlapping), are excluded from this analysis. Two-tailed Kolmogorov-Smirnov test P values are shown.

Fig 5: Sequence of intermediates during reprogramming and transformation.a, Representation of Bcl11b and Thy1 expression in single cells. The thresholds were as follows: Bcl11b < 1 and Thy1 < 2 for BLTL; Bcl11b > 2 and Thy1 < 2 for BHTL; Bcl11b < 1 and Thy1 > 4 for BLTH; and Bcl11b > 2 and Thy1 > 4 for BHTH. b, Expression of Bcl11b-tdTomato and Thy1 during reprogramming and transformation. KI: knock-in. c, Left, pictures of iPS colonies from a representative experiment. Right, quantification of AP+ colonies (n = 6 independent experiments). d, Left, pictures of foci assays from a representative experiment. Right, foci quantification (n = 5 independent experiments). e, Emergence of Bcl11b-tdTomatolow/Thy1low cells. The graph represents the distribution of BHTH, BLTH, BHTL and BLTL cells. IWP2, Wnt inhibitor; OSKM, Oct4, Sox2, Klf4, c-Myc; SNEL, Sall4, Nanog, Esrrb, Lin28. BHTH cells were FACS sorted before reprogramming/transformation. f, Emergence of BLTL cells from mouse adult ear fibroblasts. The settings were similar to those for e. g, Left, pictures of iPS colonies following reprogramming induced by OSK + IWP2, taken from a representative experiment. Right, quantification of AP+ colonies (n = 2 independent experiments). h, Left, pictures of iPS colonies from mouse adult ear fibroblasts, taken from of a representative experiment. Right, quantification of AP+ colonies (n = 3 independent experiments). i, Schematic of the experimental design. j, Left, brightfield images of tumours in a chick (as indicated by the dashed lines). Right, quantification of the tumours (n = 23 for T-BLTL cells; n = 27 for T-BHTH cells). k, Left, tumour growth curves. Right, survival curves of the mice (n = 6 animals per group). l, Left, FACS profiles. Cells harboring various levels of Bcl11b and Thy1 were FACS sorted at day 5 of reprogramming, plated back in culture and analyzed 2 days later. Right, quantification of AP+ colonies (n = 5 independent experiments). m, Left, FACS profiles. Cells harboring various levels of Bcl11b and Thy1 were FACS sorted at day 5 of transformation, plated back in culture and analyzed 2 days later. Right, quantification of foci (n = 6 independent experiments). n, Schematic of the sequence of intermediates. The corresponding efficiencies are indicated using arbitrary units. In h, j and the left panel of k, the data represent means ± s.d. Statistical significance was determined by two-tailed Student’s t-test (c, d, h and j), two-way ANOVA combined with Šidák’s multiple comparisons test (left panel in k), Gehan–Breslow–Wilcoxon test (right panel in k) or one-way ANOVA followed by Tukey’s post-hoc test (l and m).Source data