Fig 2: Bcl11b function requires its C-terminal zinc-finger motif. a Flow cytometry analyzing the expression of CD4, CD8, Thpok-GFP, and Runx3-tdTomato in peripheral T cells from Bcl11b +/+ :Thpok-Cre and Bcl1b fl/fl :Thpok-Cre mice harboring Thpok gfp and Runx3 tdTomato reporter genes. Inactivation of Bcl11b during the maturation of CD4-lineage cells by a Thpok-Cre transgene resulted in impaired Thpok-GFP expression, as well as Runx3-tdTomato de-repression. b RT-qPCR analyses showing relative amounts of Thpok (black bars) and P1-Runx3 (white bars) mRNA in splenic CD4+CD8- T cells of Bcl11b +/+ :Thpok-Cre and Bcl11b fl/fl :Thpok-Cre mice. Results in splenic CD4-CD8+ T cells of Bcl11b +/+:Thpok-Cre are shown as references. Data were summary of three experiments. Means ± SD *P < 0.05 (unpaired t-test). c Histograms showing Thpok-GFP expression from CD4+ and CD8+ T cells of Bcl11b +/+ :Thpok +/gfp (white bars) and Bcl11b fl/fl :Thpok +/gfp (gray bars) mice 4 days after retroviral Cre transduction. One representative of three experiments. d Histograms showing Runx3-tdTomato expression 3 days after retroviral transduction of Wt and hypomorphic (HM) Bcl11b into peripheral CD4+CD8- Runx3-tdTomato + cells from Bcl1b fl/fl :Runx3 +/tdTomato :Thpok-Cre mice. Expression of Runx3-tdTomato in activated control CD4+ T cells (dotted line) after transduction with an empty vector (open) are shown as controls. e Histograms showing Runx3-tdTomato expression after retroviral transduction of mutant Bcl11b proteins lacking the C-terminal zinc-finger motif are shown as in d. The dotted line represents Runx3-tdTomato expression after transduction of Wt Bcl11b. One representative of at least two experiments d, e

Fig 3: KAP1 inhibits Tat activity and cooperates with CTIP2 for this purpose (A–D) Microglial cells were transfected with LTR-Luc vector under the indicated conditions. After 48 h of transfection, the luciferase activities were measured after cell lysis and normalized under conditions where LTR-Luc vector were transfected alone. A t-test was performed on 3 independent experiments P (*P < 0.05, **P < 0.01, ***P < 0.001). (C,D) Over-expression and knockdown of the indicated proteins were validated by Western blot. Full-length blots are presented in Supplementary Figure 5.

Fig 4: 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 5: Roles of Bcl11b in ThPOK-mediated repression of Runx3. a Runx and Bcl11b ChIP-seq tracks at the Runx3 locus using total thymocytes, along with a ThPOK ChIP-chip track using CD4 SP thymocytes. Schematic structure of mutant Runx3 tdTomato reporter alleles harboring several types of deletion (bottom panel, left). Histograms (bottom panel, right) showing Runx3-tdTomato expression in CD4-CD8+ thymocytes from indicated mutant alleles (solid line) and non-transgenic allele (dotted line). Data shown are representative of at least three independent mice. b Amount of Socs1 and Socs3 mRNA in CD4 SP thymocytes and CD4+ T cells from mice of indicated genotypes. Summary of three independent experiments. Mean ± SD **P < 0.01 (unpaired t-test). c ChIP-qPCR analyzing ThPOK binding to the Sth in Thpok and the -39E and -21E regions in the Runx3 using chromatin from CD4+ T cells of Wt (white bars) and Bcl11b f/f :Cd4-Cre (gray bars) mice. Combined data of three independent ChIP experiments. Means ± SD. d Flow cytometry analyzing the effect of a Thpok transgene on Cd8 and Runx3 expression. Dot plots showing CD4/CD8 expression in splenic T cells. Histograms at the right show Runx3 tdTomato reporter expression in Wt (white) and Bcl11b-deficient (gray) CD4+CD8- subset. One representative of three individual mice. e Histograms showing CD8 and Runx3-tdTomato expression in control CD4-CD8+, Bcl11b-deficient CD4-CD8+ and CD4+CD8- T cells after retroviral transduction of Wt (open) and the non-functional HD mutant ThPOK (shaded). One representative of two independent experiments