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Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16

Nature Immunology volume 19, pages 1427–1440 (2018)Cite this article

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Abstract

Multipotent progenitor cells confirm their T cell–lineage identity in the CD4–CD8– double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.

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Fig. 1: Cellular and molecular phenotypes of in vivo deletion of Bcl11b by Vav1-iCre or Lck-Cre.
Fig. 2: Identification of Bcl11b interacting molecules in early T cells.
Fig. 3: Identification of Bcl11b-dependent cofactor peaks in DN3 cells.
Fig. 4: Bcl11b-dependent cofactor peaks around the major Bcl11b target genes.
Fig. 5: Effect of cofactor deletion on the expression of Bcl11b target genes.
Fig. 6: Id2 is involved in establishment of phenotypes of Bcl11b-deficient cells.
Fig. 7: Id2 and Zbtb16 serve key roles in the Bcl11b-mediated exclusion of alternative fates.

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Data availability

Additional data that support the findings of this study are available from the corresponding author upon request. In addition to the complete description and explanation of the methods presented here, reagent lists and some general methods are also repeated, along with statistical checklists, in the Nature Research Reporting Summary that accompanies this paper. The GEO accession codes for all the deep-sequencing data reported in this paper are GSE110305, GSE110882 and GSE115744.

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Acknowledgements

We thank D. Perez, J. Tijerina, and R. Diamond for cell sorting and advice; I. Soto for mouse colony care; V. Kumar for library preparation and sequencing; H. Amrhein and D. Trout for computational assistance; I. Antoshechkin for sequencing management; X.Wang for related exploratory experiments; and members of the Rothenberg group for discussions and reagents. Supported by the Manpei Suzuki Diabetes Foundation (H.H.), the US Public Health Service (R01AI083514 and R01HD076915 to E.V.R.), Grants-in-Aid for Advanced Research and Development Programs for Medical Innovation (T.T.), the Takeda Science Foundation (T.T.), the SENSHINE Medical Research Foundation (T.T.), the Swedish Research Council (J.U.), the California Institute of Regenerative Medicine Bridges to Stem Cell Research Program (Pasadena City College and Caltech; M.R.-W.), the L. A. Garfinkle Memorial Laboratory Fund and the Al Sherman Foundation, special project funds from the Provost and Division of Biology & Biological Engineering of Caltech, and the Albert Billings Ruddock Professorship (E.V.R.). This work was performed in part in the Collaborative Research Project Program of the Medical Institute of Bioregulation, Kyushu University.

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  1. These authors contributed equally: Hiroyuki Hosokawa, Maile Romero-Wolf.

Authors and Affiliations

  1. Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Hiroyuki Hosokawa, Maile Romero-Wolf, Mary A. Yui, Jonas Ungerbäck, Maria L. G. Quiloan & Ellen V. Rothenberg

  2. Division of Molecular Hematology, Lund University, Lund, Sweden

    Jonas Ungerbäck

  3. Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan

    Masaki Matsumoto & Keiichi I. Nakayama

  4. Department of Molecular Diagnosis, Chiba University, Chuo-ku, Chiba, Japan

    Tomoaki Tanaka

  5. AMED-CREST, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan

    Tomoaki Tanaka

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Contributions

H.H. designed the study, performed experiments, analyzed data and wrote the manuscript; M.R.-W. performed experiments, analyzed data and wrote the manuscript; M.A.Y., J.U., M.L.G.Q., M.M., K.I.N., T.T. performed experiments, analyzed data and provided discussions; and E.V.R. designed and supervised the study, analyzed data and wrote the manuscript.

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Correspondence to Ellen V. Rothenberg.

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Integrated supplementary information

Supplementary Figure 1 Characterization of Bcl11b deletion systems in vivo.

(a), Phenotypic timing of the activity of Lck-Cre (Lck-Crecw) in vivo based on activation of ROSA26R-YFP. For each Bcl11b genotype characterized in (a) and (b), results are representative of the following numbers of animals: WT, N=5; HE, N=6; KO, N=6. (b), Most CD25+ thymocytes in mice with Bcl11b deleted by Lck-Cre have the c-Kithi+ phenotype of normal DN2a cells but the YFP+CD44low phenotype similar to normal DN2b/DN3a cells. (c), (Left) Bcl11b deletion in vivo severely reduces thymocyte cellularity whether deleted before commitment by Vav1-iCre or after commitment by Lck-Cre. WT, HE, and KO refer to Bcl11b genotypes in the indicated Cre backgrounds: Bcl11b+/+, Bcl11b+/fl, and Bcl11bfl/fl, respectively. Each symbol represents a different animal and the short bars indicate geometric means. N of individual animals counted for each genotype: Vav1-iCre WT N=3; Vav1-iCre HE N=10; Vav1-iCre KO N=12; Lck-Cre WT N=8; Lck-Cre HE N=5; Lck-Cre KO N=7. (Right) Representative photographs of thymuses from age-matched Lck-Cre;ROSA26R-YFP mice with Bcl11b+/+ (WT), Bcl11b fl/+ (HE), and Bcl11bfl/fl (KO) genotypes at the Bcl11b locus.

Supplementary Figure 2 Representative RNA-seq tracks showing Bcl11b deletion effects.

Bcl11b, Id2, Zbtb16, and Cd6 transcript levels are shown as determined by RNA-seq in the thymocyte samples used for the heat maps in Fig. 1b-e. All samples shown were from Lin- (DN) CD25+ cells that were positive for expression of the ROSA26R-YFP Cre reporter. Gene models (Refseq) are shown in schematics at the top. Directions of transcription are from right to left. For each gene shown, all RNA-seq tracks have y-axis adjusted to the same scale in reads/million. The deletion of the coding region in exon 4 of Bcl11b leads to read-though transcription and splicing to a cryptic downstream exon in these samples too, as reported previously7.

Supplementary Figure 3 Identification of consensus sets of Bcl11b target genes.

(a), Experimental scheme is shown. BM-derived precursors from Cas9-Bcl2-tg mice were cultured on OP9-DL1 for 7 days, then they were infected with sgRNA. Seven days after infection, CD25+ sgRNA transduced cells were purified and subjected to RNA-seq analysis. (b), Overlaps between gene sets differentially expressed when Bcl11b is disrupted by Lck-Cre or Vav1-iCre in vivo, compared with those differentially expressed when Bcl11b is deleted acutely by Cas9 and sgRNA in bone marrow cell precursors in vitro. See Supplementary Table 1 and 2 for rpkm values and gene lists. Conditions for deletion by Cas9 are described in detail below (Fig. 5) and in Methods, and rpkm values are given in Supplementary Table 3. (c), Overlaps between gene sets differentially expressed by Bcl11b deletion in the present study with gene sets previously reported to be differentially expressed by Bcl11b deletion in fetal liver-derived precursors differentiating in vitro7. DEGs affected by Lck-Cre deletion in vivo, Vav1-iCre deletion in vivo, and Cas9-mediated deletion in vitro in the present study (“consensus set”) are compared with those previously reported using retroviral Cre transduction to delete Bcl11b in fetal liver precursors (“Gold standard” minimal sets of responding genes in Longabaugh et al.7). DEG numbers in the earlier study are lower because fetal liver-derived precursors differentiate faster and the conditions used gave less consistent differentiation extents, reducing statistical significance. The consensus set includes a much higher fraction of the previously reported targets than those genes affected by Cas9-mediated deletion in vitro but not affected in vivo (a control for cell culture effects). Note that agreement among Bcl11b-repressed DEGs in all four sample types is higher than agreement among Bcl11b-dependent DEGs.

Supplementary Figure 4 Functional characteristics of Bcl11b binding in pro-T cells.

(a), Transcriptional responses to Bcl11b perturbation of genes directly bound by Bcl11b in pro-T cells. Bcl11b-deficient CD25+ cells prepared as shown in Fig. 5a were subjected to RNA-seq analysis and differentially expressed genes (DEGs) and controls were assessed for local Bcl11b binding by ChIP-seq. Pie charts show frequency of genes bound by Bcl11b among Bcl11b-repressed genes (left), Bcl11b -dependent genes (middle), and non-DEGs (right). (b), Bcl11b peaks at CpG islands preferentially linked to non-DEGs in Bcl11b-deficient cells. The numbers of genes having Bcl11b peaks at CpG islands, non-CpG islands or both Bcl11b peaks at CpG and non-CpG islands in DEGs and non-DEGs are shown. P value is determined by two-sided Fisher’s exact test. (c), Tamoxifen-induced deletion of the Bcl11b gene in pro-T cells. Experimental scheme. BM-derived LSK cells from Bcl11bfl/fl;Cre-ERT2 mice were cultured on OP9-DL1 cells for 7 days. Then, they were treated with 4-OHT (120 nM) for 2 days and cultured an additional 5 days. (d), Flow cytometry analysis of BM-derived precursors after 7 days of OP9-DL1 culture (before 4-OHT treatment) are shown. (e), Flow cytometric analysis of BM-derived precursors after 14 days of OP9-DL1 culture are shown. (f), Reproducibility of Bcl11b ChIP peaks. Venn diagram shows the numbers of Bcl11b ChIP peaks in this study (Fig. 3b) and previously published data7. (g), Nuclear lysates from BM-derived WT and Bcl11b-deficient cells described in (Fig. 3a) were subjected to immunoblotting against Bcl11b, Mta2, and LaminB. Sections of western blots around the indicated size markers are shown. Data are based on reproducible ChIP-seq peaks in two replicate samples and four replicates of RNA-seq results (a, b, f,), or are representative of two (g) or four (d, e) independent experiments.

Supplementary Figure 5 Motif analysis of Bcl11b-dependent co-factor peaks.

(a), Top three enriched sequence motifs of Bcl11b peaks in DN3 cells (Fig. 3b) are shown. (b), Top three enriched sequence motifs of Bcl11b-dependent co-factor peaks in DN3 cells (Fig.3c) are shown. (c), Top three enriched sequence motifs of new co-factor peaks in Bcl11b-deficient cells (Fig. 3c, 4) are shown. Note that most of these new peaks do not coincide with sites that Bcl11b normally occupies in wildtype cells: see Mta2, Rest, LSD1 and Runx1 Venn diagrams in Fig. 3c. (d), Venn diagrams show the numbers of ChIP peaks in each category overlapping with Bcl11b peaks at CpG islands. Data are based on reproducible ChIP-seq peaks in two independent replicate samples.

Supplementary Figure 6 Bcl11b-dependent cofactor peaks around major Bcl11b target genes.

(a-d) Similar to the binding data from primary DN3 cells shown in Fig. 4a-d, this figure shows supporting data from Scid.adh.2c2 cells for binding of cofactors (Chd4, Hdac2, Mta2 and Rest) in control (shCont.) and Bcl11b-knockdown cells. Browser tracks show the Id2 (a), Zbtb16 (b), Tnni1 (c) and Cd163l1 (d) loci. Sites of Bcl11b-dependent cofactor peaks in Bcl11b-knockdown cells are labeled with magenta rectangles. This cell-line experiment was performed once to add corroboration to the primary-cell data in Fig. 4. (e, f), The binding patterns of Bcl11b and cofactors at the Cd3 and Cd6 loci in WT and Bcl11b KO DN3 cells with H3K27Ac ChIP-seq and RNA-seq tracks are shown. Bcl11b-dependent Runx1 peaks were labeled with magenta rectangles. Data are representative of two independent experiments.

Supplementary Figure 7 Candidate Bcl11b-dependent silencer regions for the Id2 and Tnni1 loci.

(a), The binding patterns of Bcl11b and cofactors at the Id2 locus in WT and Bcl11b-deficient DN3 cells are shown. Bcl11b-dependent cofactor peaks are labeled with magenta or light blue rectangles. We chose the two regions most clearly occupied by Bcl11b-dependent cofactor assemblies (+40k and −600k, magenta rectangles) for further analysis. (b, c), The genomic regions bound by Bcl11b and its cofactors can act as silencers for Id2 and Tnni1. Scid.adh.2c2 cells were first infected with Cas9-GFP and GFP+ cells were expanded. They were then transduced with sgRNA-CFP and sgRNA-hNFGR retroviruses, with the two sgRNA vector pools designed respectively against sequences on the two-sided of the targeted silencer regions. After two days of culture, CFP+ hNGFR+ infected cells were isolated by single cell sorting, and individual clones were expanded for two weeks. Genomic DNA from each clone was then isolated followed by quantitative PCR analysis for targeted genomic regions at the Id2 (b) (magenta rectangles in (a)) and Tnni1 (c) (magenta rectangles in Fig. 4c) loci. Each clone was also subjected to RT-qPCR analysis for transcripts from Id2 (b) and Tnni1 (c). The relative expression (/Actb) is shown. *P<0.05, **P<0.01 by two-sided Student’s t-test. ND, not detected. Data are representative of two independent experiments (a) or show individual values and averages of three biological replicates with mean +s.d. (b, c).

Supplementary Figure 8 Acute Cas9-dependent deletion of Bcl11b and its cofactors.

(a), Cas9-GFP introduced Scid.adh.2c2 cells were infected with sgRNA-CFP retroviruses. Two days after infection, nuclear lysates of the cells were prepared and subjected to IB with anti-Bcl11b, anti-Chd4, anti-Mta2, anti-Rest, anti-Ring1b, anti-LSD1 and anti-LaminB Abs. (b), Flow cytometry analysis of BM-derived precursors after 7 days of OP9-DL1 culture (before retrovirus infection) is shown. (c), Flow cytometric analysis of sgRNA transduced BM-derived pro-T cells after a total of 14 days of OP9-DL1 culture (7 d after transduction) is shown. Note the high percentage of sgRNA-transduced CFP+ cells. (d), Efficient disruption of Bcl11b cofactors by CRISPR-Cas9 system in primary DN cells. RNA-seq tracks for the cofactor loci are shown. Red arrowheads and dotted lines show sites against which sgRNA was designed. Data are representative of two independent experiments (a, d) or three independent experiments (b, c).

Supplementary Figure 9 Bcl11b-dependent Runx1 recruitment distinguishes the Tcrb and Tcrg genetic loci in primary pro-T cells.

The binding patterns of Runx1 and Bcl11b at the Tcrb locus (a) and Tcrg loci (b) in WT and Bcl11b KO DN3 cells are shown, with RNA-seq tracks below. Bcl11b-dependent Runx1 peaks are labeled with magenta rectangles. Data are representative of two independent experiments.

Supplementary Figure 10 Upregulation of Zbtb16 in absence of Bcl11b depends on upregulation of Id2.

sgRNA transduced pro-T cells cells in Fig. 6a were purified and the levels of mRNA of Id2 and Zbtb16 were measured by RT-qPCR. The relative expression (/Actb) is shown. **P<0.01, *P<0.05 by two-sided Student’s t-test. Data are individual values and averages of three biological replicates with mean +s.d.

Supplementary Figure 11 Generation of NK1.1+ and CD11c+ cells from Bcl11b-deficient pro-T cells.

(a), Experimental scheme is shown. BM-derived precursors were cultured on OP9-DL1 for 7 days, then they were transduced with sgRNA. Three days after transduction, at day 10 overall, they were passaged onto OP9-DL1 (upper) or OP9-Mig (lower) and cultured for 4 more days before analysis. (b), Flow cytometric analyses of sgControl or sgBcl11b transduced pro- T cells were performed on day 14 (a). The percentages of Lin marker-positive cells among CFP+ sgRNA transduced cells are indicated. **P<0.01 by two-sided Student’s t-test. Note change of scale for OP9-Mig cocultured samples as compared to OP9-DL1 cocultured samples. (c), Comparison of NK1.1 and CD11c expression in Bcl11b-deficient pro-T cells as compared to mature NK and DC cells in spleen. Flow cytometric analyses of sgRNA transduced BM-derived pro-T cells after 10 days of OP9-DL1 culture and 4 days of OP9-Mig culture in (a) are shown, compared with staining of fresh splenocytes from B6 mice. The percentages of NK1.1+CD11c-, NK1.1+CD11c+ and NK1.1-CD11c+ cells in CFP+ sgRNA transduced cells are indicated. Note differences in NK1.1 and CD11c fluorescence intensities relative to splenocytes. (d), sgRNA transduced BM-derived precursors after 14 days of OP9-DL1 (upper) or OP9-Mig (lower) co-culture were subjected to flow cytometric analysis. Representative profiles of Forward Scatter (FSC) vs. Lin, NK1.1 or CD11c in CFP+ cells are shown with the percentages of cells in rectangles. (e), Absolute cell numbers of Lin+, NK1.1+ and CD11c+ in CFP+ sgRNA transduced cells in (d) are indicated. **P<0.01, *P<0.05 by two-sided Student’s t-test. Data are average of three biological replicates with mean +s.d. (b, e) or are representative of two (c) or three (d) independent experiments.

Supplementary Figure 12 Effects of Bcl11b target genes Id2 and Zbtb16 on lineage diversion are cell-autonomous.

(a), Experimental scheme is shown. BM-derived precursors were cultured on OP9-DL1 for 7 days, then they were infected with the indicated sgRNA in a CFP vector, the indicated sgRNA in an hNGFR vector, or with pMxs-GFP empty vector, separately. Three days after infection, they were re-combined, transferred to OP9-Mig and cultured 4 more days (left). Representative FCS/CFP, hNGFR and GFP profiles on day 14 are shown with the percentages of cells in the rectangles (right). (b) Bar graph summarizing flow cytometric analysis in Fig. 7b. Data shown are individual values and averages of three biological replicates with mean +s.d.

Supplementary information

Supplementary Figures 1-12

Reporting Summary

Supplementary Table 1

Differentially expressed genes in Vav1-iCre or Lck-Cre Bcl11b deleted primary thymocytes ex vivo. RNA-seq values (rpkm) are given for gene expression in sorted DN2 and DN3 thymocytes from the indicated genotypes. Columns B-E: Bcl11b WT (+Lck-Cre) DN2. Columns F-J: Bcl11b Heterozygous (+Lck-Cre) DN2. Columns K-O: Bcl11b WT (+Lck-Cre) DN3. Columns P-U: Bcl11b Heterozygous (+Lck-Cre) DN3. Columns V, W: Bcl11b WT (+Vav1-iCre) CD25+ (mostly DN3). Columns X-AA: Bcl11b Heterozygous (+Vav1-iCre) CD25+. Columns AB-AF: Bcl11b Homozygous knockout CD25+ (+Lck-Cre). Columns AG-AI: Bcl11b Homozygous knockout CD25+ (+Vav1-iCre). Genes shown were those differentially expressed in Bcl11b-KO primary cells as compared to the average of control (Bcl11b WT) DN2 samples, using EdgeR with criteria of FDR < 0.05, |log2FC|>1, and RPKM>1. These data are the same as represented in the heat maps of Fig. 1b,c, and UCSC genome browser tracks of these samples are also shown in Supplementary Fig. 2. Genes inferred to be Bcl11b-repressed (UpLck+Vav) and genes inferred to be Bcl11b-dependent (DownLck+Vav) are shown in separate worksheets.

Supplementary Table 2

Differentially expressed genes after Cas9-mediated disruption of Bcl11b gene in BM-derived DN cells differentiating in vitro. RNA-seq data from all genomic loci, P values, and adjusted P values are shown for gene expression from a total of four pairs of Bcl11b deleted and control samples, generated as shown in Supplementary Fig. 3. Two pairs of Bcl11b-deleted and control samples were from experiments comparing effects of Bcl11b deletion with those of deletion of cofactors (Fig. 5b), and two pairs were from experiments comparing single Bcl11b deletion with double knockout of Bcl11b together with Id2 or Zbtb16 (Fig. 6c). Genes inferred to be Bcl11b-repressed and genes inferred to be Bcl11b-dependent are shown in separate worksheets.

Supplementary Table 3

Differentially expressed genes defined in common by Vav1-iCre-mediated, Lck-Cre-mediated, and Cas9-mediated disruptions of Bcl11b in DN2/3 pro-T cells. Table worksheets show the overlaps between gene lists of significant DEGs from Supplementary Tables 1 and 2. Lists of genes that were significantly differentially expressed in consequence of Vav1-iCre deletion in vivo, in consequence of Lck-Cre deletion in vivo, and in consequence of Cas9-mediated acute deletion in vitro were compared. Bcl11b-repressed and Bcl11b-dependent genes are shown in separate worksheets. Overlaps were identified using an online Venn diagram maker from the Bioinformatics and Evolutionary Genomics program of the University of Ghent < http://bioinformatics.psb.ugent.be/webtools/Venn/>. Venn diagrams are shown in columns W-AA of each worksheet. These lists also form the membership of the Venn diagrams shown in Supplementary Fig. 3b.

Supplementary Table 4

Representation of a novel Bcl11b binding motif in Bcl11b occupancy sites and in Bcl11b-dependent cofactor peaks of primary pro-T cells. Table presents frequency of the Bcl11b motifs newly defined by Liu et al.37 in Bcl11b peaks and Bcl11b-dependent cofactor recruitment peaks, with threshold match criteria calculated as described in Methods. Frequencies of sequences with scorable matches to these motifs at the indicated classes of sites are compared among all peaks, peaks linked to DEGs, and peaks linked to non-DEGs (Figs. 3c, 4e,f; Supplementary Figs. 4a, 5).

Supplementary Table 5

Bcl11b-interacting molecules in Scid.adh.2c2 cells identified by LC-MS/MS: protein names and Mascot scores. Purified Bcl11b complexes from Scid.adh.2C2 cells (Fig. 2b) were subjected to LC-MS/MS analysis. The resulting data set was analyzed using the Mascot software program (Matrix Science, http://www.matrixscience.com/). The table worksheet shows all of the Bcl11b-interacting proteins detected by LC-MS/MS analysis with Mascot scores >100. Bcl11b: magenta highlight. “Co-repressor complex” components: yellow highlight. BAF complex component: green highlight.

Supplementary Table 6

Effects of cofactor deletion on expression of genes that were differentially expressed in Bcl11b-deficient DN cells and directly bound by Bcl11b in WT cells. Table shows expression changes in Bcl11b-regulated genes (p.adj < 0.05) as they are affected by acute deletion of each of the indicated cofactors. A. Bcl11b-dependent genes. B. Bcl11b-repressed genes. C. Patterns of expression of genes differentially expressed (p.adj <0.1) in response to sgRNA against Mta1_2 or by sgRNA against Runx1. All results are from two complete independent experiments in which aliquots of the cells were transduced in parallel with Control sgRNA, sgRNA against Bcl11b, sgRNA against Chd4, sgRNA against Mta1 and Mta2, sgRNA against REST, sgRNA against Rung1a and Ring1b, sgRNA against LSD1, and sgRNA against Runx1, in separate samples.

A and B: Columns D-K show average rpkm for Bcl11b-regulated genes in the indicated samples from two biological replicates. Subsequent columns are in groups of three for each treatment, showing respectively Log2FC, raw P values, and adjusted P values for each treatment relative to the control. Significantly Bcl11b-regulated genes were defined by linked Bcl11b binding in ChIP-seq and differential expression between control and sgBcl11b-transduced samples in the same two experiments in which parallel samples were transduced with sgRNAs targeting the cofactors. These are two of the four pairs of Bcl11b knockout and control samples included in Supplementary Table 2.

C: Genes with significant responses to disruption of Mta1,2 or Runx1 are tabulated, focusing on their RNA-seq values (rpkm) and adjusted P values. Highlighted columns show basis of selection. Gene names in black: genes regulated concordantly by cofactors as by Bcl11b. Gene names in red: genes regulated non-concordantly. Underlined gene names: genes showing an expression pattern in normal thymocytes consistent with implied role of Bcl11b. Note the preponderance of these most likely physiological Bcl11b targets among the genes doubly responsive to Runx1 as well as Bcl11b.

Supplementary Table 7

Effects of Id2 or Zbtb16 deletion on genes differentially expressed in Bcl11b-deficient DN cells. Table shows expression changes in significantly Bcl11b-regulated genes (p.adj < 0.05) as they are affected by Cas9-mediated disruption of Id2 or Zbtb16, alone or in combination with disruption of Bcl11b. Columns show rpkm, log2FC, raw P and adjusted P values measured as in Supplementary Table 6, from two independent experiments in which control sgRNA, sgRNA against Bcl11b, sgRNA against Id2, sgRNA against Zbtb16, and combinations of sgRNAs against Bcl11b and Id2 or Zbtb16 were transduced into primary pro-T cells in parallel. Genes shown in the table are those differentially expressed with |Log2FC|>1, p.adj<0.05, RPKM>1, in response to Bcl11b deletion in these experiments, and are presented sorted from highest to lowest log2FC value. A. Effects on Bcl11b-dependent genes. B. Effects on Bcl11b-repressed genes. C. Changes in tallies of Bcl11b-regulated genes that remain expressed differentially from control, with false discovery rate (p. adj) <0.05, when Bcl11b is disrupted alone (single KO) or together with Id2 (Bcl11b-Id2 DKO) or Zbtb16 (Bcl11b-Zbtb16 DKO). Note that the Bcl11b-deficient DEGs shown here are defined only based on the two independent replicate sets of control and sgBcl11b samples in these experiments where single and double knockouts are compared. They represent two of the four control and sgBcl11b sample pairs included in Supplementary Table 2.

Supplementary Table 8

List of reagents. Antibodies: list of antibodies used for flow cytometry and for biochemistry

Chemicals, peptides, and recombinant proteins: includes tissue culture media components and additives, transfection reagents, protease inhibitor, crosslinking reagents, and enzymes

Kits: includes DNA preparation and sequencing library kits, nucleic acid purification kits, protein purification kits, and western blotting reagents.

sgRNA target sequences: lists sites targeted in sgRNA design.

qPCR primers: primers used to measure deletion of targeted genomic sites

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Hosokawa, H., Romero-Wolf, M., Yui, M.A. et al. Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16. Nat Immunol 19, 1427–1440 (2018). https://doi.org/10.1038/s41590-018-0238-4

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