NuRD complex recruitment to Thpok mediates CD4+ T cell lineage differentiation

Abstract

Although BTB-zinc finger (BTB-ZF) transcription factors control the differentiation of multiple hematopoietic and immune lineages, how they function is poorly understood. The BTB-ZF factor Thpok controls intrathymic CD4⁺ T cell development and the expression of most CD4⁺ and CD8⁺ lineage genes. Here, we identify the nucleosome remodeling and deacetylase (NuRD) complex as a critical Thpok cofactor. Using mass spectrometry and coimmunoprecipitation in primary T cells, we show that Thpok binds NuRD components independently of DNA association. We locate three amino acid residues within the Thpok BTB domain that are required for both NuRD binding and Thpok functions. Conversely, a chimeric protein merging the NuRD component Mta2 to a BTB-less version of Thpok supports CD4⁺ T cell development, indicating that NuRD recruitment recapitulates the functions of the Thpok BTB domain. We found that NuRD mediates Thpok repression of CD8⁺ lineage genes, including the transcription factor Runx3, but is dispensable for Cd4 expression. We show that these functions cannot be performed by the BTB domain of the Thpok-related factor Bcl6, which fails to bind NuRD. Thus, cofactor binding critically contributes to the functional specificity of BTB-ZF factors, which control the differentiation of most hematopoietic subsets.

Figure 1.. Thpok binds NuRD.

(A) SDS-PAGE and colloidal Coomassie blue staining of streptavidin pull down from activated Thpok^fl/fl Ox40-Cre⁺ Rosa26^BirA+ CD4⁺ T cells retrovirally transduced to express Thpok-Bio or with the empty retroviral vector (pMRx, Ctrl). Molecular markers are shown on the left. (B) Thpok-associated NuRD-related peptides identified by mass spectrometry. (C) Immunoblot analysis of streptavidin pull down (left panel) or whole cell lysates (right panel) from activated CD4⁺ T cells obtained from Thpok^fl/fl Ox40-Cre⁺ Rosa26^BirA+ that had been retrovirally transduced to express Thpok-Bio or with empty pMRx as a control vector (−). EtBr indicates treatment of cell lysates with Ethidium Bromide. Protein blots were probed with antibodies as indicated. (D) Immunoblot analysis of streptavidin pull down (left panel) or whole cell lysates (right panel) from activated CD4⁺ T cells obtained from Thpok^+/+ or Thpok^Bio/Bio Rosa26^BirA+. Protein blots were probed with antibodies as indicated. Data are representative of three independent experiments.

Figure 2.. Thpok enforces chromatin closure at CD8⁺-lineage genes.

scATACseq comparison of (i) Ctrl (Thpok^fl/fl Cd4-Cre⁻) Thpok^GFP+, CD8⁺ SP and DP thymocytes and (ii) Thpok-deficient (Thpok^fl/fl Cd4-Cre⁺) Thpok^GFP+ and DP thymocytes, all from mice carrying a Thpok^GFP BAC reporter. Data integrates two biological replicates from each genotype. (A) Schematic CD4-CD8 expression plot showing developmental trajectories of Thpok-sufficient and -deficient thymocytes, and indicating previously identified scRNAseq transcriptomic clusters (43) used for scATACseq analyses. Note that the cluster order shown here, derived from pseudo-time analysis, is consistent with the experimentally determined CD8⁺-lineage potential of immature Thpok-deficient CD4⁺ SP thymocytes (109). Imm: immature; Mat: mature; R indicates MHC II-restricted thymocytes undergoing lineage redirection (43). (B) UMAP dimensional reduction plot displaying cells separated by genotype and color-coded as in (A) according to the closest transcriptomic cluster match (43). DP thymocytes were omitted for clarity. Outlines indicate positions of Ctrl CD4⁺- and CD8⁺-lineage cells. (C-E) Genome browser tracks show scATACseq signals at indicated genes (bottom), displayed as scaled sequence read density averaged for all cells sharing the indicated transcriptome cluster projection, separated by genotype (noted at the far right) and color-coded as in (A). DP thymocyte tracks are shown for Ctrl cells only. MHC restriction is indicated on the left side of each panel. The positions of Cd8 enhancers E8_I-V and of the Thpok silencer (S) are indicated below the gene track (C, D). Stars indicate lineage specific peaks, including CD8⁺-lineage-specific peaks at Cd8 enhancers E8_I (red) E8_VI (blue). Open boxes above gene tracks indicate Thpok binding sites (GEO reference GSE148976) (43).

Figure 3.. The Thpok BTB domain binds NuRD and is needed for CD4⁺ T cell development.

(A) Schematic diagram of Thpok and mutants thereof. The Thpok BTB domain and zinc finger motifs are depicted by red and purple boxes; the Leucine Zipper sequence (LZ) by the orange box. *: R389G mutation in the Thpok^HD mutant (22). (B) Immunoblot analysis of anti-Flag immunoprecipitates (left panel) or whole cell lysates (right panel) from HEK293T cells transfected with control vector (pcDNA3, Ctrl), Flag-tagged Thpok, Thpok-HD, ΔBTB_LZ or Thpok-ΔC. Protein blots were probed with antibodies as indicated. Data are representative of more than three independent experiments. (C) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from Thpok^fl/fl Cd4-Cre⁺ mice expressing the indicated Thpok-derived transgene or no transgene (−). (D) Numbers of CD44^lo TCRβ^hi CD24^lo CD4⁺ SP thymocytes (top) and of CD44^lo TCRβ^hi CD4⁺ splenocytes (bottom) in mice shown in (C). (E) Expression of transgenic Thpok or ΔBTB_LZ in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes was assessed by intra-cellular staining and flow cytometry and is presented relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes in Thpok^fl/fl Cd4-Cre⁻ littermates (Ctrl), set as 1 in each experiment. (F) Overlaid histograms show expression of transgenic ΔBTB_LZ in TCRβ^hi CD24^lo CD4⁻CD8⁺ thymocytes from Thpok^fl/fl Cd4-Cre⁺ ΔBTB_LZ⁺ mice (plain line) and of endogenous Thpok in CD4⁺ or CD8⁺ SP thymocytes from Thpok^fl/fl Cd4-Cre⁻ (Ctrl) littermates (dotted line and grey-shaded trace, respectively). Data (C-F) are representative of 4 independent experiments totaling n= 7 (Thpok^fl/fl Cd4-Cre⁻), 12 (Thpok^fl/fl Cd4-Cre⁺), 8 (Thpok^fl/fl Cd4-Cre⁺ Thpok transgenic) or 14 (Thpok^fl/fl Cd4-Cre⁺ ΔBTB_LZ⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests. ***p<0.001, ****p<0.0001. Error bars indicate standard deviation.

Figure 4.. Functional specificity of BTB domains.

(A) Schematic diagram of Thpok and chimeric constructs. The Thpok BTB domain and zinc finger motifs are depicted by red and purple boxes; the Leucine Zipper sequence (LZ) by the orange box. The Bcl6 and Lrf BTB domain are depicted by green and cyan box, respectively. (B) Immunoblot analysis of anti-HA immunoprecipitates (left panel) or whole cell lysates (right panel) from HEK293T cells transfected with control vector (pcDNA3, Ctrl), HA-tagged Thpok, ΔBTB_LZ, B-Thpok or L-Thpok. Protein blots were probed with antibodies as indicated. * indicates non-specific bands. Data are representative of more than three independent experiments. (C) Venn diagram showing B-Thpok binding partners and Thpok 35-set as detected by mass spectrometry after streptavidin pull down in activated CD4⁺ T cells. Proteins binding Thpok but not B-Thpok are listed in the box. Peptides from NCoR1 detected in the B-Thpok pull-down are depicted by red lines along the NCoR1 schematic (bottom). RD1–3: Repression Domain; DAD: Deacetylase Activity Domain; HID: Histone Interaction Domain; RID: Receptor Interacting Domain. (D) Schematic diagram of retrogenic mice generation. (E) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from retrogenic mice generated with Ctrl (empty pPMGfIT vector), or Thpok-, ΔBTB_LZ- or B-Thpok-expressing pPMGfIT retroviruses. The contour plots are representative of three independent experiments totaling n= 13 (WT, C57BL/6 mice), 12 (Ctrl), 16 (Thpok), 9 (ΔBTB_LZ) or 8 (B-Thpok) retrogenic mice. (F) The percentages of CD4⁺ SP cells in CD44^lo CD24^lo TCRβ^hi thymocytes (top) and in CD44^lo TCRβ⁺ splenocytes (bottom) in mice shown in (E). (G) Flow cytometric expression of transduced Thpok, ΔBTB_LZ and B-Thpok in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes, expressed relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ in WT mice, set as 1 in each experiment. The data are summarized from two independent experiments totaling n= 8 (WT), 4 (Ctrl), 9 (Thpok), 4 (ΔBTB_LZ) or 7 (B-Thpok) retrogenic mice. Two-way ANOVA *p<0.05, ***p<0.001, ns: p>0.05. Error bars indicate standard deviation.

Figure 5.. A NuRD-interacting segment of the Thpok BTB domain is needed for CD4 T cell development.

(A) Schematic diagram of Thpok-SVIN (orange line box) and Thpok-RKF (cyan line box) mutations. (B) Immunoblot analysis of anti-HA immunoprecipitates (left) or whole cell lysates (right) from HEK293T cells transfected with control vector (pcDNA3, Ctrl), HA-tagged Thpok, ΔBTB_LZ, Thpok-SVIN or Thpok-RKF. Protein blots were probed with antibodies as indicated. * indicates non-specific bands. Data are representative of three independent experiments. (C) Venn diagram showing Thpok-RKF binding partners and Thpok 35-set as detected by mass spectrometry after streptavidin pull down in activated CD4⁺ T cells. Proteins binding Thpok but not Thpok-RKF complex are listed in the box. (D) ChIP-qPCR was performed on Thpok and Cd4 silencer elements, and on a region of Pax5 (negative control), from in vitro activated CD4⁺ T cells from Thpok^fl/fl Ox40-Cre⁺ Rosa26^BirA+ that had been retrovirally transduced with empty pMRx (Ctrl), or pMRx expressing Thpok-Bio or Thpok-RKF-Bio. Data are expressed as percent of input DNA. Each symbol represents a separate determination, and the figure summarizes three independent experiments. (E) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from indicated mice. (F) Numbers of CD44^lo CD24^lo TCRβ^hi CD4⁺ SP thymocytes (top) and of CD44^lo TCRβ⁺ CD4⁺ splenocytes (bottom) in mice shown in (E). (G) Flow cytometric expression of transgenic Thpok-RKF in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes, shown relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ in Thpok^fl/fl Cd4-Cre⁻ littermates (Ctrl), set as 1 in each experiment. (H) Overlaid histograms show expression of transgenic Thpok-RKF in CD24^lo TCRβ^hi CD4⁻ CD8⁺ thymocytes from Thpok^fl/fl Cd4-Cre⁺ Thpok-RKF⁺ mice (plain line) and of endogenous Thpok in CD4⁺ or CD8⁺ SP thymocytes from Thpok^fl/fl Cd4-Cre⁻ (Ctrl) littermates (dotted line and grey-shaded trace, respectively). Data (E-H) are representative of three independent experiments totaling n= 6 (Thpok^fl/fl Cd4-Cre⁻ littermates), 6 (Thpok^fl/fl Cd4-Cre⁺) or 8 (Thpok^fl/fl Cd4-Cre⁺ Thpok-RKF⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests ****p<0.0001. Error bars indicate standard deviation. (I) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from Thpok^fl/fl B2m^−/− mice expressing or not the Thpok-RKF transgene. (J) Numbers of CD44^lo CD24^lo TCRβ^hi thymocytes, CD4⁺ CD8⁻ (top left) or CD8⁺ (including CD4⁺ and CD4⁻, top right) and of CD44^lo TCRβ⁺ CD4⁺ CD8⁻ splenocytes (bottom) in mice shown in (I). Data (I-J) are representative three independent experiments totaling n= 6 (Cd4-Cre⁻ littermates), 8 (Cd4-Cre⁺) or 10 (Cd4-Cre⁺ Thpok-RKF⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests ****p<0.0001, ns: P>0.05. Error bars indicate standard deviation.

Figure 6.. NuRD recruitment recapitulates the Thpok BTB domain functions during CD4 T cell development.

(A) Schematic diagram of ΔBTB_LZ and Mta2-ΔBTB_LZ. (B) Immunoblot analysis of anti-Flag immunoprecipitates (left panel) or whole cell lysates (right panel) from HEK293T cells transfected with empty pcDNA3 (Ctrl), or vectors expressing Flag-tagged versions of Thpok, Mta2, ΔBTB_LZ or Mta2-ΔBTB_LZ. Protein blots were probed with antibodies as indicated. * indicates endogenous Mta2 protein. Data are representative of three independent experiments. (C) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from retrogenic mice generated with empty pMGfIT (Ctrl), or pMGfIT expressing ΔBTB_LZ, Mta2, Thpok, or Mta2-ΔBTB_LZ. The contour plots are representative of three independent experiments with 5 mice per transduced vector in each experiment. (D) Percentages of CD4⁺ CD8⁻ (CD4⁺ SP) cells among CD44^lo CD24^lo TCRβ^hi thymocytes (left panel) and or CD44^lo TCRβ⁺ splenocytes (right panel) from one of three independent experiments with 5 mice per group. (E) Flow cytometric expression of retroviral ΔBTB_LZ, Thpok or Mta2-ΔBTB_Lz in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes, presented relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ in WT mice, set as 1 in each experiment. (F) Scatter plots comparing gene expression (log₂ RPM [Reads Per Million]) in CD4⁺ or CD8⁺ SP thymocytes from wild type mice (WT CD4 and WT CD8, respectively) and in CD4⁺ SP thymocytes from retrogenic mice (Thpok_CD4 and Mta2-ΔBTB_Lz_CD4), all purified as CD44^lo CD24^lo CD69^lo TCRβ^hi as shown in Fig. S7B. Data is from three biological replicates processed separately up to sequencing. Reads mapping to Thpok (endogenous or retrovirally expressed) were omitted from the analyses. Genes with > 4-fold differential expression and FDR < 0.05 between indicated subsets are shown in purple or blue; numbers in corner indicate the count of differentially expressed genes (gene numbers in parentheses). (G) Graphs display expression (RPM) of indicated genes in each cell subset analyzed in (F). Each symbol represents a distinct sample. One-way ANOVA followed with Tukey multiple comparison tests *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate standard deviation.

Figure 7.. Thpok-NuRD binding is needed for Runx3 repression.

(A) Flow cytometric expression of intra-cellular Runx3 and Thpok in CD44^lo CD24^lo TCRβ^hi CD4⁺ SP (orange traces) or CD8⁺ SP (blue traces) thymocytes from indicated mice. (B) (Left panel) Expression of Runx3 in CD44^lo CD24^lo TCRβ^hi CD4⁺ SP or CD8⁺ SP thymocytes is presented relative to that of CD44^lo CD24^lo TCRβ^hi CD8⁺ SP in WT mice, set as 1 in each experiment. (Right panel) Expression of transgenic Thpok-RKF in CD44^lo CD24^lo TCRβ^hi CD4⁺ SP or CD8⁺ SP thymocytes is presented relative to that of endogenous Thpok in CD44^lo CD24^lo TCRβ^hi CD4⁺ SP in WT mice, set as 1 in each experiment. One-way ANOVA followed with Tukey multiple comparison tests. ****p<0.0001, ns: p>0.05. Error bars indicate standard deviation. Data (A, B) are representative of three independent experiments totaling n= 3 (WT), 6 (Cd4-Cre⁻ Thpok^fl/fl B2m^−/−), 7 (Cd4-Cre⁺ Thpok^fl/fl B2m^−/−) or 6 (Cd4-Cre⁺ Thpok^fl/fl B2m^−/− Thpok-RKF⁺) mice. (C) (Top) Thpok or Ctrl ChIP-seq traces on the Runx3 loci in activated CD4 T cells (42); the red bar schematizes the PCR probe in experiments below. (Bottom) Bar graph quantifies streptavidin-ChIP of activated CD4⁺ T cells from Thpok^fl/fl Ox40-Cre⁺ Rosa26^BirA+ mice that had been retrovirally transduced with empty pMRx (Ctrl) or with pMRx expressing Thpok-Bio, ΔBTB_LZ-Bio or Thpok-RKF-Bio. Data shows the amount of PCR-amplified DNA, expressed as percent of input. Each symbol represents a separate determination, and the figure summarizes three distinct experiments. Unpaired t test. *p<0.05, ***p<0.001. Error bars indicate standard deviation.

Figure 8.. Runx3-independent function of Thpok-NuRD complexes.

(A) Generation of mixed bone marrow chimeras from CD45.2⁺ tester (of indicated genotype) and CD45.1⁺ CD45.2⁺ (wild-type competitor) cells, analyzed in panels B-G. (B) Expression of CD4 and CD8 in CD44^lo TCRβ^hi CD24^lo thymocytes from B2m^−/− mixed chimeras generated as in (A). (C, D) Percentage of CD4⁺ SP (left panel) or CD4⁺ CD8⁺ (right panel) cells among tester-derived CD44^lo TCRβ^hi CD24^lo thymocytes (C) or CD44^lo TCRβ⁺ splenocytes (D). Data (B-D) are from one set of 4 chimera and are representative of a total of 2 independently generated sets of chimeras with 4–5 mice per group. One-way ANOVA followed with Tukey multiple comparison tests ***p<0.001, ****p<0.0001. Error bars indicate standard deviation. (E-G) RNA-seq analyses of CD44^lo CD24^lo CD69^lo TCRβ^hi thymocytes of indicated genotypes and purified as in Fig. S8, that were CD4⁺ SP, CD8⁺ SP, or CD4⁺ CD8⁺. Data is representative of three separate RNA samples for each population (two biological replicates one of which split into two RNA samples subsequently processed separately). (E) Principal-component analysis (PCA) displays cell subsets according to the first two components. Each symbol represents an individual RNAseq sample. (F) Scatter plots comparing CD4 lineage (41 genes) and CD8 lineage signatures (121 genes) (defined as WT CD4⁺ SP vs WT CD8⁺ SP, log₂ (Fold Change) >2 or <−2, FDR < 0.05, Table S2) in Thpok-RKF⁻ and Thpok-RKF⁺ CD4⁺ CD8⁺ thymocytes. X-axis and y-axis present log₂ RPM. Genes with 2-fold or greater differential expression between subsets (and FDR < 0.05) are shown in blue and red (gene numbers are shown in parentheses), respectively. (G) Graphs display expression (RPM) of indicated genes in each analyzed cell subset (symbols on top). Each symbol represents a distinct sample. Error bars indicate standard deviation. (H) GFP expression (indicative of Cd4 promoter activity) in RLM-11 cells transfected with (i) a GFP-based reporter plasmid for Cd4 gene expression (schematic at bottom) (ii) a vector expressing Cd8α as an internal control and (iii) expression vectors encoding Runx1, Thpok or Thpok-RKF as indicated. Enh, Pr and Sil indicate the Cd4 proximal enhancer, promoter and silencer, respectively (76, 110). Data are expressed relative to transfection with neither Thpok nor Runx1 vector (leftmost bar) and summarize more than three independent experiments. Each symbol represents a separate transfection. One-way ANOVA followed with Tukey multiple comparison tests. Unpaired t test. **p<0.01, ****p<0.0001, ns, p>0.05. Error bars indicate standard deviation.