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Nat Immunol. Author manuscript; available in PMC 2016 October 25. Published in final edited form as: Nat Immunol. 2016 June ; 17(6): 695–703. doi:10.1038/ni.3456.
Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity Shaojun Xing1,11, Fengyin Li1,11, Zhouhao Zeng2,11, Yunjie Zhao2, Shuyang Yu3, Qiang Shan1, Yalan Li12, Farrah C. Phillips1,4,9, Peterson K. Maina5, Hank H. Qi5, Chengyu Liu6, Jun Zhu7, R. Marshall Pope12, Catherine A. Musselman8, Chen Zeng2, Weiqun Peng2,10, and Hai-Hui “Howard” Xue1,9,10
Author Manuscript
1Department
of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
2Department
of Physics, The George Washington University, Washington DC, 20052
3State
Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, P. R. China 100193 5Department
of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
6Transgenic 7Systems
Core Facility, NHLBI, NIH, Bethesda, MD 20892
Biology Center, NHLBI, NIH, Bethesda, MD 20892
8Department
of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
Author Manuscript
9Interdisciplinary 12Proteomics
Immunology Graduate Program, University of Iowa, Iowa City, IA 52242
Facility, University of Iowa, Iowa City, IA 52242
Abstract The CD4+ and CD8+ T cell dichotomy is essential for effective cellular immunity. How the individual T cell identity is established remains poorly understood. Here we show that the high mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4+ lineage-associated genes including Cd4, Foxp3 and Rorc in CD8+ T cells. Tcf1- and Lef1deficient CD8+ T cells exhibit histone hyperacetylation, which is ascribed to an unexpected intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutating five conserved amino
Author Manuscript
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms 10 Corresponding authors: Hai-Hui “Howard” Xue, Tel: 319-335-7937, Fax: 319-335-9006, ; Email: [email protected], Weiqun Peng, Tel: 202-994-0129, Fax: 202-994-3001, ; Email: [email protected] 4Current affiliation: St. Jude Children’s Research Hospital, Memphis, TN 38105 11These authors contributed equally to this work Accession numbers. RNA-Seq and ChIP-Seq data are deposited at GEO, under accession number GSE73240. Author contributions. S.X. and F. L. performed most of the experiments with the help of S.Y., Q.S., and F.C.P.; Z.Z. analyzed the high throughput data under the supervision of W.P.; Y.Z. performed homology modelling and coevolution analyses under the supervision of C.Z.; Y.L. and R.M.P. performed the proteomics analysis; P.K.M., H.H.Q., and C.A.M. helped purification of recombinant proteins; C.L. directed gene targeting; J.Z. directed high throughput sequencing; W.P. and H.H.X supervised the overall study, analyzed the data, and wrote the paper. All authors edited the manuscript. Competing financial interests: The authors declare no competing financial interests.
Xing et al.
Page 2
Author Manuscript
acids in the Tcf1 HDAC domain diminishes the HDAC activity and the ability to suppress CD4+ lineage genes in CD8+ T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes.
Author Manuscript
Cell identity is established by lineage-determining transcription factors, which initiate and sustain expression of cell type-specific genes while repressing those in alternative 1 2 3 4 lineages , , , . Important insights and extrapolatable paradigms have been derived from hematopoietic cells. Transcription factors (TFs) play instructive roles in lineage determination; for examples, GATA-1 and PU.1 antagonistically control development of 5 6 erythroid-megakaryocytes and myeloid cells, respectively , . Lineage-committed cells remain dependent on key TFs to guard cell identity. Deletion of Pax5 in mature B cells 7 causes dedifferentiation to uncommitted progenitors, which generate T-lineage cells . Loss 8 of Bcl11b induces T cells to acquire properties of natural killer cells . During and after a cell identity is established, TFs are assisted by epigenetic mechanisms, i.e., covalent modification of DNA and/or nucleosomal histones, to ensure heritable expression of lineage2 4 appropriate genes , .
Author Manuscript
CD4+ and CD8+ T cells are functionally distinct lymphocytes that play essential roles in immune defense against foreign pathogens and transformed cells. Whereas CD8+ T cells are dedicated cytotoxic cells, CD4+ T cells provide versatile help tailored to specific pathogen types. In spite of the functional disparity, naïve CD4+ and CD8+ T cells show remarkably 9 similar transcriptomes, with few genes exhibiting lineage-restricted expression . Among the 10 CD4+ signature genes, transcription factor ThPOK protects the CD4+-lineage integrity . Naïve CD4+ T cells lacking ThPOK exhibit increased expression of CD8 coreceptors and 10 Runx3, and abundant upregulation of cytotoxicity-associated genes . Two histone deacetylase (HDAC) proteins, HDAC1 and HDAC2, although not exclusively expressed in CD4+ T cells, have a similar CD4+-lineage protective effect. Deleting HDAC1-HDAC2 in 11 CD4+ T cells causes induction of a CD8+ effector-like program . In contrast, it remains unknown how CD8+ T cell identity is regulated.
Author Manuscript
Tcf1 and Lef1 (encoded by Tcf7 and Lef1, respectively) are HMG transcription factors that 12 13 14 15 are required for early stages of thymocyte maturation , , , . Induction of Tcf1 in early 16 17 thymic progenitors by Notch signaling is essential for T cell lineage specification , . In T lineage-committed thymocytes, Tcf1 prevents malignant transformation and promotes β18 19 selection at the CD4−CD8− double negative (DN) 3 stage , . Upon maturation of DN thymocytes to the CD4+CD8+ double positive (DP) stage, the DP cells face a lineage choice 20 decision of becoming either CD4+ or CD8+ T cells . In DP thymocytes, Tcf1 and Lef1 act 13 21 upstream of ThPOK and regulate CD4+-lineage decision , . Therefore, Tcf1 and Lef1 have versatile, developmental stage-specific roles during T cell maturation. Here we report that although not required for DP thymocytes to choose the CD8+ fate, Tcf1 and Lef1 are essential for establishing CD8+ T cell identity, and this function is mediated by intrinsic HDAC activity embedded in these two factors. Our data suggest that Tcf1 and Lef1 directly bridge genetic and epigenetic regulation to establish a gene expression pattern appropriate for CD8+-lineage T cells. Nat Immunol. Author manuscript; available in PMC 2016 October 25.
Xing et al.
Page 3
Author Manuscript
Results Ablating Tcf1-Lef1 induces CD4+ lineage genes in CD8+ T cells 21
Author Manuscript Author Manuscript
Tcf1 and Lef1 are required for CD4+-lineage decision . Compared to WT mice, CD4Cre+Tcf7FL/FL Lef1FL/FL mice (called Tcf7−/−Lef1−/− hereafter) had very few CD4+ mature 21 thymocytes but increased CD8+ mature thymocytes (Supplementary Fig. 1a) . The Tcf7−/−Lef1−/− CD8+ T cells retained CD8+ T cell features such as production of interferon21 γ and perforin and expression of CD8+-specific genes including Runx3d, Nkg7 and Itgae (Supplementary Fig. 1b), but aberrantly expressed the CD4 coreceptor (Supplementary Fig. 21 1a) . To assess the global impact of Tcf1 and Lef1 deficiency, we performed RNA-Seq analysis on sort-purified CD69−CD24−TCRβ+CD8+ mature thymocytes from Tcf7−/−, Tcf7−/−Lef1−/−, and their control littermates (CD4-Cre+Tcf7+/+Lef1+/+, CD4Cre+Tcf7FL/+Lef1FL/+, or CD4-Cre− mice). By a setting of ≥2 fold expression changes and false discovery rate (FDR)
Nat Immunol. Author manuscript; available in PMC 2016 October 25. Published in final edited form as: Nat Immunol. 2016 June ; 17(6): 695–703. doi:10.1038/ni.3456.
Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity Shaojun Xing1,11, Fengyin Li1,11, Zhouhao Zeng2,11, Yunjie Zhao2, Shuyang Yu3, Qiang Shan1, Yalan Li12, Farrah C. Phillips1,4,9, Peterson K. Maina5, Hank H. Qi5, Chengyu Liu6, Jun Zhu7, R. Marshall Pope12, Catherine A. Musselman8, Chen Zeng2, Weiqun Peng2,10, and Hai-Hui “Howard” Xue1,9,10
Author Manuscript
1Department
of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
2Department
of Physics, The George Washington University, Washington DC, 20052
3State
Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, P. R. China 100193 5Department
of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
6Transgenic 7Systems
Core Facility, NHLBI, NIH, Bethesda, MD 20892
Biology Center, NHLBI, NIH, Bethesda, MD 20892
8Department
of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
Author Manuscript
9Interdisciplinary 12Proteomics
Immunology Graduate Program, University of Iowa, Iowa City, IA 52242
Facility, University of Iowa, Iowa City, IA 52242
Abstract The CD4+ and CD8+ T cell dichotomy is essential for effective cellular immunity. How the individual T cell identity is established remains poorly understood. Here we show that the high mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4+ lineage-associated genes including Cd4, Foxp3 and Rorc in CD8+ T cells. Tcf1- and Lef1deficient CD8+ T cells exhibit histone hyperacetylation, which is ascribed to an unexpected intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutating five conserved amino
Author Manuscript
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms 10 Corresponding authors: Hai-Hui “Howard” Xue, Tel: 319-335-7937, Fax: 319-335-9006, ; Email: [email protected], Weiqun Peng, Tel: 202-994-0129, Fax: 202-994-3001, ; Email: [email protected] 4Current affiliation: St. Jude Children’s Research Hospital, Memphis, TN 38105 11These authors contributed equally to this work Accession numbers. RNA-Seq and ChIP-Seq data are deposited at GEO, under accession number GSE73240. Author contributions. S.X. and F. L. performed most of the experiments with the help of S.Y., Q.S., and F.C.P.; Z.Z. analyzed the high throughput data under the supervision of W.P.; Y.Z. performed homology modelling and coevolution analyses under the supervision of C.Z.; Y.L. and R.M.P. performed the proteomics analysis; P.K.M., H.H.Q., and C.A.M. helped purification of recombinant proteins; C.L. directed gene targeting; J.Z. directed high throughput sequencing; W.P. and H.H.X supervised the overall study, analyzed the data, and wrote the paper. All authors edited the manuscript. Competing financial interests: The authors declare no competing financial interests.
Xing et al.
Page 2
Author Manuscript
acids in the Tcf1 HDAC domain diminishes the HDAC activity and the ability to suppress CD4+ lineage genes in CD8+ T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes.
Author Manuscript
Cell identity is established by lineage-determining transcription factors, which initiate and sustain expression of cell type-specific genes while repressing those in alternative 1 2 3 4 lineages , , , . Important insights and extrapolatable paradigms have been derived from hematopoietic cells. Transcription factors (TFs) play instructive roles in lineage determination; for examples, GATA-1 and PU.1 antagonistically control development of 5 6 erythroid-megakaryocytes and myeloid cells, respectively , . Lineage-committed cells remain dependent on key TFs to guard cell identity. Deletion of Pax5 in mature B cells 7 causes dedifferentiation to uncommitted progenitors, which generate T-lineage cells . Loss 8 of Bcl11b induces T cells to acquire properties of natural killer cells . During and after a cell identity is established, TFs are assisted by epigenetic mechanisms, i.e., covalent modification of DNA and/or nucleosomal histones, to ensure heritable expression of lineage2 4 appropriate genes , .
Author Manuscript
CD4+ and CD8+ T cells are functionally distinct lymphocytes that play essential roles in immune defense against foreign pathogens and transformed cells. Whereas CD8+ T cells are dedicated cytotoxic cells, CD4+ T cells provide versatile help tailored to specific pathogen types. In spite of the functional disparity, naïve CD4+ and CD8+ T cells show remarkably 9 similar transcriptomes, with few genes exhibiting lineage-restricted expression . Among the 10 CD4+ signature genes, transcription factor ThPOK protects the CD4+-lineage integrity . Naïve CD4+ T cells lacking ThPOK exhibit increased expression of CD8 coreceptors and 10 Runx3, and abundant upregulation of cytotoxicity-associated genes . Two histone deacetylase (HDAC) proteins, HDAC1 and HDAC2, although not exclusively expressed in CD4+ T cells, have a similar CD4+-lineage protective effect. Deleting HDAC1-HDAC2 in 11 CD4+ T cells causes induction of a CD8+ effector-like program . In contrast, it remains unknown how CD8+ T cell identity is regulated.
Author Manuscript
Tcf1 and Lef1 (encoded by Tcf7 and Lef1, respectively) are HMG transcription factors that 12 13 14 15 are required for early stages of thymocyte maturation , , , . Induction of Tcf1 in early 16 17 thymic progenitors by Notch signaling is essential for T cell lineage specification , . In T lineage-committed thymocytes, Tcf1 prevents malignant transformation and promotes β18 19 selection at the CD4−CD8− double negative (DN) 3 stage , . Upon maturation of DN thymocytes to the CD4+CD8+ double positive (DP) stage, the DP cells face a lineage choice 20 decision of becoming either CD4+ or CD8+ T cells . In DP thymocytes, Tcf1 and Lef1 act 13 21 upstream of ThPOK and regulate CD4+-lineage decision , . Therefore, Tcf1 and Lef1 have versatile, developmental stage-specific roles during T cell maturation. Here we report that although not required for DP thymocytes to choose the CD8+ fate, Tcf1 and Lef1 are essential for establishing CD8+ T cell identity, and this function is mediated by intrinsic HDAC activity embedded in these two factors. Our data suggest that Tcf1 and Lef1 directly bridge genetic and epigenetic regulation to establish a gene expression pattern appropriate for CD8+-lineage T cells. Nat Immunol. Author manuscript; available in PMC 2016 October 25.
Xing et al.
Page 3
Author Manuscript
Results Ablating Tcf1-Lef1 induces CD4+ lineage genes in CD8+ T cells 21
Author Manuscript Author Manuscript
Tcf1 and Lef1 are required for CD4+-lineage decision . Compared to WT mice, CD4Cre+Tcf7FL/FL Lef1FL/FL mice (called Tcf7−/−Lef1−/− hereafter) had very few CD4+ mature 21 thymocytes but increased CD8+ mature thymocytes (Supplementary Fig. 1a) . The Tcf7−/−Lef1−/− CD8+ T cells retained CD8+ T cell features such as production of interferon21 γ and perforin and expression of CD8+-specific genes including Runx3d, Nkg7 and Itgae (Supplementary Fig. 1b), but aberrantly expressed the CD4 coreceptor (Supplementary Fig. 21 1a) . To assess the global impact of Tcf1 and Lef1 deficiency, we performed RNA-Seq analysis on sort-purified CD69−CD24−TCRβ+CD8+ mature thymocytes from Tcf7−/−, Tcf7−/−Lef1−/−, and their control littermates (CD4-Cre+Tcf7+/+Lef1+/+, CD4Cre+Tcf7FL/+Lef1FL/+, or CD4-Cre− mice). By a setting of ≥2 fold expression changes and false discovery rate (FDR)
