The Experts below are selected from a list of 1251 Experts worldwide ranked by ideXlab platform
Koji Yasutomo - One of the best experts on this subject based on the ideXlab platform.
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regulation of membrane phospholipid asymmetry by notch mediated flippase expression controls the number of intraepithelial tcrαβ cd8αα t cells
PLOS Biology, 2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Intrinsic RBPJ in T cells is required for TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Bone marrow cells (1.5 × 107) and a 1:1 ratio from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were transplanted into irradiated (9.5 Gy) C57BL/6 mice (CD45.1+CD45.2+). (A) The ratio of intraepithelial CD45+ cells between RBPJ−/− and RBPJ+/+ mice. (B) After 6 wk posttransplantation, the number and frequency of TCRαβ+CD8αα+ IELs from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were evaluated. The data are shown as mean ± S.D., and *** indicates p < 0.001. The data in (A) and (B) are representative of three independent experiments with n = 5 mice in each experiment. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Atp8a2 is a Notch target gene in TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) Enriched gene ontology of genes in TCRαβ+CD8αα+ IELs. Genes for which expression is more than four times less in RBPJ−/− compared with RBPJ+/+ mice were analyzed. A heat map of genes (>5 times reduction for RBPJ deficiency) in TCRαβ+CD8αα+ IELs in RBPJ−/− mice compared with RBPJ+/+ mice is shown. (B) Expression of Heyl, Dtx1, and Atp8a2 in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (C) Expression of Dtx1 and Atp8a2 in DO.11.10 T-cell hybridoma cells infected with control retrovirus (“EV”) or retrovirus carrying the intracellular domain of Notch1 (“N1IC”). (D) Expression of Atp11a and Atp11c in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (E) Bone marrow cells of RBPJ−/− mice infected with control retrovirus or retrovirus carrying Atp8a2 were transplanted in irradiated wild-type mice (n = 4). Six weeks after transplantation, the development of TCRαβ+CD8αα+ IELs was evaluated by flow cytometry. The data in (A–E) are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01 (n = 4). (F) Atp8a2, Atp11a, and Atp11c expression in TCRαβ+CD8αα+ IELs, TCRγδ+CD8αα+ IELs, and splenic CD4 or CD8 T cells from RBPJ+/+ or RBPJ−/− mice evaluated by PrimeFlow analysis. Control (“Ctrl.”); shadow; control staining, RBPJ+/+ mice; solid line, RBPJ−/− mice; dotted line. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαβ+CD8αα+ T cells
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Notch deficiency does not affect the development of precursors of TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) The frequency and total number of PD-1+ and PD-1− cells in CD25−CD1d-tetramer−CD4−CD8α−TCRβ+CD5+ thymocytes in RBPJ+/+ and RBPJ−/− mice were evaluated by flow cytometry. The data are representative of three independent experiments and are shown as mean ± S.D. (B) The B220−NK1.1−CD4−CD8α−TCRγδ−TCRβ+CD5+ thymocytes from RBPJ+/+ and RBPJ−/− mice were cultured in the presence of IL-15 (50 ng/ml), and the differentiation of CD8α cells was tested by flow cytometry after 8 d of culture. (C) The expression of α4β7, CD103, and CD69 on TCRαβ+CD8αα+ IELs in RBPJ−/− and RBPJ+/+ mice. Shadow: isotype control; solid: RBPJ+/+; dotted: RBPJ−/−. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; PD-1, programmed death-1; RBPJ, recombination signal binding protein for immunoglobulin kappa J region.
Chieko Ishifune - One of the best experts on this subject based on the ideXlab platform.
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regulation of membrane phospholipid asymmetry by notch mediated flippase expression controls the number of intraepithelial tcrαβ cd8αα t cells
PLOS Biology, 2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Intrinsic RBPJ in T cells is required for TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Bone marrow cells (1.5 × 107) and a 1:1 ratio from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were transplanted into irradiated (9.5 Gy) C57BL/6 mice (CD45.1+CD45.2+). (A) The ratio of intraepithelial CD45+ cells between RBPJ−/− and RBPJ+/+ mice. (B) After 6 wk posttransplantation, the number and frequency of TCRαβ+CD8αα+ IELs from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were evaluated. The data are shown as mean ± S.D., and *** indicates p < 0.001. The data in (A) and (B) are representative of three independent experiments with n = 5 mice in each experiment. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Atp8a2 is a Notch target gene in TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) Enriched gene ontology of genes in TCRαβ+CD8αα+ IELs. Genes for which expression is more than four times less in RBPJ−/− compared with RBPJ+/+ mice were analyzed. A heat map of genes (>5 times reduction for RBPJ deficiency) in TCRαβ+CD8αα+ IELs in RBPJ−/− mice compared with RBPJ+/+ mice is shown. (B) Expression of Heyl, Dtx1, and Atp8a2 in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (C) Expression of Dtx1 and Atp8a2 in DO.11.10 T-cell hybridoma cells infected with control retrovirus (“EV”) or retrovirus carrying the intracellular domain of Notch1 (“N1IC”). (D) Expression of Atp11a and Atp11c in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (E) Bone marrow cells of RBPJ−/− mice infected with control retrovirus or retrovirus carrying Atp8a2 were transplanted in irradiated wild-type mice (n = 4). Six weeks after transplantation, the development of TCRαβ+CD8αα+ IELs was evaluated by flow cytometry. The data in (A–E) are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01 (n = 4). (F) Atp8a2, Atp11a, and Atp11c expression in TCRαβ+CD8αα+ IELs, TCRγδ+CD8αα+ IELs, and splenic CD4 or CD8 T cells from RBPJ+/+ or RBPJ−/− mice evaluated by PrimeFlow analysis. Control (“Ctrl.”); shadow; control staining, RBPJ+/+ mice; solid line, RBPJ−/− mice; dotted line. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαβ+CD8αα+ T cells
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Notch deficiency does not affect the development of precursors of TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) The frequency and total number of PD-1+ and PD-1− cells in CD25−CD1d-tetramer−CD4−CD8α−TCRβ+CD5+ thymocytes in RBPJ+/+ and RBPJ−/− mice were evaluated by flow cytometry. The data are representative of three independent experiments and are shown as mean ± S.D. (B) The B220−NK1.1−CD4−CD8α−TCRγδ−TCRβ+CD5+ thymocytes from RBPJ+/+ and RBPJ−/− mice were cultured in the presence of IL-15 (50 ng/ml), and the differentiation of CD8α cells was tested by flow cytometry after 8 d of culture. (C) The expression of α4β7, CD103, and CD69 on TCRαβ+CD8αα+ IELs in RBPJ−/− and RBPJ+/+ mice. Shadow: isotype control; solid: RBPJ+/+; dotted: RBPJ−/−. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; PD-1, programmed death-1; RBPJ, recombination signal binding protein for immunoglobulin kappa J region.
Rhett A. Kovall - One of the best experts on this subject based on the ideXlab platform.
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transcription factor RBPJ as a molecular switch in regulating the notch response
Advances in Experimental Medicine and Biology, 2021Co-Authors: Benedetto Daniele Giaimo, Rhett A. Kovall, Ellen K Gagliani, Tilman BorggrefeAbstract:The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.
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Structural and Functional Studies of the RBPJ-SHARP Complex Reveal a Conserved Corepressor Binding Site
Cell Reports, 2019Co-Authors: Zhenyu Yuan, Franz Oswald, Tilman Borggrefe, Bradley D. Vanderwielen, Benedetto Daniele Giaimo, Courtney E. Collins, Aleksandra Turkiewicz, Kerstin Hein, Rhett A. KovallAbstract:Summary Notch is a conserved signaling pathway that is essential for metazoan development and homeostasis; dysregulated signaling underlies the pathophysiology of numerous human diseases. Receptor-ligand interactions result in gene expression changes, which are regulated by the transcription factor RBPJ. RBPJ forms a complex with the intracellular domain of the Notch receptor and the coactivator Mastermind to activate transcription, but it can also function as a repressor by interacting with corepressor proteins. Here, we determine the structure of RBPJ bound to the corepressor SHARP and DNA, revealing its mode of binding to RBPJ. We tested structure-based mutants in biophysical and biochemical-cellular assays to characterize the role of RBPJ as a repressor, clearly demonstrating that RBPJ mutants deficient for SHARP binding are incapable of repressing transcription of genes responsive to Notch signaling in cells. Altogether, our structure-function studies provide significant insights into the repressor function of RBPJ.
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RBPJ direct regulation of atoh7 transcription in the embryonic mouse retina
Scientific Reports, 2018Co-Authors: Joel B Miesfeld, Rhett A. Kovall, Amy N Riesenberg, Nadean L Brown, Myung Soon Moon, Ashley N ContrerasAbstract:In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, RBPJ and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate RBPJ protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that RBPJ simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.
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structural and functional studies of the RBPJ sharp complex reveal conserved corepressor binding site
Social Science Research Network, 2018Co-Authors: Zhenyu Yuan, Franz Oswald, Tilman Borggrefe, Bradley D. Vanderwielen, Benedetto Daniele Giaimo, Courtney E. Collins, Aleksandra Turkiewicz, Kerstin Hein, Leiling Pan, Rhett A. KovallAbstract:The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit.
Stephen C Blacklow - One of the best experts on this subject based on the ideXlab platform.
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extension of the notch intracellular domain ankyrin repeat stack by nrarp promotes feedback inhibition of notch signaling
Science Signaling, 2019Co-Authors: Sanchez M Jarrett, Tom C M Seegar, Mark Andrews, Guillaume Adelmant, Jarrod A Marto, Jon C Aster, Stephen C BlacklowAbstract:Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 A resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.
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genome wide analysis reveals conserved and divergent features of notch1 RBPJ binding in human and murine t lymphoblastic leukemia cells
Blood, 2011Co-Authors: Jon C Aster, Stephen C Blacklow, Bo Zhao, James Zou, Hongfang Wang, Eric Johannsen, Jonathan Schug, Elliott Kieff, Yumi Yashiroohtani, Warren S PearAbstract:Abstract 5236 Activated Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is an important oncoprotein in murine and human T cell acute lymphoblastic leukemia/lymphoma (T-ALL), yet the interplay between Notch1 and other factors that regulate the transcriptional output of T-ALL cells is poorly understood. Using ChIP-Seq and starting with Notch1-dependent human and murine T-ALL cell lines, we find that Notch1 binds preferentially to promoters, to RBPJ binding sites, and near sites for ZNF143, as well as Ets and Runx factors. By ChIP-Seq, ZNF143 binds to ∼40% of Notch1 sites, whereas Ets1 binding is observed within 100 basepairs of ∼70% of genomic Notch1 binding sites. Notch1/ZNF143 “co-sites” have high Notch1 and ZNF143 signals, frequent co-binding of RBPJ to sites embedded within ZNF143 motifs, strong promoter bias, and low mean levels of “activated” chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in T-ALL cell lines. In contrast, Ets1 binding sites flank RBPJ/Notch1 binding sites and are associated with high levels of activated chromatin marks, whereas Runx sites are predominantly intergenic. Although Notch1 predominantly binds promoters, ∼75% of direct Notch1 target genes lack promoter binding and appear to be regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R and the GIMAP gene cluster. Both Ets1 and Notch1 binding to an intronic enhancer located in DTX1 were required for expression of this well characterized Notch1 target gene, suggesting that these two factors coordinately regulate DTX1 expression. Although the association of Notch1 binding with ZNF143, Ets, and Runx sites was highly conserved, binding near certain important genes showed substantial divergence. For example, in human T-ALL lines Notch1/RBPJ bind a 39 enhancer near the IL7R gene, whereas in murine T-ALL lines no binding was observed near Il7r. Similarly, in human T-ALL lines Notch1/RBPJ bound an enhancer located ∼565 kb 59 of MYC, whereas in murine T-ALL cells Notch1/RBPJ bound an enhancer located ∼1 Mb 39 of Myc. Human and murine T-ALL genomes also have many sites that bind only RBPJ. Murine RBPJ “only” sites are highly enriched for imputed sites for the corepressor REST, whereas human RPBJ “only” sites lack REST motifs and are more highly enriched for imputed CREB binding sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in T-ALL cell lines, as well as novel classes of divergent RBPJ “only” sites that also likely regulate transcription. To extend these findings to normal and pathophysiologic tissues, ChIP-Seq was used to identify RBPJ/Notch1 binding sites in primary murine thymocytes and primary murine T-ALL associated with Notch1 gain-of-function mutations. Early findings appear to indicate that primary T-ALLs closely resemble normal DN3a thymocytes in terms of the distribution of Notch1 binding sites and associated chromatin marks. These data suggest that Notch1-driven T-ALLs epigenetically resemble the DN3a stage of T cell development, during which Notch1 signaling is high and cells are rapidly proliferating. Disclosures: No relevant conflicts of interest to declare.
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epstein barr virus exploits intrinsic b lymphocyte transcription programs to achieve immortal cell growth
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Bo Zhao, James Zou, Hongfang Wang, Eric Johannsen, Chih Wen Peng, John Quackenbush, Jessica C Mar, Cynthia C Morton, Matthew L Freedman, Stephen C BlacklowAbstract:Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5′ of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.
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genome wide analysis reveals conserved and divergent features of notch1 RBPJ binding in human and murine t lymphoblastic leukemia cells
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Hongfang Wang, Stephen C Blacklow, Bo Zhao, James Zou, Eric Johannsen, Todd Ashworth, Hoifung Wong, Warren S Pear, Jonathan Schug, K L ArnettAbstract:Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, we find that in human and murine T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to ∼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, ∼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription.
Yoichi Maekawa - One of the best experts on this subject based on the ideXlab platform.
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regulation of membrane phospholipid asymmetry by notch mediated flippase expression controls the number of intraepithelial tcrαβ cd8αα t cells
PLOS Biology, 2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Intrinsic RBPJ in T cells is required for TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Bone marrow cells (1.5 × 107) and a 1:1 ratio from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were transplanted into irradiated (9.5 Gy) C57BL/6 mice (CD45.1+CD45.2+). (A) The ratio of intraepithelial CD45+ cells between RBPJ−/− and RBPJ+/+ mice. (B) After 6 wk posttransplantation, the number and frequency of TCRαβ+CD8αα+ IELs from RBPJ−/− (CD45.2) and RBPJ+/+ (CD45.1) mice were evaluated. The data are shown as mean ± S.D., and *** indicates p < 0.001. The data in (A) and (B) are representative of three independent experiments with n = 5 mice in each experiment. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Atp8a2 is a Notch target gene in TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) Enriched gene ontology of genes in TCRαβ+CD8αα+ IELs. Genes for which expression is more than four times less in RBPJ−/− compared with RBPJ+/+ mice were analyzed. A heat map of genes (>5 times reduction for RBPJ deficiency) in TCRαβ+CD8αα+ IELs in RBPJ−/− mice compared with RBPJ+/+ mice is shown. (B) Expression of Heyl, Dtx1, and Atp8a2 in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (C) Expression of Dtx1 and Atp8a2 in DO.11.10 T-cell hybridoma cells infected with control retrovirus (“EV”) or retrovirus carrying the intracellular domain of Notch1 (“N1IC”). (D) Expression of Atp11a and Atp11c in TCRαβ+CD8αα+ IELs of RBPJ+/+ or RBPJ−/− mice evaluated by real-time PCR. (E) Bone marrow cells of RBPJ−/− mice infected with control retrovirus or retrovirus carrying Atp8a2 were transplanted in irradiated wild-type mice (n = 4). Six weeks after transplantation, the development of TCRαβ+CD8αα+ IELs was evaluated by flow cytometry. The data in (A–E) are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01 (n = 4). (F) Atp8a2, Atp11a, and Atp11c expression in TCRαβ+CD8αα+ IELs, TCRγδ+CD8αα+ IELs, and splenic CD4 or CD8 T cells from RBPJ+/+ or RBPJ−/− mice evaluated by PrimeFlow analysis. Control (“Ctrl.”); shadow; control staining, RBPJ+/+ mice; solid line, RBPJ−/− mice; dotted line. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
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Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαβ+CD8αα+ T cells
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (RBPJ) or Notch1 and Notch2 in T cells. RBPJ-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. RBPJ-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in RBPJ-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in RBPJ-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.
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Notch deficiency does not affect the development of precursors of TCRαβ+CD8αα+ IELs.
2019Co-Authors: Chieko Ishifune, Shin-ichi Tsukumo, Yoichi Maekawa, Katsuto Hozumi, Doo Hyun Chung, Chihiro Motozono, Sho Yamasaki, Hiroyasu Nakano, Koji YasutomoAbstract:(A) The frequency and total number of PD-1+ and PD-1− cells in CD25−CD1d-tetramer−CD4−CD8α−TCRβ+CD5+ thymocytes in RBPJ+/+ and RBPJ−/− mice were evaluated by flow cytometry. The data are representative of three independent experiments and are shown as mean ± S.D. (B) The B220−NK1.1−CD4−CD8α−TCRγδ−TCRβ+CD5+ thymocytes from RBPJ+/+ and RBPJ−/− mice were cultured in the presence of IL-15 (50 ng/ml), and the differentiation of CD8α cells was tested by flow cytometry after 8 d of culture. (C) The expression of α4β7, CD103, and CD69 on TCRαβ+CD8αα+ IELs in RBPJ−/− and RBPJ+/+ mice. Shadow: isotype control; solid: RBPJ+/+; dotted: RBPJ−/−. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; PD-1, programmed death-1; RBPJ, recombination signal binding protein for immunoglobulin kappa J region.
