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Does ribosomal protein-Mdm2 interaction mediate the p53-dependent hematopoietic defect in diamond-blackfan anemia?
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
during haematopoietic specification and previously unidentified separable roles of the Ets and Gata motifs during this process.
S71
P1185 - THE CKROX TRANSCRIPTION FACTOR AFFECTS THE DEVELOPMENT AND FUNCTION OF MULTIPLE T CELL TYPES Yuh-Ching Twu1 and Hung-Sia Teh2 1 Department of Biotechnology and Laboratory Science in Medicine, National YangMing University, Taipei, Taiwan; 2Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada The mature T cell population is divided into two main lineages that are defined by the expression of CD4 and CD8 surface molecules. CD8+ T cells are restricted by MHC I molecules and possess cytotoxic activity by virtue of expression of molecules such as perforin and granzyme. By contrast, CD4+ T cells are restricted by MHC II molecules and either provide help or suppress other immune cells through either cytokine secretion and/or expression of specific cell surface molecules. During T cell development the strength and duration of TCR signaling play key roles in CD4/CD8 lineage choice. In addition to TCR signaling, a number of transcription factors have recently been shown to play important roles in CD4/CD8 lineage choice. The zinc finger transcription factor cKrox plays a crucial role in CD4 T cell development and CD4/CD8 lineage decision. In cKrox-deficient mice, developing T cells expressing MHC class II-restricted TCRs are redirected into the CD8 T cell lineage. In this study, we investigated whether the cKrox transgene affected the development and function of two types of regulatory T cells, namely self-specific CD8 and CD4+FoxP3+ T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The cKrox transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4+ T cells are no longer self-reactive, lost the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the cKrox transgene promoted the development of CD4+FoxP3+ regulatory T cells resulting in an increase recovery of CD4+FoxP3+ regulatory T cells that expressed higher TGF-b-dependent suppressor activity. These studies indicate that the cKrox transgene differentially affect the development and function of self-specific CD8 T cells and CD4+FoxP3+ regulatory T cells.
P1184 - PERTURBING HAEMATOPOIETIC TRANSCRIPTION FACTOR NETWORKS AND CELL FATE DECISIONS USING TRANSCRIPTIONAL ACTIVATOR-LIKE EFFECTORS Viviane Kawata1,2,3, Adam Wilkinson1,2, Xuefei Gau4, Cheuk-Ho Tsang4, Hidetoshi Shimauchi3, Pentao Liu4, and Berthold Gottgens1,2 1 Haematology, University of Cambridge, Cambridge, UK; 2Wellcome Trust & MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK; 3Oral Biology, Tohoku University, Sendai, Miyagi, Japan; 4Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK Transcription factors are key determinants of cell identity and fate. Numerous TFs are known to play critical roles in haematopoiesis, including Scl/Tal1, PU.1 and Gata1. Our laboratory, along with others, has identified many transcriptional cis-regulatory elements within gene loci of key haematopoietic TFs, often distal to gene promoters. Haematopoietic TFs interact via these regulatory elements to form TF circuits and interconnected networks that act to antagonise or reinforce cell fate decisions. Transcriptional Activator-Like Effector (TALE) proteins have modular and predictable DNA binding domains, a feature that allows their de novo assembly as synthetic sequence-specific TFs. Fusion of transcriptional activator (VP64) or repressor (KRAB) domains to TALEs targeting promoters has recently been shown to modulate gene expression. We have designed TALE activators and repressors to target several haematopoietic TF regulatory elements, including the Scl+40kb, PU.1-14kb and Gata1-3.7kb enhancers. Initial testing in haematopoietic cell lines confirmed their ability to modulate target gene expression from these distal regulatory elements. Interestingly, expression of neighbouring genes can also affected suggesting their co-regulation. We have further applied TALEs to haematopoietic programming from embryonic stem cells and analysed their perturbation of TF networks in haematopoietic progenitor cell fate decisions. We will present validation of TALE activators and repressors targeting distal regulatory elements as an efficient method of perturbing haematopoietic TF networks and the novel insights into cell fate decisions gained.
P1186 - DOES RIBOSOMAL PROTEIN-MDM2 INTERACTION MEDIATE THE P53-DEPENDENT HEMATOPOIETIC DEFECT IN DIAMONDBLACKFAN ANEMIA? Pekka Jaako1,2, Shubhranshu Debnath1, Karin Olsson1, and Stefan Karlsson1 1 Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden; 2Section for Immunology, Lund University, Lund, Sweden Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia. Mutations in genes encoding ribosomal proteins (RPs) have been identified in 60-70 % of DBA patients. Among these genes, RPS19 is the most common DBA gene. We and others have demonstrated that the defective ribosome biogenesis in DBA results in the activation of p53, which is largely responsible for the hematopoietic symptoms. However, despite its therapeutic potential, direct interference with p53 pathway raises concerns given its role as tumor suppressor. Therefore, identification of disease-specific mechanisms upstream of p53 could reveal more promising therapeutic targets. In vitro studies have shown that p53 response upon ribosomal stress is due to the nuclear accumulation of free RPL5 and RPL11, which bind and inhibit Mdm2, the main negative regulator of p53. However, the physiological relevance of the RP-Mdm2-p53 pathway in DBA is unclear. We have recently generated a mouse model for RPS19-deficient DBA containing Rps19-targeting shRNA under a doxycycline-responsive promoter, allowing inducible and graded downregulation of Rps19 (Jaako, Blood 2011). Mice with severe Rps19 deficiency develop lethal bone marrow failure that is rescued in p53-deficient background. To assess whether the RP-Mdm2-p53 pathway mediates the activation of p53 in Rps19-deficient mice, we have crossed these mice with the Mdm2 C305F knockin mice. The C305F mutation prevents the binding of RPL5 and RPL11 to Mdm2, and subsequent p53 response to perturbed ribosome biogenesis. Our in vitro studies show that the presence of the C305F mutation decreases the expression of p53 target genes in dose-dependent manner improving the proliferation defect of Rps19-deficient hematopoietic progenitors. However, even the homozygosity for C305F does not completely abolish p53 response suggesting that additional pathways may contribute to p53 activation. Consistently, our preliminary in vivo results suggest that in contrast to p53-deficient background, the homozygosity for C305F is not sufficient to rescue the lethal bone marrow failure caused by severe Rps19 deficiency. We are currently studying whether the impact of C305F depends on the level of Rps19 downregulation and whether additional pathways operate upstream of p53.
during haematopoietic specification and previously unidentified separable roles of the Ets and Gata motifs during this process.
S71
P1185 - THE CKROX TRANSCRIPTION FACTOR AFFECTS THE DEVELOPMENT AND FUNCTION OF MULTIPLE T CELL TYPES Yuh-Ching Twu1 and Hung-Sia Teh2 1 Department of Biotechnology and Laboratory Science in Medicine, National YangMing University, Taipei, Taiwan; 2Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada The mature T cell population is divided into two main lineages that are defined by the expression of CD4 and CD8 surface molecules. CD8+ T cells are restricted by MHC I molecules and possess cytotoxic activity by virtue of expression of molecules such as perforin and granzyme. By contrast, CD4+ T cells are restricted by MHC II molecules and either provide help or suppress other immune cells through either cytokine secretion and/or expression of specific cell surface molecules. During T cell development the strength and duration of TCR signaling play key roles in CD4/CD8 lineage choice. In addition to TCR signaling, a number of transcription factors have recently been shown to play important roles in CD4/CD8 lineage choice. The zinc finger transcription factor cKrox plays a crucial role in CD4 T cell development and CD4/CD8 lineage decision. In cKrox-deficient mice, developing T cells expressing MHC class II-restricted TCRs are redirected into the CD8 T cell lineage. In this study, we investigated whether the cKrox transgene affected the development and function of two types of regulatory T cells, namely self-specific CD8 and CD4+FoxP3+ T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The cKrox transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4+ T cells are no longer self-reactive, lost the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the cKrox transgene promoted the development of CD4+FoxP3+ regulatory T cells resulting in an increase recovery of CD4+FoxP3+ regulatory T cells that expressed higher TGF-b-dependent suppressor activity. These studies indicate that the cKrox transgene differentially affect the development and function of self-specific CD8 T cells and CD4+FoxP3+ regulatory T cells.
P1184 - PERTURBING HAEMATOPOIETIC TRANSCRIPTION FACTOR NETWORKS AND CELL FATE DECISIONS USING TRANSCRIPTIONAL ACTIVATOR-LIKE EFFECTORS Viviane Kawata1,2,3, Adam Wilkinson1,2, Xuefei Gau4, Cheuk-Ho Tsang4, Hidetoshi Shimauchi3, Pentao Liu4, and Berthold Gottgens1,2 1 Haematology, University of Cambridge, Cambridge, UK; 2Wellcome Trust & MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK; 3Oral Biology, Tohoku University, Sendai, Miyagi, Japan; 4Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK Transcription factors are key determinants of cell identity and fate. Numerous TFs are known to play critical roles in haematopoiesis, including Scl/Tal1, PU.1 and Gata1. Our laboratory, along with others, has identified many transcriptional cis-regulatory elements within gene loci of key haematopoietic TFs, often distal to gene promoters. Haematopoietic TFs interact via these regulatory elements to form TF circuits and interconnected networks that act to antagonise or reinforce cell fate decisions. Transcriptional Activator-Like Effector (TALE) proteins have modular and predictable DNA binding domains, a feature that allows their de novo assembly as synthetic sequence-specific TFs. Fusion of transcriptional activator (VP64) or repressor (KRAB) domains to TALEs targeting promoters has recently been shown to modulate gene expression. We have designed TALE activators and repressors to target several haematopoietic TF regulatory elements, including the Scl+40kb, PU.1-14kb and Gata1-3.7kb enhancers. Initial testing in haematopoietic cell lines confirmed their ability to modulate target gene expression from these distal regulatory elements. Interestingly, expression of neighbouring genes can also affected suggesting their co-regulation. We have further applied TALEs to haematopoietic programming from embryonic stem cells and analysed their perturbation of TF networks in haematopoietic progenitor cell fate decisions. We will present validation of TALE activators and repressors targeting distal regulatory elements as an efficient method of perturbing haematopoietic TF networks and the novel insights into cell fate decisions gained.
P1186 - DOES RIBOSOMAL PROTEIN-MDM2 INTERACTION MEDIATE THE P53-DEPENDENT HEMATOPOIETIC DEFECT IN DIAMONDBLACKFAN ANEMIA? Pekka Jaako1,2, Shubhranshu Debnath1, Karin Olsson1, and Stefan Karlsson1 1 Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden; 2Section for Immunology, Lund University, Lund, Sweden Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia. Mutations in genes encoding ribosomal proteins (RPs) have been identified in 60-70 % of DBA patients. Among these genes, RPS19 is the most common DBA gene. We and others have demonstrated that the defective ribosome biogenesis in DBA results in the activation of p53, which is largely responsible for the hematopoietic symptoms. However, despite its therapeutic potential, direct interference with p53 pathway raises concerns given its role as tumor suppressor. Therefore, identification of disease-specific mechanisms upstream of p53 could reveal more promising therapeutic targets. In vitro studies have shown that p53 response upon ribosomal stress is due to the nuclear accumulation of free RPL5 and RPL11, which bind and inhibit Mdm2, the main negative regulator of p53. However, the physiological relevance of the RP-Mdm2-p53 pathway in DBA is unclear. We have recently generated a mouse model for RPS19-deficient DBA containing Rps19-targeting shRNA under a doxycycline-responsive promoter, allowing inducible and graded downregulation of Rps19 (Jaako, Blood 2011). Mice with severe Rps19 deficiency develop lethal bone marrow failure that is rescued in p53-deficient background. To assess whether the RP-Mdm2-p53 pathway mediates the activation of p53 in Rps19-deficient mice, we have crossed these mice with the Mdm2 C305F knockin mice. The C305F mutation prevents the binding of RPL5 and RPL11 to Mdm2, and subsequent p53 response to perturbed ribosome biogenesis. Our in vitro studies show that the presence of the C305F mutation decreases the expression of p53 target genes in dose-dependent manner improving the proliferation defect of Rps19-deficient hematopoietic progenitors. However, even the homozygosity for C305F does not completely abolish p53 response suggesting that additional pathways may contribute to p53 activation. Consistently, our preliminary in vivo results suggest that in contrast to p53-deficient background, the homozygosity for C305F is not sufficient to rescue the lethal bone marrow failure caused by severe Rps19 deficiency. We are currently studying whether the impact of C305F depends on the level of Rps19 downregulation and whether additional pathways operate upstream of p53.