Regulation of Monocyte and Granulocyte Lineage Specification

单核细胞和粒细胞谱系规范的调节

• 批准号: 7577119
• 负责人: ALAN D FRIEDMAN
• 金额: $ 41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7577119
• 关键词: Acids; Acute Myelocytic Leukemia; Acute leukemia; Binding; Biological Assay; Bone Marrow Cells; Breeding; CSF3 gene; CSF3R gene; Carbon; Cell Cycle Inhibition; Cell Line; Cell Transplants; Cells; Common Lymphoid Progenitor; Complex; Consensus; DNA; DNA Binding; Data; Development; Distal; Dominant-Negative Mutation; Electrophoretic Mobility Shift Assay; Enhancers; Erythroid; Estradiol; FOS Protein; FOS gene; Family member; Future; Generations; Genes; Genetic; Genetic Transcription; Granulocyte-Macrophage Colony-Stimulating Factor; Granulopoiesis; Hematopoiesis; Homo; Infection; JUN gene; Lentivirus Vector; Leucine Zippers; Leukocytes; Liquid substance; Lymphoid; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Maps; Marrow; Mediator of activation protein; Megakaryocytes; Methods; Methylcellulose; Modeling; Mus; Mutation; Myelogenous; Myeloid Cells; Normal Cell; Oligonucleotides; Pathway interactions; Patients; Phosphorylation; Proteins; Proto-Oncogene Proteins c-fos; Puromycin; RNA analysis; Regulation; Retroviral Vector; Role; S100A8 gene; STAT3 gene; Signal Transduction; Site; T-Lymphocyte; Testing; Tetanus Helper Peptide; Time; To specify; Transcription Factor AP-1; Transgenic Mice; Transplantation; bZIP Domain; base; cellular transduction; fighting; granulocyte; in vivo; mRNA Expression; macrophage; monocyte; novel; novel strategies; prevent; progenitor; promoter; public health relevance; research study; response; Acids; Acute Myelocytic Leukemia; Acute leukemia; Binding; Biological Assay; Bone Marrow Cells; Breeding; CSF3 gene; CSF3R gene; Carbon; Cell Cycle Inhibition; Cell Line; Cell Transplants; Cells; Common Lymphoid Progenitor; Complex; Consensus; DNA; DNA Binding; Data; Development; Distal; Dominant-Negative Mutation; Electrophoretic Mobility Shift Assay; Enhancers; Erythroid; Estradiol; FOS Protein; FOS gene; Family member; Future; Generations; Genes; Genetic; Genetic Transcription; Granulocyte-Macrophage Colony-Stimulating Factor; Granulopoiesis; Hematopoiesis; Homo; Infection; JUN gene; Lentivirus Vector; Leucine Zippers; Leukocytes; Liquid substance; Lymphoid; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Maps; Marrow; Mediator of activation protein; Megakaryocytes; Methods; Methylcellulose; Modeling; Mus; Mutation; Myelogenous; Myeloid Cells; Normal Cell; Oligonucleotides; Pathway interactions; Patients; Phosphorylation; Proteins; Proto-Oncogene Proteins c-fos; Puromycin; RNA analysis; Regulation; Retroviral Vector; Role; S100A8 gene; STAT3 gene; Signal Transduction; Site; T-Lymphocyte; Testing; Tetanus Helper Peptide; Time; To specify; Transcription Factor AP-1; Transgenic Mice; Transplantation; bZIP Domain; base; cellular transduction; fighting; granulocyte; in vivo; mRNA Expression; macrophage; monocyte; novel; novel strategies; prevent; progenitor; promoter; public health relevance; research study; response

DESCRIPTION (provided by applicant): C/EBPa is a key mediator of myeloid development, and mutation of C/EBPa is common on AML. Mice lacking C/EBPa are defective in the CMP to GMP transition, but the subsequent role of C/EBPa in monopoiesis versus granulopoiesis is not defined. We find that exogenous C/EBPa directs lineage-negative marrow progenitors along the monocytic pathway and that C/EBPa has the capacity to zipper and bind DNA as a heterodimer with c-Jun or c-Fos but not c-Maf or MafB. Endogenous C/EBPa co-ips with endogenous c-Jun or c-Fos. To study the role of specific heterodimers, we developed acid and basic leucine zippers (LZE, LZK) and find that C/EBPa:c-Jun or C/EBPa:c-Fos induce monopoiesis with far greater potency than does C/EBPa:C/EBPa homodimers or c-Jun:c-Fos. Oligonucleotide selection identified a consensus C/EBPa:AP-1 DNA element, and C/EBPa:c-Jun binds and activates the PU.1 promoter via a related site. Microarray studies using an inducible cell line, combined with ChIP data, suggest that the EGR2 promoter is an additional C/EBPa:AP-1 genetic target. Egr-2, like elevated PU.1, favors monopoiesis. Comparing MCSFR with GCSFR signals in lineage- negative murine marrow myeloid cells, we find that while G-CSF specifically activates STAT3, M-CSF preferentially activates ERK and thereby stabilizes c-Fos. ERK signaling is also known to induce the FOS and EGR1/2 genes and to favor myeloid over lymphoid development. We hypothesis that monocyte lineage commitment results from synergy between transcriptional induction of PU.1 and EGR1/2 by C/EBPa:c-Jun and C/EBPa:c-Fos combined with MCSFR signals that activate ERK to induce c-Fos and EGR1/2. To test this model, we propose: AIM 1. To determine whether C/EBPa:c-Jun or C/EBPa:c-Fos heterodimers specifically induce monocytic commitment of myeloid progenitors in vivo, either by transplanting cells transduced with retroviral vectors or with tet-regulated lentiviral vectors followed by analysis 1-4 months later or be generation and breeding of MRP8 transgenic mice. AIM 2: To determine whether C/EBPa:AP-1 heterodimers directly regulate transcription of the EGR2 or related EGR1 genes and whether EGR1/2 or PU.1 induction is required for C/EBPa:AP-1 to direct monopoiesis. Direct interaction with and induction of the EGR1 or EGR2 promoters will be assessed and mapped, including studies with normal cells. The effect of dominant-inhibition of Egr-1 and Egr-2 or of diminished expression of PU.1 in mice lacking the PU.1 distal enhancer on the ability of C/EBPa:AP-1 complexes to induce monopoiesis will be assessed. AIM 3. To determine whether MCSFR signals stabilize c-Fos and induce FOS and EGR1/2 transcription via ERK activation more effectively than GCSFR signals. Lineage-negative marrow cells will be used to compare M-CSF and G-CSF for ERK activation, c-Fos phosphorylation and protein stabilization, and FOS and EGR1/2 RNA expression at various time points and in response to ERK inhibition. The ability of ERK inhibition to alter myeloid lineage specification using FACS and RNA analysis of lineage markers will also be assessed. PUBLIC HEALTH RELEVANCE: Monocytes and granulocytes are different types of white blood cells that help fight infections and also can become transformed into acute leukemias. Proposed experiments using mouse bone marrow cells will determine how C/EBPa, c-Jun, c-Fos, Egr-2 and the M-CSF receptor direct development of monocytes instead of granulocytes from a common ancestor cell. These studies will guide future efforts to develop novel methods to provide white blood cells to patients with infections and to treat acute myeloid leukemias.

描述（由申请人提供）：C/EBPA是髓样发育的关键介体，C/EBPA的突变在AML上很常见。缺乏C/EBPA的小鼠在CMP到GMP过渡中有缺陷，但是C/EBPA随后在Monopoiesis与粒藻植物中的作用尚未定义。我们发现，外源C/EBPA指导沿单核细胞途径的谱系阴性骨髓祖细胞，并且C/EBPA具有拉链和DNA作为与C-Jun或C-Fos的异二聚体的能力，而不是C-JUN或C-FOS，而不是C-MAF或MAFB。内源性C/EBPA二副置，带有内源性C-JUN或C-FOS。为了研究特定异二聚体的作用，我们开发了酸和碱性亮氨酸拉链（LZE，LZK），并发现C/EBPA：C-JUN或C/EBPA：C-FOS诱导远比C/EBPA：C/EBPA同型或C-Jun或C-Jun：C-Jun：C-FOS的单opoiesis。寡核苷酸选择确定了共识C/EBPA：AP-1 DNA元素，C/EBPA：C-Jun通过相关位点结合并激活PU.1启动子。使用诱导细胞系列的微阵列研究与芯片数据结合使用，表明EGR2启动子是另一种C/EBPA：AP-1遗传靶标。 Egr-2，例如高高的pu.1，有利于单播。将MCSFR与谱系 - 阴性鼠骨髓髓样细胞中的MCSFR与GCSFR信号进行比较，我们发现虽然G-CSF专门激活STAT3，但M-CSF优先激活ERK，从而稳定C-FOS。众所周知，ERK信号传导诱导FOS和EGR1/2基因，并有利于髓样的淋巴发育。我们假设单核细胞谱系的承诺是由C/EBPA的PU.1和EGR1/2的转录诱导之间的协同作用引起的：C-JUN和C/EBPA：C-FOS：C-FOS结合MCSFR信号，与激活ERK激活ERK以诱导C-FOS和EGR1和EGR1/2。要测试该模型，我们提出：目标1。确定C/EBPA：C-JUN或C/EBPA：C-FOS异二聚体是否特异性地诱导体内髓样祖细胞的单核细胞承诺，要么通过将递离体移植或通过TET调节的Lentiviral vector and tean-ther-extranter and the-Beenter and the-telriviral vectors 1-4个月1-4个月1-4个月1-4个月1-4个月份。老鼠。目标2：确定C/EBPA：AP-1异二聚体是否直接调节EGR2或相关的EGR1基因的转录以及C/EBPA：AP-1是否需要EGR1/2或PU.1诱导才能引导单播。将评估和映射与EGR1或EGR2启动子的诱导直接相互作用，包括对正常细胞的研究。在缺乏PU.1远端增强子的小鼠中，EGR-1和EGR-2的显性抑制作用对C/EBPA：AP-1络合物诱导单oiSIS的能力的影响。目标3。确定MCSFR是否比GCSFR信号更有效地通过ERK激活稳定C-FOS并通过ERK激活诱导FOS和EGR1/2转录。谱系阴性细胞将用于比较M-CSF和G-CSF的ERK激活，C-FOS磷酸化和蛋白质稳定，以及在不同时间点的FOS和EGR1/2 RNA表达，并响应ERK抑制。还将评估ERK抑制使用FACS改变髓样谱系规范的能力和谱系标记的RNA分析。公共卫生相关性：单核细胞和粒细胞是不同类型的白细胞，有助于抵抗感染，也可以转化为急性白血病。提出的使用小鼠骨髓细胞的实验将确定C/EBPA，C-JUN，C-FOS，EGR-2和M-CSF受体直接发育单核细胞而不是来自共同祖先细胞的粒细胞。这些研究将指导未来的努力开发新方法，以向感染患者提供白细胞并治疗急性髓样白血病。