Regulation of Monocyte and Granulocyte Lineage Specification

单核细胞和粒细胞谱系规范的调节

基本信息

批准号：
8386588
负责人：
ALAN D FRIEDMAN
金额：
$ 38.64万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8386588
关键词：
Acids Acute Myelocytic Leukemia Acute leukemia B-Lymphocytes Binding Biological Assay Bone Marrow Cells Breeding CCAAT-Enhancer-Binding Proteins 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 IL3 gene IL6 gene Infection JUN gene Lentivirus Vector Leucine Zippers Leukocytes Liquid substance Lymphoid Macrophage Colony-Stimulating Factor Maps Marrow Mediator of activation protein Megakaryocytes Messenger RNA Methods Methylcellulose Modeling Mus Mutation Myelogenous Myeloid Cells Normal Cell Oligonucleotides Pathway interactions Patients Phosphorylation Proteins 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 induced pluripotent stem cell mRNA Expression macrophage monocyte novel novel strategies prevent progenitor promoter receptor research study response

项目摘要

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.

C/EBPA是髓样发育的关键介体，C/EBPA的突变在AML上很常见。小鼠缺乏 C/EBPA在CMP到GMP过渡中有缺陷，但是C/EBPA在单播中的作用与单播的作用粒子尚未定义。我们发现外源C/EBPA指导谱系阴性骨髓祖细胞沿着单核细胞途径，C/EBPA具有拉链和将DNA作为异二聚体与DNA结合的能力 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。学习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），发现 C/EBPA：C-Jun或C/EBPA：C-Fos诱导单opoiesis，其效力比C/EBPA高得多：C/EBPA 同型二聚体或C-Jun：C-Fos。寡核苷酸选择确定了共识C/EBPA：AP-1 DNA元素，并且 C/EBPA：C-Jun通过相关位点结合并激活PU.1启动子。使用诱导的微阵列研究细胞系结合芯片数据，表明EGR2启动子是另一种C/EBPA：AP-1遗传目标。 Egr-2，例如高高的pu.1，有利于单播。将MCSFR与谱系中的GCSFR信号进行比较阴性鼠骨髓髓样细胞，我们发现虽然G-CSF专门激活STAT3，M-CSF 优先激活ERK，从而稳定C-Fos。众所周知，ERK信号传导会诱导FOS和 EGR1/2基因，并有利于髓样的淋巴样发育。我们假设单核细胞谱系 C/EBPA的转录诱导和EGR1/2的转录诱导之间的协同作用结果：C-Jun和 C/EBPA：C-FOS与MCSFR信号相结合，激活ERK诱导C-FOS和EGR1/2。测试这个模型，我们建议：目标1。确定C/EBPA是：C-Jun还是C/EBPA：C-FOS异二聚体专门诱导体内髓样祖细胞的单核细胞承诺，是通过移植与转导的细胞逆转录病毒载体或TET调节的慢病毒载体，然后进行1-4个月后分析或生成和MRP8转基因小鼠的繁殖。目标2：确定C/EBPA是否直接直接调节EGR2或相关EGR1基因的转录以及是否需要EGR1/2或PU.1诱导用于C/EBPA：AP-1指导单播。与EGR1或EGR2启动子的直接互动和诱导将评估和映射，包括对正常细胞的研究。 EGR-1显性抑制的影响和Egr-2或在缺乏PU.1远端增强子的小鼠中PU.1表达的表达降低。 C/EBPA：将评估诱导单噬菌的AP-1复合物。目标3。确定MCSFR是否信号稳定C-FOS并通过ERK激活诱导FOS和EGR1/2转录，而不是比 GCSFR信号。谱系阴性细胞将用于比较ERK的M-CSF和G-CSF 在各种时间点和对ERK抑制作用的响应。 ERK抑制改变髓样谱系规范的能力还将评估使用FACS和谱系标记的RNA分析。