Regulation of Monocyte and Granulocyte Lineage Specification

C/EBPA是髓系发育的关键调节因子，C/EBPA突变在急性髓系白血病中较为常见。小鼠缺乏 C/EBPA在cMP向GMP的转变过程中存在缺陷，但随后C/EBPA在单核细胞生成中的作用与 粒细胞生成没有被定义。我们发现外源性C/EBPA能定向谱系阴性的骨髓祖细胞 C/EBPA具有拉链并作为异源二聚体与DNA结合 C-Jun或c-Fos，但不是c-Maf或MafB。内源性C/EBPA与内源性c-jun或c-Fos共表达。学习 对于特定的杂二聚体的作用，我们开发了酸性和碱性亮氨酸拉链(LZE，LZK)，并发现 C/EBPA：C-jun或C/EBPA：C-Fos比C/EBPA：C/EBPA更有效地诱导单核细胞生成 同源二聚体或c-jun：c-Fos。寡核苷酸筛选鉴定出一个共识的C/EBPA：AP-1 DNA元件，以及 C/EBPA：C-jun通过相关位点结合并激活PU.1启动子。使用诱导剂的微阵列研究 结合芯片数据，细胞株提示EGR2启动子是一个额外的C/EBPA：AP-1基因 目标。EGR-2和升高的PU1一样，有利于单核细胞生成。比较MCSFR和GCSFR信号在谱系中的差异 阴性的小鼠骨髓髓系细胞，我们发现G-CSF特异性地激活STAT3，M-CSF 优先激活ERK，从而稳定c-Fos。ERK信号也是已知的诱导FOS和 Egr1/2基因和偏爱髓系而不是淋巴系发育。我们假设单核细胞系 C/EBPA：C-Jun和C-Jun对PU1和Egr1/2转录诱导的协同作用 C/EBPA：c-Fos与激活ERK的MCSFR信号相结合，诱导c-Fos和Egr1/2。 模型，我们提出：目的1.确定C/EBPA：C-Jun或C/EBPA：C-Fos杂二聚体是否特异 在体内诱导髓系祖细胞的单核细胞承诺，或者通过移植转导了 逆转录病毒载体或与tet调节的慢病毒载体在1-4个月后或BE代后进行分析 MRP8转基因小鼠的选育。目的2：直接测定C/EBPA：AP-1异源二聚体 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 对于C/EBPA：AP-1引导单核细胞生成。Egr1或EGR2启动子的直接相互作用和诱导 将进行评估和绘图，包括对正常细胞的研究。Egr-1的显性抑制效应 和Egr-2或在缺乏PU1末端增强子的小鼠中PU.1表达减弱对小鼠的 C/EBPA：将评估AP-1复合体诱导单核细胞生成的作用。目的3.确定MCSFR是否 信号通过ERK激活更有效地稳定c-Fos和诱导Fos和Egr1/2转录 GCSFR信号。谱系阴性的骨髓细胞将用于比较M-CSF和G-CSF的ERK C-Fos的激活、c-Fos的磷酸化和蛋白的稳定以及不同时期Fos和Egr1/2的RNA表达 时间点和对ERK抑制的反应。ERK抑制改变髓系谱系的能力 使用流式细胞仪和RNA分析谱系标记物也将得到评估。

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.