Regulation of Monocyte and Granulocyte Lineage Specification

描述（申请人提供）：C/EBPa是骨髓发育的关键介质，C/EBPa突变在AML中常见。缺乏C/EBPa的小鼠在CMP到GMP的转变中存在缺陷，但C/EBPa在单细胞生成与粒细胞生成中的后续作用尚未确定。我们发现外源性C/EBPa引导谱系阴性骨髓祖细胞沿着单核细胞途径，并且C/EBPa具有拉链和结合DNA作为异二聚体与c-Jun或c-Fos而不是c-Maf或MafB的能力。内源性C/EBPa与内源性c-Jun或c-Fos共表达。为了研究特异性异源二聚体的作用，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诱导单细胞生成的效力远大于C/EBPa：C/EBPa同源二聚体或c-Jun：c-Fos。寡核苷酸选择鉴定了共有C/EBPa：AP-1 DNA元件，并且C/EBPa：c-Jun通过相关位点结合并激活PU. 1启动子。使用诱导型细胞系的微阵列研究结合ChIP数据表明，EGR 2启动子是额外的C/EBPa：AP-1遗传靶点。Egr-2和升高的PU.1一样，有利于单细胞生成。在谱系阴性的鼠骨髓髓样细胞中比较MCSFR与GCSFR信号，我们发现虽然G-CSF特异性激活STAT 3，但M-CSF优先激活ERK，从而稳定c-Fos。ERK信号传导还已知诱导FOS和EGR 1/2基因，并有利于髓样而非淋巴样发育。我们假设单核细胞谱系定型是由C/EBPa：c-Jun和C/EBPa：c-Fos与MCSFR信号结合激活ERK诱导c-Fos和EGFR 1/2的PU.1和EGFR 1/2转录诱导之间的协同作用引起的。为了测试这个模型，我们提出：AIM 1。为了确定C/EBPa：c-Jun或C/EBPa：c-Fos异二聚体是否特异性地诱导体内髓系祖细胞的单核细胞定型，通过移植用逆转录病毒载体或tet调节的慢病毒载体转导的细胞，然后在1-4个月后进行分析，或者通过产生和繁殖MRP 8转基因小鼠。目标2：确定C/EBPa：AP-1异二聚体是否直接调节EGFR 2或相关EGFR 1基因的转录，以及C/EBPa：AP-1是否需要EGFR 1/2或PU. 1诱导来指导单细胞生成。将评估和绘制与EGR 1或EGR 2启动子的直接相互作用和诱导，包括正常细胞的研究。将评估Egr-1和Egr-2的显性抑制或缺乏PU.1远端增强子的小鼠中PU.1表达减少对C/EBP α：AP-1复合物诱导单细胞生成能力的影响。AIM 3.确定MCSFR信号是否比GCSFR信号更有效地稳定c-Fos并通过ERK激活诱导FOS和EGR 1/2转录。将使用谱系阴性骨髓细胞比较M-CSF和G-CSF在不同时间点的ERK激活、c-Fos磷酸化和蛋白稳定化以及FOS和EGR 1/2 RNA表达以及对ERK抑制的反应。还将使用谱系标志物的FACS和RNA分析评估ERK抑制改变骨髓谱系特化的能力。公共卫生相关性：单核细胞和粒细胞是不同类型的白色血细胞，有助于对抗感染，也可以转化为急性白血病。使用小鼠骨髓细胞的拟议实验将确定C/EBPa、c-Jun、c-Fos、Egr-2和M-CSF受体如何指导单核细胞而不是来自共同祖先细胞的粒细胞的发育。这些研究将指导未来努力开发新的方法，为感染患者提供白色血细胞，并治疗急性髓性白血病。

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.