Regulation of Monocyte and Granulocyte Lineage Specification

描述(申请人提供)：C/EBPA是髓系发育的关键介质，C/EBPA突变在AML中很常见。缺乏C/EBPA的小鼠在从CMP到GMP的转变中存在缺陷，但C/EBPA在单系和粒系系中的后续作用尚不明确。我们发现，外源性C/EBPA通过单核细胞途径引导非血统骨髓祖细胞，并且C/EBPA具有拉链并作为异源二聚体与DNA结合c-Jun或c-Fos的能力，但不能与c-Maf或MafB结合。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)，发现C/EBPA：C-Jun或C/EBPA：C-Fos诱导单核细胞生成的效力远远高于C/EBPA：C/EBPA同源二聚体或c-Jun：C-Fos。寡核苷酸筛选鉴定出一个共有的C/EBPA：AP-1 DNA元件，C/EBPA：C-Jun通过相关位点结合并激活PU.1启动子。利用可诱导细胞系进行的微阵列研究，结合芯片数据，表明EGR2启动子是额外的C/EBPA：AP-1基因靶点。EGR-2和升高的PU1一样，有利于单核细胞生成。在谱系阴性的小鼠骨髓细胞中，比较MCSFR和GCSFR信号，我们发现G-CSF特异性地激活STAT3，而M-CSF优先激活ERK，从而稳定c-Fos。ERK信号也被认为可以诱导Fos和Egr1/2基因，并且有利于髓系而不是淋巴系的发育。我们假设C/EBPA：C-Jun和C/EBPA：C-Fos转录诱导PU.1和Egr1/2是协同作用的结果，结合MCSFR信号激活ERK诱导c-Fos和Egr1/2。为了验证这一模型，我们提出：目的1.确定C/EBPA：C-Jun或C/EBPA：c-Fos异源二聚体是否在体内特异性地诱导髓系祖细胞的单核细胞承诺，无论是通过将逆转录病毒载体转导的细胞移植到体内，还是通过tet调节的转基因慢病毒载体移植，然后在1-4个月后进行分析。目的：确定C/EBPA：AP-1异源二聚体是否直接调控EGR2或相关Egr1基因的转录，以及C/EBPA：AP-1是否需要Egr1/2或PU1诱导才能直接产生单核细胞。将评估和绘制与EGR1或EGR2启动子的直接相互作用和诱导，包括对正常细胞的研究。在缺乏PU1末端增强子的小鼠中，显性抑制Egr-1和Egr-2或降低PU.1的表达对C/EBPA：AP-1复合体诱导单核细胞生成的能力的影响将被评估。目的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Egr1/2转录。谱系阴性的骨髓细胞将被用来比较M-CSF和G-CSF在不同时间点和对ERK抑制的反应中ERK激活、c-Fos磷酸化和蛋白稳定性以及Fos和Egr1/2RNA的表达。使用流式细胞仪和谱系标记物的RNA分析也将评估ERK抑制改变髓系谱系特征的能力。与公共卫生相关：单核细胞和粒细胞是不同类型的白细胞，有助于对抗感染，也可以转化为急性白血病。拟议中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开发新的方法，为感染患者提供白细胞，并治疗急性髓系白血病。

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.