C/EBPE--STUDY OF A NOVEL MYELOID TRANSCRIPTION FACTOR

我们最近克隆了一个新的人类基因（C/EB肽）， 称为CCAAT的转录因子大家族成员 增强子结合蛋白（C/EBP）。 C/EB肽是唯一表达的 在粒细胞前体细胞中，这种表达明显 被类维生素A强化 C/EBP蛋白具有较强的特异性， 与含有共有C/EBP位点的双链DNA结合。 正义或反义C/EB肽表达的共转染 构建体以及含有髓样- 特异性c-mim或人髓过氧化物酶启动子提示了 C/EBPepsilon调节骨髓特异性基因子集。 同样，在骨髓细胞系中C/EB肽的过度表达和表达不足， (NB4)增加和减少细胞的克隆生长， 分别 具体目标1将研究表达的调节 C/EB肽在骨髓细胞中的表达，包括：A.）哪些骨髓细胞 表达C/EB肽; B.）类维生素A的作用机制是什么 诱导C/EB肽表达; C.）如何表达C/EB肽 监管.在具体目标2中，我们将确定C/EB Peptide的合作伙伴 以及这些伙伴如何影响C/EB肽的生物学。 使用 酵母双杂交选择系统，我们已经确定CREB 2作为一个 C/EBPeptide的合作伙伴。 CREB 2与C/EB肽的结合 将通过几种额外的体外和体内技术进行证实 并探讨其对C/EB肽功能的影响。 其他 还将鉴定和研究C/EB肽的伙伴。 具体 目的3阐明C/EB肽的体外生物学功能， 对小鼠 使用瞬时和稳定的正义（S）和反义（AS）- C/EB肽的表达载体和寡聚核苷酸， C/EB肽在髓系细胞组合过程中的作用 将使用人髓样细胞系探索分化， 小鼠32 D骨髓细胞。 体内实验将包括：1.）研究 CMV驱动的C/EB肽S-和AS-表达转基因小鼠。 2.） C/EB肽基因种系敲除小鼠和C/EB肽的分析 缺失嵌合体具体目标4将剖析转录 C/EB肽分子的活性并鉴定其靶基因。 第一组实验，将定义转录区域， C/EB肽分子的激活和抑制。 进一步 实验中，我们将定义的共识结合序列 C/EBPeptide使用CAST和全基因组PCR，以便开始 鉴定新C/EB肽靶序列和基因。 此外，本发明还提供了一种方法， 我们将详细研究C/EB肽反式激活 髓过氧化物酶和中性粒细胞弹性蛋白酶基因。综上所述各项 研究将提供对C/EB肽的理解， 可诱导骨髓特异性转录激活因子。

We have recently cloned a novel human gene (C/EBPepsilon) which is a member of large family of transcriptional factors known as the CCAAT enhancer-binding proteins (C/EBP). C/EBPepsilon is uniquely expressed in granulocytic precursor cells, and this expression is markedly enhanced by retinoids. C/EBP protein exhibits strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of either the sense or anti-sense C/EBPepsilon expression constructs together with CAT-reporter vectors containing myeloid- specific c-mim or human myeloperoxidase promoters suggest a role for C/EBPepsilon in the regulation of a subset of myeloid-specific genes. Likewise, over- and under-expression of C/EBPepsilon in a myeloid line (NB4) increased and decreased the clonal growth of the cells, respectively. Specific Aim 1 will study the modulation of expression of C/EBPepsilon in myeloid cells including: A.) Which myeloid cells express C/EBPepsilon; B.) What is the mechanism by which retinoids induce expression of C/EBPepsilon; C.) How is expression of C/EBPepsilon regulated. In Specific Aim 2, we will determine partners of C/EBPepsilon and how these partners influence the biology of C/EBPepsilon. Using the yeast two hybrid selection system, we have identified CREB2 as one of the frequent partners of C/EBPepsilon. Binding of CREB2 to C/EBPepsilon will be confirmed by several additional in vitro and in vivo techniques and its influence on C/EBPepsilon function will be explored. Other partners of C/EBPepsilon will also be identified and studied. Specific Aim 3 will elucidate biological function of C/EBPepsilon in vitro and in mice. Using both transient and stable sense (S)-and antisense (AS)- expression vectors and oliogonucleotides of C/EBPepsilon, the role that C/EBPepsilon plays in the combinatorial process of myeloid differentiation will be explored using human myeloid cell lines and the murine 32D myeloid cells. In vivo experiments will include: 1.) Study of CMV-driven C/EBPepsilon S- and AS-expressing transgenic mice. 2.) Analysis of C/EBPepsilon germline knock-out mice and C/EBPepsilon deletional chimeras. Specific Aim 4 will dissect the transcriptional activity of the C/EBPepsilon molecule and identify its target genes. The first set of experiments, will define regions of transcriptional activation and repression of the C/EBPepsilon molecule. In further experiments, we will define the consensus binding sequences of C/EBPepsilon using CASTing and whole genome PCR in order to begin to identify novel C/EBPepsilon target sequences and genes. In addition, we will study in detail the ability of C/EBPepsilon to transactivate the myeloperoxidase and neutrophil elastase genes. Taken together, these studies will provide an understanding of C/EBPepsilon, a retinoid inducible, myeloid specific transcriptional activator.