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

我们最近克隆了一种新的人类基因(C/EBPepsilon)，它是一种 被称为CCAAT的转录因子大家族的成员 增强子结合蛋白(C/EBP)。C/EBPepsilon是唯一表达的 在粒细胞前体细胞中，这种表达明显 被维甲酸强化了。C/EBP蛋白表现出较强的特异性 与含有共同C/EBP位点的双链DNA结合。 正、反义C/EBPepsilon共表达的研究 与含有髓系的CAT报告载体一起构建- 特异性c-mim或人类髓过氧化物酶启动子提示 C/EBPepsilon在调节髓系特异基因子集中的作用。 同样，C/EBPepsilon在髓系中的过度和低表达 (NB4)促进和抑制细胞克隆生长， 分别进行了分析。特定目标1将研究表达的调节 C/EBPepsilon在髓系细胞中的表达包括：A.)哪些髓系细胞 Express C/EBPepsilon；B.)维甲酸的作用机制是什么？ 诱导表达C/EBPepsilon；C.)C/EBPepsilon的表达情况如何 受监管的。在具体目标2中，我们将确定C/EBPepsilon的合作伙伴 以及这些伙伴如何影响C/EBPepsilon的生物学。使用 酵母双杂交选择系统，我们已将CREB2鉴定为 C/EBPepsilon的常客合作伙伴。CREB2与C/EBPepsilon的结合 将通过几种额外的体外和体内技术来证实 并探讨其对C/EBPepsilon功能的影响。其他 还将确定和研究C/EBPepsilon的合作伙伴。特定的 目的3阐明C/EBPepsilon的体外生物学功能和 在老鼠身上。使用瞬时和稳定的正义(S)-和反义(AS)- C/EBPepsilon的表达载体和寡核苷酸的作用 C/EBPepsilon在髓系细胞组合过程中的作用 将使用人类髓系细胞系和 小鼠32D髓系细胞。活体实验将包括：1)学习 巨细胞病毒驱动的C/EBPepsilon S和AS表达转基因小鼠。2.) C/EBPepsilon种系敲除小鼠和C/EBPepsilon基因敲除小鼠的分析 删除嵌合体。《特定目标4》将剖析转录 研究C/EBPepsilon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/EBPepsilon分子的激活和抑制。在进一步的 实验中，我们将定义共识结合序列 C/EBPepsilon用铸型和全基因组聚合酶链式反应开始 鉴定新的C/EBPepsilon靶序列和基因。此外, 我们将详细研究C/EBPepsilon反式激活 髓过氧化物酶和中性粒细胞弹性蛋白酶基因。这些加在一起， 研究将提供对C/EBPepsilon的了解，这是一种维甲酸 可诱导的髓系特异性转录激活剂。

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.