HOMEOBOX GENES IN HEMATOPOIETIC DIFFERENTIATION AND MATURATION

(摘自申请者的摘要)这些研究的目的是 确定多系造血生长因子，粒细胞- 巨噬细胞集落刺激因子(GM-CSF)刺激 祖细胞向特定髓系细胞的增殖和成熟 元素。我们的研究将集中在一类已知的基因上 在协调胚胎发生和分化中的重要作用 果蝇和非洲爪哇，同源盒基因。这些基因编码 含有高度保守的60个氨基酸DNA的转录因子- 结合基序称为同源结构域。同源异型盒基因可以 正向和负向调节其他基因的转录，以及 因此，它们在前轴或后轴中起关键作用。 形成、分割和协调协调表达 产生复杂结构所需的基因。这笔赠款的重点是 应用是确定与同源异型盒相关的基因 髓系分化及其与GM-CSF相互作用的研究 髓系祖细胞影响这些基因的表达和作用 同源盒基因。 特定目标1--识别和表征同源框基因 人类的骨髓生成。逆转录聚合酶链式反应 (RT-PCR)，使用识别同源结构域的简并引物，是 用于鉴定在正常骨髓中表达的同源异型盒基因 GM-CSF刺激的祖细胞在半固体培养液中经历两次 或者三次细胞分裂。同源异型盒基因在人体内的表达 髓系祖细胞将使用定量RT-PCR(和 荧光激活细胞分选(FACS)骨的特异性引物 骨髓亚群。支持这些功能作用的证据 同源盒基因将使用反义寡核苷酸(ODN)获得 以阻断它们在体外实验中的表达。我们已经成功地 利用这些策略识别了三个HOX基因，到目前为止，已经 获得的生物学数据支持它们中的两个在 骨髓再生。具体目标2--使用生物测试来确定 特定同源异型盒基因在人类骨髓生成中的功能作用。 逆转录病毒载体将用于强制表达特定的 造血细胞系和原代造血细胞中的同源异型盒基因 祖细胞。细胞株将被检测它们对 多种分化诱导剂。转导CD34祖细胞 将在长期培养试验中检测它们的分化情况 和增殖潜能。特定目标3-识别目标基因 在骨髓生成过程中受特定同源框蛋白调控的基因。 同源异型盒基因在目标1中被发现，并被证明在生理上起作用 在AIM 2中描述的研究中的骨髓生成将被用于分离 靶基因。引导A基因表达的可诱导质粒载体 特异性HOX基因将稳定地转入造血细胞模型 细胞系；“代表性差异分析”(RDA)将用于 识别被激活或抑制的靶基因 这种同源异型盒基因的表达。

(Adapted from the applicant's abstract) The goal of these studies is to determine how the multilineage hematopoietic growth factor, granulocyte- macrophage colony-stimulating factor (GM-CSF), stimulates the proliferation and maturation of progenitor cells into specific myeloid elements. Our studies will focus on a class of genes known to play an important role in coordinating embryogenesis and differentiation in Drosophila and Xenopus, the homeobox genes. These genes encode transcription factors that contain a highly conserved 60-amino acid DNA- binding motif known as the homeodomain. Homeobox genes can both positively and negatively regulate the transcription of other genes, and consequently, they play critical roles in anteriorposterior axis formation, segmentation, and orchestrate the coordinated expression of genes required to generate complex structures. The focus of this grant application is to identify the homeobox genes that are involved in myeloid differentiation and to elucidate how interaction of GM-CSF with the myeloid progenitor cells affects the expression and action of these homeobox genes. Specific Aim 1 - Identify and characterize homeobox genes involved in human myelopoiesis. Reverse transcription polymerase chain reaction (RT-PCR), using degenerate primers recognizing the homeodomain, is employed to identify the homeobox genes expressed in normal bone marrow progenitors stimulated by GM- CSF, in semi-solid media, to undergo two or three cell divisions. The expression of specific homeobox genes in myeloid progenitors will be confirmed using quantitative RT-PCR (and specific primers) of fluorescence-activated cell sorted (FACS) bone marrow subsets. Evidence supporting a functional role for these homeobox genes will be obtained using antisense oligonucleotides (ODN) to block their expression in in vitro assays. We have successfully employed these strategies to identify three HOX genes, and thus far, have obtained biological data supporting the role of two of them in myelopoiesis. Specific Aim 2 - Employ biological assays to determine the functional role of specific homeobox genes in human myelopoiesis. Retroviral vectors will be used to enforce expression of specific homeobox genes in hematopoietic cell lines and primary hematopoietic progenitor cells. Cell lines will be assayed for their responses to multiple differentiation inducers. Transduced CD34+ progenitor cells will be assayed in long-term culture assays for their differentiation and proliferative potentials. Specific Aim 3 - Identify target genes that are regulated by specific homeobox proteins during myelopoiesis. Homeobox genes identified in Aim 1 and shown to play a physiologic role in myelopoiesis in studies described in Aim 2 will be used to isolate target genes. Inducible plasmid vectors directing the expression of a specific HOX gene will be stably transfected into a model hematopoietic cell line; "representational difference analysis" (RDA) will be used to identify target genes that are either activated or repressed by the expression of this homeobox gene.