HOMEOBOX GENES IN HEMATOPOIETIC DIFFERENTIATION AND MATURATION

（摘自申请人摘要）这些研究的目标是 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 祖细胞增殖和成熟为特定的髓样细胞 元素我们的研究将集中在一类已知发挥作用的基因上。 在协调胚胎发生和分化中的重要作用， 果蝇和非洲爪蟾，同源异型盒基因。这些基因编码 含有高度保守的60个氨基酸的DNA的转录因子， 称为同源结构域的结合基序。同源异型盒基因既可以 积极和消极地调节其他基因的转录， 因此，它们在前后轴中起着关键作用 形成，分割，并协调表达 产生复杂结构的基因。本次资助的重点 应用程序是确定同源盒基因，参与 骨髓分化，并阐明GM-CSF与 骨髓祖细胞影响这些细胞的表达和作用， 同源异型盒基因 具体目标1 -确定和表征参与的同源盒基因 人类骨髓生成逆转录聚合酶链反应 使用识别同源结构域的简并引物的RT-PCR， 用于鉴定正常骨髓中表达的同源异型盒基因 在半固体培养基中，用GM-CSF刺激祖细胞， 或三次细胞分裂。特异性同源异型盒基因的表达， 将使用定量RT-PCR（和 特异性引物）的荧光激活细胞分选（FACS）骨 骨髓亚群支持这些功能作用的证据 同源框基因将使用反义寡核苷酸（ODN）获得， 以在体外测定中阻断它们的表达。我们已经成功 采用这些策略来识别三个HOX基因，到目前为止， 获得的生物学数据支持其中两个在 骨髓生成具体目标2 -采用生物测定法， 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加强特异性表达。 造血细胞系和原发性造血干细胞中的同源盒基因 祖细胞将测定细胞系对以下物质的反应： 多分化诱导剂。转导的CD 34+祖细胞 将在长期培养试验中测定其分化 和增殖潜能。具体目标3 -确定靶基因 在骨髓生成过程中由特定的同源异型盒蛋白调节。 在Aim 1中鉴定的同源框基因，并显示其发挥生理作用 在目的2所述的研究中， 靶基因指导表达的诱导型质粒载体 特异性HOX基因将被稳定转染到模型造血细胞中 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， 这个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.