REGULATION OF NEUTROPHIL-SPECIFIC GENE EXPRESSIONS

次级颗粒蛋白(SGP)基因是中性粒细胞成熟后期表达的阶段特异性基因的一部分。我们研究了Sgp基因，特别是乳铁蛋白(LF)的协同转录调控，并鉴定了与其表达有关的正负调控蛋白。我们和其他人已经证明C/EBPepsilon对于SGP基因的协调上调至关重要。我们还发现，CCAAT置换蛋白(CDP)是Sgp基因的一个共有的抑制子。最后，我们发现在中性粒细胞成熟过程中，C/EBPepsilon本身受到CDP的负调节。在理解髓系成熟过程中的一个无法解释的悖论是，已发现几个关键的转录因子在多个分化阶段以一种不完全由其基因表达模式预测的方式调控广泛的髓系基因。例如，C/EBPalpha的过度表达上调了LF基因 启动子，但在非允许白血病细胞和特异性颗粒缺乏症(SGD)中， 尽管C/EBPalpha表达水平很高，但没有表达。我们用了染色质 免疫沉淀(CHIP)显示与髓系基因启动子结合的特定C/EBP因子的调节影响反式激活。相反，CDP抑制Sgp基因和gp91Phox基因的表达，gp91Phox基因是在髓系分化早期表达的基因。初步研究和其他人的工作表明，CDP通过结合位点依赖的转录抑制和CDP自身翻译后修饰引起的结合变化来调节基因的表达。我们之前已经证明，CDP的过表达抑制了Sgp的表达，这种表型类似于白血病细胞系、C/EBPepsilon-/-小鼠和SGD患者。我们建议进一步研究这些正负调控因子在正常骨髓生成、白血病细胞和SGD中的相互作用。我们的具体目标是：1)进一步确定C/EBPalpha和epsilon在髓系分化中的作用；2)确定CDP抑制LF启动子的方式，并将结果与gp91-Phox和C/EBPepsilon启动子的抑制进行比较；3)研究C/EBPepsilon、CDP在SGD Sgp表达失败中的作用及其相互作用。

The secondary granule protein (SGP) genes are a subset of stage-specific genes expressed during late neutrophil maturation. We have investigated the coordinate transcriptional regulation of SGP genes, particularly lactoferrin (LF), and have identified both positive and negative regulatory proteins that contribute to their expression. We and others have shown that C/EBPepsilon is critical for coordinate upregulation of the SGP genes. We have also shown that CCAAT displacement protein (CDP) is a shared repressor of SGP genes. Finally, we have shown that C/EBPepsilon is itself negatively regulated by CDP during neutrophil maturation. An unexplained paradox in understanding myeloid maturation is the finding that several key transcription factors have been shown to regulate a wide range of myeloid genes at multiple stages of differentiation in a manner that is not entirely predicted by their pattern of gene expression. For example, C/EBPalpha overexpression upregulates the LF gene promoter, but in non-permissive leukemia cells and in specific granule deficiency (SGD), LF expression is absent despite high levels of C/EBPalpha expression. We have used chromatin immunoprecipitation (ChIP) to show that modulation of the specific C/EBP factors binding to myeloid gene promoters influences transactivation. Conversely, CDP represses expression of both SGP genes and gp91 phox, a gene expressed earlier in myeloid differentiation. Preliminary studies and work by others suggest that CDP modulates gene expression both by binding-site dependent transcriptional repression and by changes in binding induced by post-translational modification of CDP itself. We have previously demonstrated that overexpression of CDP blocks SGP expression a phenotype resembling that of leukemic cell lines, C/EBPepsilon-/- mice, and SGD patients. We propose to further study the interaction of these positive and negative regulatory factors in normal myelopoiesis, in leukemic cells, and in SGD. Our specific aims are: 1) To further define the role of C/EBPalpha and epsilon during myeloid differentiation; 2) To determine the means by which CDP represses the LF promoter and to contrast the findings with the repression of the gp91-phox and C/EBPepsilon promoters; 3) To characterize the role of C/EBPepsilon, CDP, and the interaction of the two in the failure of SGP expression in SGD.