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.
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