The neutrophil differentiation program is postulated to occur via a series of maturational events directed primarily by transcriptional regulation of sequentially expressed genes. Recent studies have identified a large number of transcriptional regulators, both positive and negative, which appear to act in concert in the developing neutrophil. These genes drive the complex and delicately timed sequence of genetic events which takes the cell from a progenitor to a functionally active neutrophil. Many of these genes are expressed throughout neutrophil differentiation, however, and the ongoing challenge is to elucidate how the function of these factors is modulated to allow the induction of sequential gene expression. We have investigated the transcriptional regulation of the neutrophil secondary granule protein genes, a cassette of unlinked genes which are coordinately regulated at the transcriptional level during myeloid differentiation. We have identified a region within the human lactoferrin (LF) gene promoter which confers a silencing effect on the expression of reporter gene plasmids in LF non-expressing cell lines. We have confirmed that the silencing sequence binds CCAAT displacement protein (CDP). CDP has been shown to inhibit expression of the gp9l- phox gene by binding to the promoter. CDP is constitutively expressed in myeloid cells, but shows decreased binding associated with gp9l-phox expression in maturing neutrophils. CDP is also widely expressed in other tissues, and has been implicated in regulating expression of widely disparate genes such as NCAM, g-globin, and c-myc. The common characteristic of all these genes is that they exhibit tightly regulated expression that is tissue and stage specific. To confirm its role in the repression of LF expression, we overexpressed CDP in 32D cells to determine whether CDP overexpression would block LF expression upon induction. 32D cells overexpressing CDP maintained their ability to undergo morphologic differentiation with G-CSF, but expressed no LF upon induction. Furthermore, CDP blocked expression of all secondary granule content protein genes. Since CDP can control the expression of both early (gp9l phox) and late (secondary granule proteins) gene expression, we hypothesize that CDP plays a major role in stage-specific gene expression throughout neutrophil differentiation. More recently, we have established that the transcriptional regulator CCAAT enhancer binding protein alpha (C/EBPa), a transcriptional activator known to upregulate expression of many myeloid genes, binds to the LF promoter and upregulates LF gene expression. We have studied U937 cells that overexpress C/EBPa, in which C/EBPa overexpression induces expression of LF and other secondary granule protein genes in these non-permissive cells. LF expression in 32D cells is associated with reciprocal changes in binding of CDP and C/EBPa to their respective sites in the LF promoter. We postulate that these positive and negative regulators of myeloid-specific gene expression interact to induce stage- specific coordinate expression of the secondary granule protein genes, as well as other genes expressed throughout neutrophil differentiation. However, both CDP and C/EBPa are expressed throughout neutrophil differentiation. Therefore, the mechanism by which binding of these constitutively present factors is modulated to produce stage-specific gene expression remains unclear. We propose to further characterize the interaction of CDP and C/EBP in regulating stage specific gene expression during myelopoiesis. We will also determine the role of CDP and C/EBPa in abnormal states characterized by global failure of secondary granule protein gene expression (leukemic cell lines, PU.1 knockout mice, and specific granule deficiency). Finally, since the expression of CDP is constant throughout differentiation, we will investigate the posttranslational mechanism(s) by which CDP interacts with promoters at different stages of maturation.
