Transcriptional Regulation of the Nuclear Envelope Lamin B Receptor: New Roles for GA Binding Protein (GABP) in Neutrophil Development.

Abstract 1464

Poster Board I-487

The essential roles that neutrophils play in innate immune responses require that these phagocytes rapidly migrate to sites of infection, adhere to and escape capillary epithelium, and then engulf and destroy invading pathogens. The capacity of neutrophils to perform these complicated functions is largely dependent on the actions of multiple hematopoietic transcription factors that coordinate the expression of critical functional genes during neutrophil development. These transcription factors include CCAAT/enhancer binding proteins C/EBPα and C/EBPε, the ETS family protein PU.1, and the GA binding protein, GABP, which is also an ETS transcription factor that acts as an obligate heterotetramer comprised of GABPα and GABPβ. In addition to the activation of functional genes, neutrophil progenitors in the bone marrow undergo profound morphologic changes that include the formation of lobulated nuclei. Although it is still unclear as to the precise purpose of nuclear lobulation, we and others have demonstrated that the nuclear envelope protein called the lamin B receptor (LBR) is essential to neutrophil nuclear lobulation and that loss of Lbr expression in mouse neutrophils leads to decreased functional responses, including a reduced respiratory burst and abnormal chemotaxis. We also have shown that Lbr gene expression is upregulated during neutrophil development, indicating that the transcriptional control of Lbr expression may play a critical role in both neutrophil morphologic maturation and function. To identify the transcriptional regulators that control Lbr activation during neutrophil development, we have isolated the promoter of the mouse Lbr gene and have assessed the roles that different regulators of neutrophil development play on Lbr activation. Previous studies demonstrated that C/EBPε directly activates the Lbr promoter, but we found that myeloid C/EBPε-/- cells exhibit normal Lbr expression. We therefore focused on identifying alternative regulators that may control Lbr gene activation. We first identified two putative ets binding sites in the Lbr gene promoter that are located near two previously identified C/EBP binding sites. We then focused on analyzing transcriptional activities of two ETS family members known to play roles in myeloid gene activation, PU.1 and GABP, on Lbr promoter sequences that contain these ets binding sites. Using promoter expression constructs that contain different sized regions of the Lbr gene promoter and Cos cell transfections, we confirm that C/EBPε alone does indeed drive Lbr expression, but that either C/EBPα or PU.1 alone fails to activate the Lbr promoter. In contrast, expression of GABP, via co-expression of GABPα and GABPβ, transcriptionally activates the Lbr promoter to levels observed with C/EBPε alone. Interestingly, GABP activates a short Lbr promoter that contains only one ets binding site but that lacks C/EBPε binding sites, indicating that a single ets site is sufficient for GABP to drive Lbr expression in the absence of C/EBPε. Furthermore, co-expression of GABP plus PU.1 significantly increased activation of the Lbr promoter to levels above that observed for either GABP alone or C/EBPε alone. This result indicates that GABP and PU.1 synergistically activate the Lbr promoter. Ongoing analyses of mutant forms of these promoter constructs will address the importance of ets binding sites to Lbr gene activation, and binding assays will be used to identify in vivo interactions between GABP and/or PU.1 with the Lbr promoter. We are also generating an EML cell line that contains flox binding sites that flank GABP gene exons, which will be used to generate myeloid progenitors that lack GABP expression. Together our studies are identifying novel mechanisms that regulate the expression of a nuclear envelope protein that is essential for neutrophil development, and may reveal important insight into how morphologic maturation of neutrophils is closely linked to neutrophil functions.