C/EBPalpha Is a Key Transcriptional Regulator of the Triggering Receptor on Myeloid Cells-2 Gene

Triggering receptor on myeloid cells-2 (TREM-2) is a member of the innate immune signaling receptor TREM family. After differentiation of monocytes, TREM-2 is expressed on the cell surface of macrophages, monocyte-derived dendritic cells, microglia, and osteoclasts. TREM-2 is necessary for modulation of cellular activation and function, and loss of TREM-2 results in the decrease of phagocytosis of apoptotic neurons and cells and an increase in inflammation response of macrophage-lineage cells. To better understand TREM-2 gene regulation during monocyte differentiation we isolated and then analyzed the putative human TREM-2 promoter to identify transcriptional control. The 1131 bp TREM- 2 promoter contains binding sites for C/EBPα and PU.1 transcription factors, known for regulation of myeloid cell differentiation and function. A series of 5 promoter reporter deletion mutants were generated to dissect transcription regulation. We progressively deleted the full-length promoter, −1002/+129, of HIF1, STAT5, GATA1, YY1F, C/EBPα binding sites generating −866/+129, −632/+129, −526/+129, −258/+129, +37/+129 promoter reporters, respectively. All TREM-2 promoter reporters showed minimal transcriptional activity when transfected in 293T-HEK cells. In contrast, C/EBPα was necessary and sufficient for TREM-2 promoter reporter activity in co-transfection studies, demonstrating a 5-fold induction of transcriptional activity as compared to controls. Consistent with the TREM-2 reporter findings showing C/EBPα mediated activity, EMSA assays demonstrated specific C/EBPα binding to sites within the TREM-2 promoter. To determine if both C/EBPα binding sites in TREM-2 were necessary for activity we generated loss of C/EBPα binding site reporters and observed that only the −43/−28 C/EBPα binding site was essential for activity. To clarify if +73/+93 PU.1 binding site had a role in TREM-2 regulation, we co-transfected the full-length promoter with PU.1 and C/EBPα and observed a 10-fold increase of induction of TREM-2 transcriptional activity. However, PU.1 alone did not activate the full-length TREM-2 promoter or bind to the PU.1 consensus site, and loss of the PU.1 binding site did not alter transcriptional activity. These results suggest a key role for direct C/EBPα and indirect PU.1 participation in regulation of TREM-2. Studies are underway to precisely understand TREM-2 gene regulation and how TREM-2 controls macrophage-mediated phagocytosis and inflammation.