Abstract
Purpose :
Translocator Protein (18kDa; TSPO) is strongly expressed in activated microglia and serves as an attractive therapeutic target for alleviation of retinal degeneration. However, TSPO transcriptional regulation in immune cells remains largely unknown. This study sought to carry out detailed characterization of the TSPO promoter and identify elements required for microglia-specific expression
Methods :
Tspo promoter sequence was amplified by PCR and cloned into pGL4.10 luciferase vector. Plasmids containing 5’ unidirectional deletions of the promoter were generated. BV2 microglia cells were transfected with the reporter plasmids for 24 hrs, and an additional 6 hrs during lipopolysaccharide (LPS) stimulation. Sequence analysis was performed using Matinspector and substitution mutagenesis was used to confirm functional status of the putative transcription factor binding sites. In vivo binding of transcription factors was determined using chromatin immunoprecipitation (ChIP). Effect of transcription factor knockdown on promoter activity was investigated using siRNA mediated gene silencing followed by transfection with the reporter constructs and measuring luciferase activity 24hrs later
Results :
Deletion mutagenesis indicated that −845 bases upstream of the transcription start site was sufficient to reconstitute near maximal promoter activity in BV2 cells. Deletion of sequences extending -593 to -520, which harbour an AP1, Ets.2 and Nkx3.1 site led to a dramatic decrease in both basal and LPS induced promoter activity (p<0.0001). Deletion of sequences extending -168 to -39, which contains four Sp1/Sp3 sites, led to a notable but non-significant decrease in promoter activity. Point mutations of AP1, Ets.2, Nkx3.1 and Sp1/Sp3 sites led to significant decreases (p<0.01) in promoter activity. ChIP-qPCR revealed that Pu.1, cJUN, cFOS, Stat3, Sp1, Sp3 and Sp4 bind the endogenous Tspo promoter. Notably, binding of these factors was significantly (p<0.05) enhanced upon LPS treatment. Silencing Pu.1, cJUN, cFOS, Sp1, Sp3, Sp4 and Stat3 gene expression dramatically (p<0.0001) lowered Tspo promoter activity
Conclusions :
Our data shows that binding sequences for AP1, Ets.2 and Sp1/Sp3 sites must be intact for maximal basal and LPS induced Tspo promoter activity in microglia. Furthermore, LPS-mediated increase in Tspo expression is accompanied by an enhanced recruitment of Pu.1, cJUN, cFOS, Sp1, Sp3, and Sp4 factors to the Tspo promoter
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.