Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation.
Several alterations in the expression of immune-related transcripts were identified recently in the degenerating retina of the retinoschisin knockout (Rs1h(-/Y)) mouse, including the strong expression of the adaptor protein Dap12. As Dap12 is found in leukocytes, we hypothesized that its disease-related expression may be confined to activated retinal microglia cells. To test this hypothesis, we established a procedure for isolation and culture of retinal microglia cells and performed genome-wide expression profiling from Rs1h(-/Y) and control microglia. While retaining their activated state in culture, ex vivo microglia expressed high levels of Dap12 and the transcription factor PU.1. The activation-dependent induction of Dap12 was also confirmed in the microglia cell line BV-2 following in vitro stimulation. To examine the transcriptional regulation of Dap12 further, macrophage cell lines were transfected with several Dap12 reporter constructs. Promoter deletion assays and site-directed mutagenesis experiments demonstrated an essential role of evolutionarily conserved PU.1 consensus sites in the proximal -104/+118 Dap12 promoter. In vitro and in vivo binding of PU.1 to this promoter region was demonstrated using EMSA and chromatin immunoprecipitation. Knockdown of PU.1 by RNA interference caused a significant reduction of endogenous Dap12 expression and re-expression, and activation of PU.1 in PU.1(-/-) progenitor cells induced Dap12 transcription. Taken together, our results indicate that activated microglia from degenerating retinae express high levels of Dap12 and PU.1, and PU.1 controls the myeloid-specific regulation of Dap12 directly and may also play a general role in microglia gene expression during retinal degeneration.
Weigelt
Karin
K
Institute of Human Genetics, University of Regensburg, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
Ernst
Wolfgang
W
Walczak
Yana
Y
Ebert
Stefanie
S
Loenhardt
Thomas
T
Klug
Maja
M
Rehli
Michael
M
Weber
Bernhard H F
BH
Langmann
Thomas
T
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000172:deficiency*; D002469:Cell Separation; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005136:Eye Proteins; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000378:metabolism*; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D013234:Stem Cells / Q000378:metabolism; D015534:Trans-Activators / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1