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Published online before print September 7, 2007

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(Journal of Leukocyte Biology. 2007;82:1564-1574.)
© 2007 by Society for Leukocyte Biology

Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation

Karin Weigelt^*,1, Wolfgang Ernst^*,1, Yana Walczak^*, Stefanie Ebert^*, Thomas Loenhardt^*, Maja Klug, Michael Rehli, Bernhard H. F. Weber^* and Thomas Langmann^*,2

^* Institute of Human Genetics, University of Regensburg, and
Department of Hematology and Oncology, University Hospital Regensburg, Germany

²Correspondence: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. E-mail: thomas.langmann{at}klinik.uni-regensburg.de

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.

Key Words: Tyrobp • retinal degeneration • myeloid promoter

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