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Dap12 expression in activated microglia from retinoschisin‐deficient retina and its PU.1‐dependent promoter regulation
Author(s) -
Weigelt Karin,
Ernst Wolfgang,
Walczak Yana,
Ebert Stefanie,
Loenhardt Thomas,
Klug Maja,
Rehli Michael,
Weber Bernhard H. F.,
Langmann Thomas
Publication year - 2007
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.0707447
Subject(s) - microglia , biology , chromatin immunoprecipitation , gene knockdown , microbiology and biotechnology , transcription factor , cell culture , promoter , gene expression , immunology , gene , genetics , inflammation
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.

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