Spi1 -14 Kb upstream regulatory element (URE) is not required for maintenance of PU.1 expression in macrophages

Abstract

Background: Previous work suggested an upstream regulatory element (URE) of Spi1 was required to maintain constant expression of the PU.1 transcription factor in bone marrow and foetal liver cells. PU.1, encoded by Spi1, is essential for development and maintenance of myeloid and B-lymphocyte populations in mice. Deletion of this (-14 Kb) URE potentially reduces expression of PU.1 and therefore provides a way to investigate its role in myeloid populations in development and disease. This study aimed to examine the impact of removal of the -14 Kb Spi1 URE in Cx3cr1+ cells on the myeloid lineage formation and maintenance. Methods: B6;129-Spi1tm1.2Dgt/J mice, whose -14 Kb Spi1 URE mice is flanked by LoxP sites (‘floxed’), were bred to a strain with constitutively active Cre expressed under the Cx3cr1 promoter (B6J.B6N(Cg)-Cx3cr1tm1.1(cre)Jung/J) to delete the Spi1 URE in myeloid cells. The floxed mice were also bred to mice with a tamoxifen-inducible Cre expressed under the Cx3cr1 promoter (B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J) to be used as URE intact controls and to permit temporally-controlled deletion of the URE if required. PU.1 protein expression was measured in the peritoneal macrophages and microglia by flow cytometry. Additionally, a Cre-encoding lentiviral vector was used to assess the impact on PU.1 expression in bone-marrow derived macrophages from these mice in vitro. Results: Expression of the PU.1 transcription factor was not significantly altered in the peritoneal macrophages or microglia in mice lacking the -14 Kb Spi1 URE. Moreover, initial experiments utilising Cre encoding lentivirus did not reduce PU.1 protein in bone-marrow derived macrophages differentiated from the -14 Kb Spi1 URE floxed mice. Conclusions: These observations suggest that the -14 Kb URE does not play a major role in PU.1 protein expression in either mature peritoneal macrophages or microglia.