FIGURE 3.

Lhx8 directly regulates TrkA promoter activity. Placental alkaline phosphatase (PLAP) reporter constructs are schematically shown (A). The predicted Lhx-binding sequence in the TrkA promoter is shown. Its core region (ATTAATT) is shown in bold and underlined. In the TrkA mutant promoter, the Lhx core sequence was changed from ATTATTT to ATCGCGT, as shown in red. B, C, and G, PC12 cells were transiently co-transfected with reporter constructs and GFP or Lhx8 expression vector. Twenty four hours after transfection, the cells were treated with 25 ng/ml NGF, 10 μm U0126, 10 μm LY294002, 2 μm H89, or combinations as indicated. Placental alkaline phosphatase activity was assayed after 72 h of culture with or without NGF. Data are presented as the mean ± S.E. n = 5; **, p < 0.01; Student's t test. D and E, ChIP assay performed using PC12 cells transfected with GFP or Lhx8 tagged with human influenza hemagglutinin peptide (HA-Lhx8) expression vector. Soluble chromatin was immunoprecipitated with anti-HA antibody. Immunoprecipitates were subjected to PCR with a primer pair specific for the TrkA promoter (D). Quantitative PCR analysis was performed to measure the amount of DNA (E). Data are presented as the mean ± S.E. n = 3; **, p < 0.01; Student's t test in E. F, primary cultured septum neurons at 3 DIV were infected with AAV-GFP or AAV-Lhx8 with AAV-TrkA promoter placental alkaline phosphatase for 4 days, and placental alkaline phosphatase activity was assayed. Data are presented as the mean ± S.E. n = 5; **, p < 0.01; Student's t test.