Stage-Dependent and Locus-Specific Role of Histone Demethylase Jumonji D3 (JMJD3) in the Embryonic Stages of Lung Development
Figure 6
Jmjd3 coregulates SP-B expression with Nkx2.1 and Brg1.
(A) The interaction between Jmjd3 and Nkx2.1 was evaluated by coimmunoprecipitation (CoIP) analysis of 293T cells expressing HA-tagged Jmjd3 and FLAG-tagged Nkx2.1. (B) A cell-based luciferase assay was used to evaluate SP-B promoter activity in 293T cells cotransfected with Nkx2.1, Jmjd3, and mouse SP-B promoter-linked episomal luciferase vector (containing Nkx2.1 binding sites). (C) The interaction between Jmjd3 and Brg1 was determined by CoIP analysis of 293T cells expressing HA-tagged Jmjd3 and FLAG-tagged Brg1. (D) Cell-based luciferase assay was used to evaluate SP-B promoter activity in 293T cells cotransfected with Nkx2.1, Jmjd3, Brg1, and mouse SP-B promoter-linked episomal luciferase vector. *P<0.05 (Student's t test). (E) Generation of Jmjd3 deletion constructs. Numbers represent the corresponding amino acid residue position in the Jmjd3 coding region. (F) CoIP analysis of the interaction between Nkx2.1 and HA-tagged truncated Jmjd3 proteins. (G) CoIP analysis of the interaction between Brg1 and truncated Jmjd3 proteins. (H) CoIP analysis of the interaction among endogenous Nkx2.1, Brg1, and Jmjd3 in WT and Jmjd3-deficient lung tissues. (I) ChIP-PCR and ChIP-qPCR analysis of Nkx2.1 and Brg1 binding to SP-B promoter regions in WT and Jmjd3-deficient lung tissues. *P<0.05 (Student's t test).