HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression. (A) HMGA2 and GATA6 co-localized in the nuclei of the same cells of the distal lung epithelium. Fluorescence microscopy of embryonic lung sections (E14.5) after double immunostaining using HMGA2- and GATA6-specific antibodies. Nuclear staining with DAPI (blue). Square shows details at higher magnification. Scale bars, 40 μm. (B) Hmga2-KO decreased expression of Gata6 and its downstream target gene Fzd2. Expression analysis of the indicated genes as in Figure 4D. Rel nor exp, relative expression normalized to Tuba1a. Data are presented as mean ± SEM (n = 4). ***P <0.001; **P <0.01 after one-way analysis of variance. (C) Protein extracts from WT (+/+) or Hmga2−/− embryonic lung (E18.5) were analyzed as in Figure 4C using the indicated antibodies. (D) Hmga2 over-expression increased Gata6 transcription. Top, Luciferase reporter assays of MLE-12 cells transiently transfected with a Gata6-Luc reporter plasmid and control (Ctrl, empty vector) or Hmga2 expression construct. RLU, relative light units. Bottom, Gata6 expression was monitored by quantitative RT-PCR in MLE-12 cells treated as above. Rel nor exp, relative expression normalized to Gapdh. Data are presented as mean ± SEM (n = 4) and asterisks as in B. (E) Endogenous HMGA2 binds to the endogenous Gata6 promoter. Top, in silico analysis of the Gata6 gene (-631 to +1 base pairs relative to transcription initiation site) revealed several HMGA2 binding elements (squares). Arrows, position of the primers used for ChIP. Bottom, ChIP of the Gata6 promoter using HMGA2-specific antibody or IgG (negative control). ChIP, chromatin-immunoprecipitation assay; IgG, immunoglobulin G. Data are presented as mean ± SEM (n = 4) and asterisks as in B.