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. 2012 Sep 6;287(44):37282–37295. doi: 10.1074/jbc.M112.408401

FIGURE 6.

FIGURE 6.

NKX2-1 binds to Grhl2 intronic regions and increases transcription. A, shown is the binding pattern of NKX2-1 protein to the Grhl2 intron identified in a global ChIP-on-chip analysis of NKX2-1 binding profiles in E11.5 mouse lung epithelium (23). H and L identify the binding regions; H1, H2, and L1 identify the probes used in EMSAs. B, shown is qRT-PCR analysis of Nkx2-1, Grhl2, and Tert expression in MLE15 cells infected with Nkx2-1-shRNA or non-silencing-shRNA. The data set was normalized against Gapdh (n = 3). C, a representative ChIP-PCR analysis shows NKX2-1 binding to the Grhl2 intron. ChIP was performed with chromatin from E11.5 day lung bud immunoprecipitated with NKX2-1-specific antibody or its isotype control. Immunoprecipitated and input DNA fragments were amplified using primers corresponding to the Grhl2 intron. D, shown is a scheme of the Grhl2 gene depicting, first, and second exons (black boxes) and first intron (gray box). Numbers are relative to the ATG (+1). Green (H1), blue (H2), and red boxes (L1) represent probes used in EMSAs. Arrows indicate oligonucleotides used in ChIP-PCR analyses. E, left panel, EMSA analysis shows binding of MLE15 nuclear proteins to probe H1 (lane 1), competition with 100-fold unlabeled probe (lane 2), super-shift of the complex using NKX2-1 antibody (lane 3), and control with IgG. The single asterisk marks the H1-protein complex. The double asterisk marks the super-shifted complex. Right panel, EMSA analysis shows binding of MLE15 nuclear proteins to probe L1 (lane 1), competition with 100-fold unlabeled probe (lane 2), super-shift of complex formation using Nkx2-1 antibody (lane 3), and control with IgG. The single asterisk marks the L1-protein complex. The double asterisk marks the super-shifted complex. F, MLE15 cells were co-transfected with empty luciferase plasmid (pGL3; 0-Luc-plasmid), Sv40 promoter-luciferase (Sv40Luc), Grhl2 intron fragment H inserted 5′ to Sv40-luciferase plasmid (H-Sv40Luc), or Grhl2 intron fragment L inserted 5′ to Sv40-luciferase plasmid (L-Sv40Luc) with CMV-Nkx2-1 or pCMV plasmids. Firefly luciferase was normalized to Renilla luciferase levels (n = 5). Data represent the mean ± S.E. # indicates p ≤ 0.05.