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Fig. 3. HOXA13 activates transcription from the Bmp2 and Bmp7 enhancer regions, and associates with these enhancers in vivo. Co-transfection of NG108-15 cells with luciferase reporter plasmids containing forward or reverse orientations of the Bmp2 enhancer sequence (A,C), or the Bmp7 enhancer sequence (B,C), and pCMV-A13 resulted in 2.5- and 1.8-fold (Bmp2), and 1.8- and 2.0-fold (Bmp7), increases in RLA, when compared with identical transfections with the control pCMV vector. Luciferase activity was normalized for transfection efficiency using a Renilla Luciferase control plasmid in all co-transfection assays. Bars represent the standard deviation of results derived from four transfection assays. (D) Western blot analysis of protein lysates derived from Hoxa13 wild-type (+/+), heterozygous mutant (+/–) and homozygous mutant (–/–) tissues confirms that the Hoxa13 antibody recognizes proteins of the correct molecular weight for wild-type HOXA13 (43 kDa) and mutant HOXA13-GFP (64 kDa). (E) The Hoxa13 antibody immunoprecipitates HA-tagged full-length HOXA13. (F-I) Immunostaining of cultured limb mesenchyme from HOXA13-GFP mutant mice, using the Hoxa13 antibody, reveals strong nuclear co-localization (H, yellow, arrows) between the endogenous HOXA13-GFP protein (F) and the Hoxa13 antibody (G,H). Nuclei are stained with DAPI (I). (J-L) Chromatin immunoprecipitation using the Hoxa13 antibody confirms that wild-type (+/+) HOXA13 binds the Bmp2 (J) and Bmp7s1 (K) enhancer regions in the developing limb, whereas immunoprecipitates from mutant limbs (–/–) lacking the HOXA13 DNA-binding domain did not contain the Bmp2 and Bmp7s1 enhancer regions. (L) The absence of Bmp7s2 sequences in wild-type immunoprecipitates confirms HOXA13 specificity for the TAAT-containing sequences in Bmp2 and Bmp7s1. The TAAT-containing sequences present in the Bmp2, Bmp7s1 and Bmp7s2 regions are listed below panels J, K, and L. NC, negative PCR control; PC, positive PCR control.





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