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Figure 7


Fig. 7. 5' HoxD proteins are bound to and directly activate transcription from the Shh limb enhancer (ShhE). (A) ChIP analyses on E10.5 mouse fore- and hindlimbs, using specific anti-Hoxd10 ({alpha}Hoxd10), anti-Hoxd13 ({alpha}Hoxd13) and anti-Pbx ({alpha}Pbx) antisera, demonstrate their direct in vivo binding to the mouse ShhE. A 424 bp fragment of the ShhE was amplified by PCR. No PCR amplification of a 373 bp negative-control region (Co) located 1400 bp upstream of this enhancer was detected using E10.5 limb chromatin. Representative reactions of all PCR amplifications, carried out in triplicate, are shown. (B) ChIP on pooled E13.5 limbs using the {alpha}Hoxd13 antiserum revealed no amplification of the ShhE. (C) At E10.25 and E11, Pbx1 (blue) and Shh (purple) mRNA transcripts do not colocalize in wild-type and Pbx2–/– (KO) hindlimbs. (D) HoxD proteins can activate transcription from the ShhE (eightfold for Hoxd9, fourfold for Hoxd10 and threefold for Hoxd13) in P19 embryonal carcinoma cells. Luciferase activity, in arbitrary units (R.L.U.), was assayed from extracts of transiently transfected P19 cells. Co-transfection assays were performed in the presence (+) of the indicated expression vectors encoding Pbx2, Hoxd9, Hoxd10 and Hoxd13, and with a luciferase reporter construct (pT81mShhE) containing the ShhE. Bars represent the mean±s.e.m. of at least four independent experiments. (E) Simplified model (top) depicts the overlapping genetic hierarchical roles of Pbx1 and Pbx2 in controlling Hox gene spatial distribution and Hox recruitment to the limb ShhE. In addition, Pbx2, albeit not functionally essential within this network, is also recruited to this enhancer in limb cells. Intensity of the hue of Pbx2 correlates with its functional relevance. Lower left diagram illustrates the requirement of Pbx1 (violet), Pbx2 (rose) and Pbx1/Pbx2 (purple) for controlling the positioning of 5' Hox genes (i.e. Hoxa/Hoxd, orange) to future Shh-expressing domain (green). In Pbx1–/–;Pbx2+/– (mutant) limbs, Hox gene expression remains external to the ZPA, and Shh activation does not occur (red cross). Lower right diagram depicts our refinement of a previous model (Zakany et al., 2004) and highlights that Shh loss, owing to early Pbx1/Pbx2 perturbation, leads to partial disruption of 5' Hoxd reverse colinearity (yellow), but not of 5' Hoxa expression (deep orange). Proximal is towards the left; anterior is towards the top. Co, control; FL, forelimb; HL, hindlimb; I, input chromatin; NR, non-specific control antibody; NoAb, no antibody-control resin.