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Fig. 6. Nkx2-mediated derepression of the Phox2b enhancer enhances Hox-dependent regulation at the PH site. (A-D) Fold activation of luciferase activity assayed from P19 cells co-transfected with different vector combinations (as indicated below the graphs) along with P2b_0.38/Luc (A) or p3xPH/Luc (C) carrying three copies of the PH site, or their mutated versions P2b_0.38mPH/Luc (B) and p3xmPH/Luc (D). The nucleotide changes in the PH site are shown in the boxes in B and D. Cooperative activation by Hoxb1, Pbx1a and Prep1 is further enhanced by Nkx2.2 or Nkx2.2HD-EnR repressor proteins (A,C), and it requires an intact PH site (B,D). Co-transfection of P2b_0.38/Luc with Nkx2.2HD-VP16 does not activate reporter expression (A), suggesting that Nkx2.2 HD does not bind to the enhancer. Co-transfection of Nkx2.2{Delta}TN, carrying a deletion of the Groucho-interacting domain (Muhr et al., 2001), almost abolishes Nkx2-dependent transcriptional cooperation (C). (E-G) Dorsolateral views (rostral to the left) of stage 17-18 chick embryo hindbrains electroporated with p3xPH/lacZ carrying three copies of the PH site (E), and with Nkx2.2 (F), or Nkx2.2HD-EnR (G) vectors. p3xPH/lacZ is weakly active and does not display spatially restricted reporter expression (E). (F,G) p3xPH/lacZ expression is enhanced throughout the hindbrain by Nkx2.2 (F) or Nkx2.2HD-EnR (G) repressors. (H,I) Model for integration of Hox-dependent activation and Nkx2-mediated derepression on the Phox2b enhancer. (H) In the ventral neural tube, dorsal to the pMNv domain (i.e. in the absence of Nkx2 factors) Phox2b is repressed, despite the presence of Hox activators and their Pbx and Prep co-factors, through the binding of a putative repressor (R) at, or in the vicinity of, the PH site. (I) In the pMNv domain, the repressor activity of Nkx2.2/Groucho (Gro) inhibits R, either directly or transcriptionally. After derepression, a Hox-Pbx-Prep ternary complex can bind to the PH and P/M sites and stimulate high levels of Phox2b transcription. Shh, sonic hedgehog; ov, otic vesicle.