Partially Redundant Enhancers Cooperatively Maintain Mammalian Pomc Expression Above a Critical Functional Threshold
Fig 1
The Pomc neuronal enhancers nPE1 and nPE2 Share a common cis-regulatory code.
(A) Evolutionary tree of placental mammalian lineages. The relative lengths of DNA sequences in kilobases (kb) separating nPE1 from nPE2 and nPE2 from exon 1 are illustrated by purple and green bars, respectively. (B) Alignment between an almost palindromic sequence carrying canonical homeodomain binding sites (HDBS) present in nPE1core and a remarkably similar sequence present within nPE2 region 1. (C) Scheme of nPE1core and regions 1 and 3 of nPE2 showing the relative positions of conserved HDBS. nPE1core carries a pair of inverted HDBS similar to another present in nPE2 region 1 (green boxes, full sequences depicted in Fig. 1B). Another pair of HDBS is present in region 3 of nPE2 (purple boxes). Canonical HDBS of the NKX subfamily are shown (blue boxes). Red letters indicate the mutated nucleotides. Grey boxes denote the critical enhancer regions determined previously in transgenic mice. (D) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE1core. The control nPE1corePomc-EGFP carries the wild-type (wt) enhancer sequence whereas nPE1core(mut)Pomc-EGFP (mut) carries six nucleotide substitutions covering all HDBS (red letters in Fig. 1C). Coronal brain sections showing EGFP expression in hypothalamic arcuate nucleus of nPE1corePomc-EGFP (Left) but not in nPE1core(mut)Pomc-EGFP transgenic founder newborn mice (Right). (E) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE2. The control transgene nPE2Pomc-EGFP carries the wild-type (wt) enhancer whereas nPE2(mut)Pomc-EGFP (mut) carries twelve nucleotide substitutions covering all HDBS (red letters in Fig. 1C). EGFP is expressed in the arcuate nucleus of founder transgenic mice carrying nPE2Pomc-EGFP (Left) but not nPE2(mut)Pomc-EGFP (Right). 3V, third ventricle.