Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis‐acting elements

Abstract

The Hoxc8 early enhancer that controls the initiation and establishment of Hoxc8 expression in the developing mouse embryo is found in different vertebrate lineages including mammals, birds and fish. Mouse and Fugu Hoxc8 early enhancers (200 bp) have diverged in the composition of elements located towards the 3 0 region. However, they share cis-acting elements A-E located in the 5 0 region. Mutations at these elements in the context of the mouse Hoxc8 early enhancer affect reporter gene expression in the posterior neural tube, somites and lateral plate mesoderm of day 9.5 mouse embryos. Here, we demonstrate that mutations introduced at the same elements but in the context of the Fugu Hoxc8 early enhancer had different consequences on the reporter gene expression in transgenic mouse embryos. Furthermore, in contrast to the mouse enhancer the Fugu enhancer does not utilize elements D and E in achieving posterior neural tube and somite expression. These results suggest that the diverged sequences prevent regulatory interactions at conserved cis-acting elements. We propose that divergent sequences modify regulatory interactions at conserved elements by providing a ''contextual change''. Our finding that the enhancer elements do not act in a unitary fashion but function in the context of the surrounding sequence brings a new dimension to the study of cis-regulatory evolution.

Salamander Hox clusters contain repetitive DNA and expanded non-coding regions: a typical Hoxstructure for non-mammalian tetrapod vertebrates?

“…Conserved non-coding sequences (CNS) were identified from regions flanking Hox exons; these likely correspond to enhancer elements and non-coding RNAs that function in the regulation of Hox gene expression. For example, two CNSs that were identified downstream of newt hoxd11 (40 kb) correspond to enhancer elements VIII and IX from Gerard et al [26], and a CNS upstream (3 kb) of newt hoxc8 corresponds to an enhancer from Shashikant et al [27]. Also, a CNS identified downstream of hoxc10 (28 kb) corresponds to human miRNA-196a, and a canonical miR-196a seed-pairing site is predicted 268 bp from the end of newt hoxd8 [28].…”

The Crest Phenotype in Chicken Is Associated with Ectopic Expression of HOXC8 in Cranial Skin

“…We therefore propose that Crest is caused by a cis-acting regulatory mutation that underlies the ectopic expression of HOXC8 that we have confirmed in four different breeds of Crested chickens. Such a regulatory mutation may be located in a region corresponding to one of the HOXC8 regulatory elements that have been defined in other species, including a microRNA binding site, an early enhancer element, and a long-range regulatory sequence in the downstream region [27], [28], [29]. However, we have not been able to identify the causative mutation as it has not yet been possible to assemble this chromosomal region.…”

Gdf11/Smad signalling and Cdx proteins cooperate to activate the Hoxc8 early enhancer in HepG2 cells

“…A further reductionist approach is now presented in the present paper where conditions are described for activation of mouse Hoxc8 early enhancer/reporter constructs in cell culture. HepG2 cells are used since they respond to Gdf11 with activation of the Smad2/3 signalling pathway (Andersson et al, (Shashikant et al, 2007, Wang et al, 2004. Blue box, the pair of putative Smad binding elements.…”

“…expression is activated by Cdx1 in Xenopus embryos (Schyr et al, 2012). 3) Hoxc8 early enhancer/lacZ transgene expression boundaries in mouse embryos are disrupted by mutations within two enhancer Cdx binding sites (Shashikant et al, 2007, Shashikant andRuddle, 1996). 4) EMSA studies reveal binding of Cdx2 to both of these Cdx binding sites (Taylor et al, 1997).…”