Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development

Abstract

The products of Hox genes function in assigning positional identity along the anterior-posterior body axis during animal development. In mouse embryos, Hox genes located at the 3' end of HoxA and HoxB complexes are expressed in nested patterns in the progenitors of the secondary heart field during early cardiogenesis and the combined activities of both of these clusters are required for proper looping of the heart. Using Hox bacterial artificial chromosomes (BACs), transposon reporters, and transgenic analyses in mice, we present the identification of several novel enhancers flanking the HoxB complex which can work over a long range to mediate dynamic reporter expression in the endoderm and embryonic heart during development. These enhancers respond to exogenously added retinoic acid and we have identified two retinoic acid response elements (RAREs) within these control modules that play a role in potentiating their regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these control modules, spread throughout the flanking intergenic region, have regulatory activities that overlap with other local enhancers. This suggests that they function as shadow enhancers to modulate the expression of genes from the HoxB complex during cardiac development. Regulatory analysis of the HoxA complex reveals that it also has enhancers in the 3' flanking region which contain RAREs and have the potential to modulate expression in endoderm and heart tissues. Together, the similarities in their location, enhancer output, and dependence on retinoid signaling suggest that a conserved cis-regulatory cassette located in the 3' proximal regions adjacent to the HoxA and HoxB complexes evolved to modulate Hox gene expression during mammalian cardiac and endoderm development. This suggests a common regulatory mechanism, whereby the conserved control modules act over a long range on multiple Hox genes to generate nested patterns of HoxA and HoxB expression during cardiogenesis.

Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress

“…Mammals retain Nppa and Nppb, conserved among species and positioned at a distance of only 15 kb from each other in the genome (Inoue et al, 2003). Several studies of clustered genes describe coordinated regulation by shared regulatory sequences (Nolte et al, 2013;Tena et al, 2011). Given the highly similar spatiotemporal expression patterns of Nppa and Nppb, we hypothesized that these clustered genes might also share regulatory elements.…”

“…They are positioned at 10 to 15 kb distance from each other in the mammalian genome. Analysis of other gene clusters, such as Iroquois and Hox, revealed that multiple genes within a cluster have similar expression patterns and are regulated by shared regulatory sequences (Nolte et al, 2013;Tena et al, 2011). These features may underlie the evolutionary stability of such clusters.…”

Retinoic Acid and the Development of the Endoderm

“…RA is able to shift the expression pattern of Hox genes in the pharyngeal mesoderm [11,94,98,99] and thus it is likely that RA can also regulate Hox expression in the pharyngeal endoderm [101]. Hox genes are known to vary in response to different concentrations of RA in many tissues and reporter assays for RA activity suggest that such a gradient of RA signaling exists in the pharyngeal endoderm [53,99,104].…”

“…Subdivision of the Cdx expression domain into different regions such as the large and small intestine correspond to expression of different members of the Hox gene cluster and it is likely that these are essential for defining the different fates as mutations in different Hox genes do cause gut malformations [130]. Given the essential role for RA in directly regulating the Hox gene cluster [104,135], it would appear likely that subdivision of the hindgut would be regulated, at least in part, by RA.…”

Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies

“…Interestingly, we also observed derepression of yR7 enh in outer PRs in dve mutants, suggesting that yR7 enh serves as an inducible backup or 'dark' shadow enhancer in these cells. Shadow enhancers are DNA elements that drive redundant expression patterns and ensure robust gene expression in cases of genetic and environmental perturbation (Bothma et al, 2015;Frankel et al, 2010;Hong et al, 2008;Miller et al, 2014;Nolte et al, 2013;Perry et al, 2010;Wunderlich et al, 2015). yR7 enh represents an unusual 'dark' shadow enhancer as it is normally repressed and only becomes active when Dve driven by the primary outer enh is compromised.…”