For many organisms, the ability to regenerate damaged tissues diminishes with increasing maturity. The mechanisms responsible are unknown, but their identification is essential to promote tissue regeneration in adult organisms. Drosophila imaginal discs, when damaged in situ, are able to regenerate, but this ability diminishes dramatically during the third larval instar. This decline correlates with a reduction in the ability to upregulate wingless (wg, WNT1) and WNT6 expression following damage. Since WNT proteins are upregulated following injury in diverse taxa, the mechanisms that regulate WNT expression following tissue damage are likely conserved.
To understand how damage-induced wg expression is activated, we have characterized an enhancer that regulates the expression of two flanking genes, wg and WNT6, during regeneration. Deletion of this enhancer permits normal development but compromises regeneration. Dissection of the genomic region reveals a bipartite structure that includes a damage-responsive module and a silencing element. The damage-responsive module is robustly activated following various damaging stimuli, requiring the JNK pathway. Notably, this activity is undiminished with increasing maturity. In contrast, an adjacent element, which has no enhancer activity on its own, can progressively attenuate expression mediated by the damage-responsive module by nucleating highly localized epigenetic silencing at the enhancer. Cas9-mediated deletion of this silencing element abolishes silencing and restores damage-induced WNT expression in mature discs, demonstrating that the silencing element is indeed responsible for the age-related loss of damage-induced WNT expression in vivo. Importantly, this epigenetic silencing remains restricted to the enhancer, thus allowing adjacent enhancers that are regulated by developmental signals to remain accessible.
As the majority of genes implicated in regeneration also function during development, until now it has been difficult to explain how a maturing organism can limit the capacity to regenerate without compromising normal growth and differentiation. The localized silencing of damage-responsive enhancers is a mechanism that can prevent the activation of genes following injury, while still allowing those same genes to be expressed by developmental signals. In addition to wg and Wnt6, this mechanism likely regulates many other regeneration genes. Using sequence motif analysis we have identified a similar bipartite enhancer within the MMP1 gene. Alongside genome-wide chromatin structure analysis, we will use these approaches to identify other such regulatory regions, and thus comprehensively define the genetic basis for declining regenerative capacity of maturing tissue.