The limbs of tetrapod vertebrates are composed of four basic segments: the limb, forelimb, wrist/ankle, and hand/foot. The identity of these segments is determined during limb formation by specific combinations of Hox genes. As the limb bud grows away from the main body axis, two waves of Hoxd gene expression occur. The first wave specifies the limb and forelimb. Then, following a short period of low Hoxd expression, a second wave of expression specifies the hand and foot portions. Cells that are produced during the transient step between these waves will form the wrist and ankle. Remarkably, these two phases of expression result from topological changes within the HoxD gene cluster and flanking regulatory domains. During formation of the arm and forearm, enhancers in the telomeric regulatory domain (T-DOM) drive the first collinear wave of expression. Then in proliferating distal limb bud cells, the T-DOM is inactivated, followed shortly by activation of the centromeric regulatory domain enhancers (C-DOM), driving the second wave of Hoxd expression and formation of the hand. The HoxA transcription factor HOXA13 is essential to this transition, acting to repress T-DOM and then activating C-DOM. The goal of the work proposed here is to understand how changes in enhancer status drive the topological transition between the two waves of Hoxd expression. To do this, we will perform two key experiments to challenge the conversion of each regulatory domain from active to inactive, or vice-versa. Specifically, we will first disrupt the activation of C-DOM by deleting an important autopod (hand/foot) enhancer called II-1. Our preliminary data indicates that HOXA13 acts through II-1 to drive the second wave of Hoxd expression. We will then target a copy of this enhancer into T-DOM, providing HOXA13 with an enhancer element that it normally acts through to activate C-DOM, but now within the chromatin context of T-DOM, which is being silenced in the same cells. We will evaluate changes in the chromatin state induced by these genetic perturbations by monitoring chromatin modifications via histone ChIP-Seq (H3K27me3, H3K4me1, and H3K27Ac) and ATAC-Seq. We will monitor for changes in gene expression quantity by RNA-Seq, and changes in the location of expression by whole-mount in situ hybridization. Furthermore, because we expect these mutations to induce large-scale changes in chromatin topology and possibly force continued activity from native T-DOM enhancers, we will utilize Capture Hi-C to monitor changes in HoxD topology. One prediction from these experiments is that each modified regulatory domain will resist complete conversion from one state to the other, due to the influence of this enhancer, with significant changes in chromosomal topology and gene expression. This experimental approach mirrors some natural mutations in chromatin structure that influence human development and health. Together these experiments will shed light on the molecular mechanisms of limb patterning, developmental gene regulation, and the influence of enhancers on genome topology.
The goal of the proposed work is to understand how Hoxd gene expression is controlled during limb development, but the findings will also be broadly applicable to all gene expression during embryonic development. This is important because disruption of Hoxd expression leads to congenital limb malformations and disease in humans.