Understanding Hox epigenetic memory formation during motor neuron differentiation

Project Summary Hox genes are crucial for patterning the early embryo along the anterior-posterior axis in all bilateral animal species. Hox dysregulation leads to severe developmental defects, including homeotic transformations. During early developmental time points and in pluripotent stem cells, Hox genes are repressed by the Polycomb group

complexes (PcG). They are then activated by extracellular patterning signals like retinoid acid (RA) acting on RAREs (retinoic acid response elements), which activate the anterior Hox genes within the cluster. A CCCTC- binding factor (CTCF)-dependent boundary between Hox5 and Hox6 acts as an insulator, allowing for the RA

signaling to activate only anterior genes maintaining PcG repression on posterior Hox genes. Recent developments in the field have characterized a sufficient boundary element to include CTCF and MAZ, a myc- associated zinc finger protein. Thus, this project aims to study activators and repressors further, which will be

crucial for explaining the minimal elements sufficient to recapitulate epigenetic memory. By taking advantage of synthetic DNA technology developed in collaboration with the Boeke lab I can insert a highly editable synthetic Hox cluster (SynHoxA) into a pre-determined ectopic locus. This system allows for highly sensitive

transcriptional and chromatin analyses of different SynHoxA variants. Using this system, we recently showed that the ectopic SynHox cluster itself contains all the information necessary to decode patterning signals. Thus, I propose to use the SynHoxA system to dissect the remaining two regulatory components required for a Hox

cluster to respond to patterning signals. Aim 1 will tackle the relative contributions of RARE by testing whether the anterior Hox activation domain is a product of the additive RARE activity or whether each RARE activates specific Hox genes. I will measure the transcriptional output and chromatin modifications of SynHox variants

carrying mutations of RARE to compare RARE activation of the anterior cluster. Aim 2 will identify PcG recruitment elements (PREs) and nucleation sites, which have not been identified in mammalian cells nor in the HoxA cluster. Although the ectopic SynHoxA cluster recruits PRC II, we find no evidence of it interacting

with PRC II nucleation sites in trans. Therefore, I will generate overlapping constructs that can undergo a typical promoter bashing strategy to isolate “PRE-like” minimal elements. Finally, I will create a minimal construct containing all three elements required for epigenetic memory: activators (RARE), repressors (PcG),

and boundary elements (CTCF+MAZ). This study will elucidate how Hox clusters receive patterning signal information and store it into stable epigenetic memory. This model will add to previous studies that have looked at the binding behavior of the activators without the context of chromatin boundaries and the establishment of transient signals and vice

versa.