Elucidating the Role of Hox Transcription Factors in the Chromatin Context

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Many transcription factors (TFs) are expressed in multiple cell types, yet they control distinct transcriptional programs with highest precision in different cellular contexts. One of the most prominent examples of such broadly expressed but specifically acting TFs are the Hox proteins. Intriguingly, Hox TFs, despite being active in many cell types, can “reprogram” the regional identity of cells, which is under strong epigenetic control along the anterior-posterior axis of animals. This behavior suggests that Hox proteins can induce chromatin opening, allowing them to adapt lineage-specific gene expression programs to the regional requirements. However, the role of Hox proteins in the regulation of local chromatin state and configuration in vivo and the underlying mechanisms are still poorly understood. Building on our recent findings that the Hox TF Ubx interacts in a cell lineage restricted fashion with components of the Brahma chromatin remodelling complex and the fact that Hox TFs bind to open and closed chromatin to carry out dual roles in regional and lineage fate regulation, we will now explore the possibility that Hox TFs pioneer chromatin. We hypothesize that Hox chromatin pioneering is dependent on binding to low-affinity DNA motifs on nucleosomes and that generic cell lineage programs are tuned to region-specific requirements by interacting with lineage-restricted (pioneer) TFs. In order to rigorously test our hypothesis, we will resolve the modalities of Hox binding, the sequence requirements and the functional interaction with other (pioneer) TFs. To this end, we will employ state of the art genomic techniques, which will allow us to simultaneously probe TF chromatin binding and the local chromatin environment in vivo, without the need of binding site predictions, as well as complementing biochemical and genetic approaches. We will use Ubx and its lineage-specific interaction partners Tin and Grh as well as the generic interaction partner Exd, which has been shown to increase Hox binding to closed chromatin, as models, and will study their role in chromatin opening using in vitro, in cellulo and in vivo approaches. In sum, this work will be instrumental in resolving one of the major puzzles in Developmental Biology, which is how Hox TFs can function highly specifically in assigning regional identity while fulfilling at the same time generic functions in cell lineage stabilization.