The Role of Hox Transcription Factors in Cellular Plasticity

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Multicellular animals owe their complexity to their capacity to produce and maintain different lineages composed of multiple cell types that share virtually the same genomic DNA. Cell fates are controlled by networks of transcription factors (TFs) that act in the context of the genomic chromatin state to activate transcriptional programs realizing the distinct properties of cells of a given lineage. However, how TFs control different cell fates is still un unsolved question in biology. Hox TFs represent an excellent model to address this fundamental problem, since they are active in all lineages along the anterior-posterior (AP) axis of bilaterian animals. Our preliminary data using Ubx as a model show that Hox TFs, besides their well-described function in specifying regional identities along the AP axis, play a major role in the development and diversification of lineages, and that one of the mechanisms is the lineage-specific recruitment or stabilization of the Polycomb complex to Ubx chromatin sites for the repression of alternative fate and early lineage specification genes. Our results also imply that the Hox-encoded restriction of cellular and temporal plasticity is required for stably maintaining cell fates throughout the lifetime of a cell. One fundamental question arising from these findings is how an already committed lineage will develop in the absence of any functional Hox input and whether Hox-free cells are more plastic that Hox-expressing cells. We will tackle these questions by analyzing how a Hox-free cellular environment affects lineage development. To this end, we will eliminate the Hox code in the mesodermal lineage and will record the effects at the transcriptome level. In addition, we will characterize the identity and morphology of the lineage on the phenotypic level. To elucidate the mechanistic basis of the Hox-encoded suppression of multipotency, we will identify those genomic regions that are critical for lineage stabilization, as they are converted into an open and activatable status in the absence of Hox TFs. As a proof of concept, we will select candidate regions and test their function in lineage stabilization in vivo by lineage-specifically deleting these regions using CRISPR/Cas9 mediated genome engineering. And finally, we will compare the developmental potential of Hox-free and Hox-expressing cells to elucidate whether cells “stripped” of their Hox code are more plastic and can thus more easily change their identity when stimulated than cells expressing the inherent Hox code. To this end, we will mis-express a master-regulator of an alternative lineage in control and Hox-depleted mesodermal cells in vivo and will compare their phenotypic and molecular identities. In sum, we envision this study to be crucial in resolving the mechanistic basis of Hox-encoded lineage stabilization, which might prove to be highly relevant for reprogramming strategies.