TALE family transcription factors (TFs) are ubiquitously expressed and act as cofactors to several classes of essential TFs ? most prominently Hox proteins ? in embryogenesis and cellular homeostasis, but their exact functions remain enigmatic. For instance, two family members ? Prep and Meis ? bind identical sequence motifs in vitro and share the ability to dimerize with Pbx proteins, suggesting that they function interchangeably. However, loss-of-function analyses in several systems suggest that their roles diverge in vivo. Furthermore, a third TALE family member (TGIF) shares DNA binding motif preference with Prep and Meis, but it is unclear if these three TFs compete or compensate for each other in vivo. Because most known TFs belong to larger families that share DNA and protein:protein interaction properties, similar questions about shared and divergent functions vex our understanding of most TF families. Further, since TF function in vivo is subject to constraints not encountered in vitro, it is essential to evaluate their functional properties in native systems. Indeed, numerous fundamental questions about TF activity remain to be addressed in vivo. Perhaps most importantly, do TFs with similar in vitro binding selectivity select distinct motifs in vivo? What is the mechanistic basis of divergent in vivo binding selectivity and how does it impact function? The answers to these questions will have profound implications for our understanding of embryogenesis and disease, but progress in this area has been hampered by major barriers. Specifically, access to multiple genome-wide data sets for closely related TFs has been limited ? particularly during embryogenesis, when cell numbers are often small. We have addressed this by generating ChIP-seq and RNA-seq data for members of the TALE family of TFs at multiple stages of zebrafish development ? thereby establishing one of the most comprehensive sets of data available for a single TF family. In spite of containing near-identical homeodomains and binding to indistinguishable motifs in vitro, we find that Prep and Meis TFs display divergent binding preferences in vivo. Also, Prep, but not Meis, occupies a novel non-Hox related genomic element in vivo. These initial observations underscore the importance of exploring TF function in their native environment and highlight the strong technical and conceptual position of our research group to pursue these analyses further. Based on our preliminary findings, we hypothesize that emergent in vivo constraints restrict TALE TF motif selectivity and that dynamic exchange among TALE members controls transcriptional outcome. To test this hypothesis, we will first express wild-type and domain-swapped TF constructs in zebrafish to define the mechanistic basis of TALE TF binding selectivity in vivo. Second, we will use gain- and loss-of-function approaches to manipulate the balance of TALE TFs in order to define the functional consequences of different TALE TFs occupying the same genomic binding sites in vivo. Lastly, we will use loss-of-function animals and CRISPR-mediated deletions to examine the in vivo role for a novel TALE-occupied motif. Since TALE TFs are implicated in several cancers, our in vivo delineation of the dynamic interplay between TALE family members will directly impact our understanding of both embryogenesis and oncogenesis. Conceptually, our findings will also be applicable to other homeodomain TFs (constituting the 2nd largest class of TFs) and other multi-member TF families.

R01GM142158

2021-05-01

2025-03-31