In vivo motif selectivity and functionality of TALE family TFs

Parent grant Summary: TALE transcription factors (TFs) are broadly expressed and act as cofactors to several classes of essential TFs – e.g. Hox proteins – in embryogenesis and cellular homeostasis, but their exact functions remain enigmatic. 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 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 in vivo. Because most 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 prefer distinct motifs

in vivo? What is the mechanistic basis of divergent in vivo binding selectivity and how does it impact function? 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 small. We have addressed this by generating ChIP-seq and RNA-seq data for members of the TALE TF family 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 their near-identical homeodomains and binding 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 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 manipulate the balance of TALE TFs to define the functional consequences of different TALE TFs occupying the

same genomic sites in vivo. Lastly, we will 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. Our findings will also

be applicable to other homeodomain TFs (the 2nd largest class of TFs) and other multi-member TF families.