In all organisms, cell differentiation and survival depends on differential gene expression that is
tightly regulated by an intricate interplay of specialized DNA elements and regulatory proteins. One
class of regulatory proteins so called transcription factors (TFs) bind to DNA and activate the
transcription of genes from DNA to RNA, typically via the recruitment of a second layer of cofactor
proteins. Much is known about the TFs protein-DNA interactions, but little about their activating
functions. In particular, it is generally unknown where within the full-length TF protein sequences
these so-called trans-activating domains (TADs) are located and which sequence-properties are most
important for TAD function.
Here, we propose to identify TADs for all Drosophila TFs with a functional high-throughput assay
(TAD-seq) that we will develop for this purpose. We will base TAD-seq directly on the TADs defining
function, namely the activation of reporter gene transcription, and use next-generation sequencing
(NGS) combined with computational analyses as a powerful readout that allows the screening of
hundreds of TFs in a single experiment in parallel. We will perform TAD-seq using different reporter
genes, each selected to specifically assess the regulatory functions typical of strong global
activators, housekeeping activators, and combinatorial developmental regulators, different types
of TFs we characterized recently, and anticipate to identify TADs for each of these TF categories.
This aim will provide the first comprehensive annotation of TADs, which we will validate using
standard approaches and make publicly available to the scientific community. We will further search
for sequence patterns (motifs) that are repeated in the sequences of different TADs and might
therefore be particularly important for TAD functionality. We anticipate that this will allow the
identification of different motifs for the different TF categories, which can be further used to
computationally predict TADs in additional TFs, also across different species. We will validate these
predictions by experimentally changing (mutating) these motifs and test if the corresponding TADs
and TFs are still functional.
This project will provide the basis for a comprehensive annotation of Drosophila TFs and the
computational annotation of TFs more generally, as well as the prediction of how mutations in TF
sequences might alter TF functionality. Overall, our proposal will lead to novel insights into the
sequence and function of TFs and our understanding of gene expression more generally, key areas of
central significance especially today when the importance of gene expression during development
and disease is increasingly recognized and transcriptional regulation is becoming the focus of novel
therapeutic strategies.