Functional dissection of transcription factor proteins

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.

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. In this project, we developed a functional high-throughput assay (tAD-seq) and identified tADs for all Drosophila TFs. tAD-seq is based directly on the tADs defining function, namely the activation of reporter gene transcription, and use next-generation sequencing (NGS) combined with computational analyses that allows the screening of hundreds of TFs in a single experiment in parallel. We provided the first comprehensive annotation of tADs, which we validated using standard approaches and made publicly available to the scientific community. We further determined sequence composition and patterns (motifs) in the sequences of different tADs and validated the functional importance of these patterns by experimentally changing (mutating) the motifs and demonstrating that the corresponding tADs lost their functionality. This project provided the basis for a comprehensive annotation of Drosophila TFs and a high-throughput method that is used by us and others. Overall, our work lead to novel insights into the sequence and function of TFs and our understanding of gene expression more generally.