Rewiring gene regulatory networks: Characterizing the effects of TF mutations and alternative isoforms

Transcription factors (TFs) can regulate hundreds of target genes through sequence-specific DNA binding to cis-regulatory sites. Thus, changes in TF function, especially in DNA binding, can have profound effects on interactions within gene regulatory networks. Advances in sequencing technology have revealed that such TF variants are not rare: healthy individuals, surprisingly, can carry dozens of coding mutations in TF DNA binding domains (DBDs), and ~40% of currently annotated TF alternative isoforms demonstrate altered DBD sequences compared with those of reference forms. It is unclear which of these variants actually alter DNA binding. To better understand how TF mutations affect DNA binding, we have characterized dozens of TF variants in vitro using Protein Binding Microarrays in an attempt to develop a classifier geared specifically to predict the effects of mutations on TF–DNA binding. Separately, we are characterizing TF isoforms by HT-SELEX to understand how alternative splicing may be a cellular strategy to regulate TFs’ DNA-binding function.