Uncovering the Role of Intrinsically Disordered Regions in Regulating FOXG1-Chromatin Binding Dynamics

About This Grant

Project Summary/Abstract The forkhead box (FOX) family of transcription factors (TFs) regulate key biological processes across eukaryotes. One member, FOXG1, functions as a transcriptional repressor which regulates neurodevelopment within embryonic brains, and has been implicated in a range of neurodevelopmental disorders. The FOX family of TFs is defined by the presence of a conserved DNA-binding domain (DBD). The molecular mechanisms of this DBD on its own, and how mutations to this region impair its function and lead to disease, have been well studied. However, how the intrinsically disordered regions (IDRs) outside of the folded DBD influence FOX TF function remains understudied. There is a critical need to understand the role of these IDRs given that dozens of disease-associated mutations have been identified within the IDRs of FOXG1. In the proposed work, we aim to investigate the role of IDRs on FOXG1 chromatin binding, nuclear organization, and function in gene regulation. My preliminary data have revealed that FOXG1 forms nanoscale clusters in nuclei of live cells which drives its tight binding to chromatin. However, a disease-associated variant of FOXG1, which has truncations within its central IDR, no longer forms DNA-associated clusters. Additionally, I found that FOXG1 forms biomolecular condensates via phase separation which contributes to its association with DNA. Biomolecular condensates are dynamic structures often formed through IDR-mediated phase separation and have been recently implicated as an underlying mechanism for TF clustering in vivo to regulate gene expression at specific genomic loci. Intriguingly, we found that mutations within the same central IDR of FOXG1 which show a loss in nuclear clustering also show a loss of phase separation. We have identified a conserved enrichment of aromatic amino acids within the central IDR of FOXG1 using a state-of-the-art sequence analysis approach in collaboration with Dr. Rohit Pappu’s group at WashU. I have determined that these conserved aromatic residues are critical for FOXG1 phase separation, nuclear clustering, and chromatin association. From this preliminary data, I hypothesize that FOXG1 utilizes IDR-driven phase separation to form nuclear condensates which directly regulate its chromatin binding and function as a transcriptional repressor. We propose to test this hypothesis with a series of computational, biophysical, and cellular approaches designed to 1) identify critical sequence features within FOXG1 IDRs and predict their function on phase separation and chromatin binding, 2) quantify the effects that individual IDRs and critical amino acid residues have on FOXG1 phase separation, 3) determine how FOXG1 IDRs influence its DNA binding strength at a single-molecule level both in vitro and in cellulo, and 4) identify the role of FOXG1 IDRs in transcriptional repression. Collectively, the results from this study will provide critical insights from single-molecule to mesoscale levels into how IDRs regulate FOX TF activity and how mutations in these regions can lead to disease.