TWIST1-centric Protein Interactome and Gene Regulation Network in Craniofacial Development

Abstract

During craniofacial development, the morphogenesis, regionalization and patterning of primordial tissues is orchestrated by activities of key transcriptional regulators. These factors constitute the central nodes of a gene regulatory network (GRN) and signaling pathways for head development. The basic helix-loop-helix domain (bHLH) transcription factor TWIST1 is one of these key regulators. It is frequently mutated in human Saethre-Chotzen Syndrome (SCS). Protein-protein interaction including dimerization with other bHLH proteins is hypothesised to endorse and regulate the diverse roles of TWIST1 in cranial mesenchymal cell behaviour. The broad aim of this project is to identify these protein cooperates with TWIST1 at the top of the head GRN hierarchy, and understand their mode of interaction, using systems biology approach. Using the BioID proteomic approach, we captured proteins that interact with TWIST1 in native cellular environment, and identified these interactors by mass-spectrometry. Bioinformatics analysis of mass-spec data generated biologically relevant TWIST1 partners in three cell lines, the mouse cranial neural crest stem cell line, mesenchymal progenitor cell and fibroblast. Cross comparison with public datasets revealed both known and novel interacting partners. The TWIST1 interactome comprises interaction hubs featuring transcription factors, chromatin modifiers and RNA-binding proteins. Notably, mutations in key hub nodes, such as CHD7, CHD8 and WHSC1 are also associated with congenital craniofacial malformations that share features with SCS. To understand the biological meaning of these novel interactions, published RNA-seq and ChIP-seq datasets of TWIST1 and selected BioID partners were analysed. These data indicated that TWIST1 and its partners cooperatively binds to regulatory regions of target genes, including genes in cell migration and neurogenesis. The concerted function of TWIST1 and its partners in regulating these common target genes were validated in CRISPR knockout embryoid bodies. Moreover, TWIST1 and some of its protein partners collectively regulate cell migration and osteogenic differentiation in progenitor cell lines.