Collaborative Research: Out of one, many: paralog-specific regulons from a single network architecture

About This Grant

Life depends on intricate control of cellular decisions, and the sophistication of this control scales with organismal complexity. This is nicely illustrated by transcription factors (TFs), DNA-binding proteins that turn genes off and on in a carefully choreographed manner. In both plants and animals, the number and type of TF families dramatically increased throughout evolution, primarily through gene duplication. After duplication, TF family members often took on new regulatory functions, in a process termed functional divergence. This expanded the regulatory toolkit of organisms, giving them increasingly sophisticated control over decisions ranging from stress response to immunity to development. Our group recently described a mechanism of TF functional divergence operating in plants and animals which we termed ‘differential usage of shared binding sites’. This proposal will map the underlying regulatory properties of this deeply understudied mechanism using cutting-edge multi-omic techniques. A key goal of the proposal is to forge a long-term collaboration between an R1 institute (Penn) and an undergraduate teaching institution (Lincoln University (LU)). Students reap maximum benefits when research is sustained throughout the academic year at the students’ home institutes. We will model this philosophy by building local research capacity at LU and providing a paired summer research experience at Penn, enabling year-round student-led research. We believe this will reduce scientific disengagement by prioritizing retention – not just initial exposure – of students to research. Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. This proposal will begin to address this knowledge gap using CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) proteins as a model. This ancient family of TFs proliferated over the course of evolution to regulate nearly all aspects of plant development through functionally redundant and functionally divergent activities. We recently discovered that two co-expressed functionally divergent HD-ZIPIII family members – CORONA and PHABULOSA – bind to a nearly overlapping set of genes. Despite this, each paralog has hundreds of uniquely regulated direct targets. Thus, HD-ZIPIII TFs do not generate their specific transcriptional outcomes by binding to distinct sites in the genome. Instead, these outcomes emerge by paralog-specific interpretation of a commonly bound network of genes. We termed this mechanism differential usage of shared binding sites, and showed it depends in part on their lipid binding StAR-related transfer (START) domain. However, its underlying regulatory properties remain unknown. This proposal will begin to characterize this newly identified mechanism controlling HD-ZIPIII specificity in two Aims. Aim 1 will formally link differential usage of shared binding sites to HD-ZIPIII paralog divergence, then identify and functionally characterize its causative genomic enhancer sequences. Aim 2 will test whether paralog-specific transcriptional outputs are generated by interacting partners using proteomic approaches. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.