PROJECT SUMMARY Gene regulatory networks (GRNs) involving interactions between transcription factors (TFs) and cis- regulatory elements (CREs) are central to most biological processes. Research in my laboratory aims to characterize the structure and logic of human GRNs with the ultimate goal of devising therapeutic strategies. Current gaps in our understanding of GRNs include: determining how TF combinations provide transcriptional specificity, determining how GRNs are rewired in response to environmental cues and pathogens, and designing strategies to manipulate GRNs to modulate biological outcomes. Cytokines present an archetype model to study GRNs because cytokines genes are highly regulated at the transcriptional level in different cell types and stimulation conditions. We have recently delineated a large- scale cytokine GRN by determining the binding of individual TFs to cytokine promoter regions and integrating with expression datasets. Here, we will expand these studies to identify the role of heterodimeric TFs in cytokine regulation by integrating TF-wide screens using a novel heterodimer yeast one-hybrid approach we developed, and multi-CUT&tag to profile TF heterodimer binding genome-wide. This will identify the rules by which heterodimers confer transcriptional specificity and will provide targets for modulation of cytokine expression in disease. Viruses can rewire or leverage host GRNs for productive viral replication and to evade immune responses. Given the coexistence of viral genomes and host TFs and host genomes and viral TF within infected cells, this provides the opportunity for viral-host cross- transcriptional regulation. For example, CREs of DNA viruses and retroviruses can bind host TFs to regulate viral gene expression in the appropriate cell types and states to promote viral replication or latency. We will identify the viral CREs from dsDNA viruses and retroviruses that are active in different cell types and activation states using high-throughput reporter assays, as well as the human TFs that regulate these viral CREs. Viral TFs can bind to host CREs to modulate the expression of host genes to promote cell proliferation, metabolic changes, and immune suppression. We will continue to characterize the function of viral TFs including identifying targets, transcriptional activity, protein-protein interactions, and changes in transcriptional programs. Altogether, the proposed studies will identify general principles and generate a framework to study and manipulate immune GRNs, and will identify mechanisms by which viruses control host GRNs to replicate and evade immune responses.