Temporal Regulation of mRNA Biogenesis in Immune Responses

PROJECT SUMMARY The innate immune response is highly regulated and the precise timing of gene regulation is crucial for cellular survival. A primary feature of initial innate immune response cascades is the timely mounting of transcriptional and processing steps that underlie immune gene expression, with extensive rewiring of mRNA levels and compositions. Thus, the rates at which mature mRNAs are produced during immune responses might be a critical regulatory step. Published and preliminary studies suggest gene-specific kinetic regulation of mRNA biogenesis may underlie changes in gene expression levels and transcriptome complexity in the early innate immune response. Yet, it remains unclear how the temporal coordination between transcriptional and co-transcriptional processes governs the timing of immune gene expression and if this co-regulation underlies the ability to mount a productive immune response upon first encounter with a stimulus. Gene-specific co-regulation of mRNA biogenesis rates may also allow for efficient immune responses after repeated stimulation. Some innate immune cells, such as macrophages, undergo epigenetic reprogramming following initial exposure to immunogenic signals to mount long-term memory, a phenomenon called trained immunity. Trained macrophages exhibit a faster, more robust innate immune response to resist reinfection, suggesting that epigenetic changes may enable gene-specific kinetic regulation of mRNA biogenesis. How the kinetics of transcription and splicing are regulated by epigenetic changes during immune responses is unknown. This project seeks to uncover mechanistic insights into the regulation and impact of stimulus-induced immune mRNA biogenesis. This work will test the hypothesis that cellular immune responses are driven by a series of regulated changes in the kinetics of transcriptional and co-transcriptional processes, ultimately determining gene expression dynamics over response time. Aim 1 will examine coordination of transcription and splicing kinetics using lipopolysaccharide (LPS)-stimulated human monocyte-derived macrophages (MDMs). Simultaneous sequencing of nascent and mature RNA at non-overlapping 15-minute intervals over a 2-hour stimulation time course will be used to characterize how transcription and splicing rates govern the timing of early immune gene expression. The functional output of key cytokines will be measured in cell supernatants over the time course. Aim 2 will explore how trained immunity-associated epigenetic rewiring impacts transcriptional and co- transcriptional kinetics in MDMs. Trained and un-trained MDMs will be stimulated with LPS in a 2-hour time course as in Aim 1 to assess transcription and splicing rates, then harvested to assess chromatin accessibility. Cytokine output will also be assessed over time, as in Aim 1. Overall, this work will elucidate the mechanisms by which epigenetic rewiring drives resistance to reinfection in trained immunity, illuminate how kinetic regulation of transcriptional and co-transcriptional processes contributes to innate immune phenotypes, and provide the fellow with training in RNA biology, innate immune signaling, computational biology, and immunogenomics.