Our long-range goal is to elucidate the chromatin regulatory factors that establish and maintain CD8+ cytotoxic T lymphocyte (CTL) differentiation in the context of viral infection. We showed that lL-2 receptor (IL- 2R) stimulation intensity regulates effector and memory CTL development transcriptionally via differential chromatin remodeling and RNA Polymerase II (Pol 11) recruitment; and that the capacity of resting memory CTL to induce effector genes rapidly upon secondary stimulation involves unharnessing pre-recruited RNA Pol 11 complexes that are paused. Our hypothesis is that differential usage of subunits in chromatin regulatory complexes underlies both the transcriptional specificity of effector and memory CTL differentiation in response to IL-2R signaling and the poised chromatin configuration of effector genes in memory CTL. There are approximately 300 genes in the mammalian genome with potential chromatin regulatory functions. Together with the Crotty laboratory, we developed a pooled approach using shRNAmirs in vivo, to screen hundreds of genes in parallel in T cells for their roles during acute viral infections in mice. A preliminary screen identified Smarca4, one essential ATPase of BAF-chromatin remodeling complexes, as being required for effector CTL development. To address our hypothesis, our specific objectives are to dissect the requirement for all genes encoding BAF-complex subunits during CTL differentiation in vivo and to clarify their molecular and biological roles in establishing protective CTL responses (Aim 1); to identify the chromatin regulators that mediate IL-2R dependent gene regulation in CTL using an arrayed shRNAmir screen in vitro, and decipher how they control transcription and viral protection in vivo (Aim 2); and, to interrogate all potential chromatin regulators in CDS T cells in a pooled screen during viral infection to identify those that program effector versus memory CTL formation, and to begin revealing the chromatin crosstalk that underlies CTL differentiation (Aim 3). Our studies synergize with analogous approaches that will discover the conventional transcription factors that control antiviral CTL development (Project 3, Goldrath) and both transcription factors and chromatin regulators in antiviral CD4 T cells (Project 1, Crotty).
To develop protective antiviral T cells, a specific family of chromatin regulatory genes must activate expression of another class of 'effector' genes to endow naive T cells with antiviral effector functions. Using the first in vivo approach to disable hundreds of individual genes of an entire family in parallel with short hairpin RNAs, we will elucidate in T cells which regulator genes induce the antiviral program during viral infection.
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