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).

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
1U19AI109976-01
Application #
8711623
Study Section
Special Emphasis Panel (ZAI1-ZL-I (J1))
Project Start
Project End
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
$642,435
Indirect Cost
$103,645
Name
La Jolla Institute
Department
Type
DUNS #
603880287
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Shaw, Laura A; Bélanger, Simon; Omilusik, Kyla D et al. (2016) Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation. Nat Immunol 17:834-43
Martinez, Gustavo J; Hu, Joyce K; Pereira, Renata M et al. (2016) Cutting Edge: NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection. J Immunol 196:2015-9
Pedros, Christophe; Zhang, Yaoyang; Hu, Joyce K et al. (2016) A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1. Nat Immunol 17:825-33
Crotty, Shane (2015) A brief history of T cell help to B cells. Nat Rev Immunol 15:185-9
Nance, J Philip; Bélanger, Simon; Johnston, Robert J et al. (2015) Bcl6 middle domain repressor function is required for T follicular helper cell differentiation and utilizes the corepressor MTA3. Proc Natl Acad Sci U S A 112:13324-9
Martinez, Gustavo J; Pereira, Renata M; Äijö, Tarmo et al. (2015) The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells. Immunity 42:265-78
Hatzi, Katerina; Nance, J Philip; Kroenke, Mark A et al. (2015) BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms. J Exp Med 212:539-53
Choi, Youn Soo; Gullicksrud, Jodi A; Xing, Shaojun et al. (2015) LEF-1 and TCF-1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6. Nat Immunol 16:980-90
Nance, J Philip; Bélanger, Simon; Johnston, Robert J et al. (2015) Cutting edge: T follicular helper cell differentiation is defective in the absence of Bcl6 BTB repressor domain function. J Immunol 194:5599-603
Crotty, Shane; Pipkin, Matthew E (2015) In vivo RNAi screens: concepts and applications. Trends Immunol 36:315-22

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