Activators and repressors of NFkappaB and IRF3/7 in innate immunity Abstract: Much is known about pathways that activate NF-kB, a central coordinator of innate and adaptive immune responses, but there is still much to learn about how NF-kB is kept in check in unstressed cells. We will use insertional mutagenesis to identify novel inhibitors of NF-kB activation. We will also collaborate with Xiaoxia Li and Ganes Sen to uncover and study novel positive and negative regulators of pathways that activate NF-kB, IRF7 and IRF3 in response to Toll-like receptor 8 (TLR8) and TLR3, using genetic selections that their labs have already established. We have developed a novel viral vector that introduces new promoters into many loci in the genome. In the resulting dominant mutants, the promoter, which drives the over-expression of a protein encoded by downstream DNA, can be removed at will, proving that the mutation was caused by the insertion. We have used this method to identify the lysine demethylase FBXL11 as a novel negative regulator of NF-kB whose expression is regulated by NF-kB.
Specific Aim 1 A: we will determine the mechanism of action of FBXL11.
Specific Aim 1 B: we will study additional mutants in which the expression of different negative regulators has been driven by inserted promoters.
Specific Aim 2 : we will study signaling pathways that respond to TLR8, which is normally activated by single-stranded RNA.
Specific Aim 2 A: we will isolate dominant mutants in which the activation of TLR8-dependent promoters is suppressed by negative regulators.
Specific Aim 2 B: we will isolate dominant mutants in which the constitutive activation of an IRF7-dependent promoter is stimulated by positive regulators.
Specific Aim 2 C: we will isolate recessive mutants in which the expression of a protein required for signaling has been ablated, using a gene-trap vector in combination with a strategy to promote loss or inactivation of the remaining unmutated allele of the targeted gene.
Specific Aim 2 D: we will isolate recessive mutants in which NF-kB has been activated constitutively.
Specific Aim 2 E: we will investigate how the novel components or negative regulators of the TLR8 pathway that we have newly identified function in signaling.
Specific Aim 3 : we will study signaling pathways that respond to double-stranded RNA (dsRNA), which activates NF-kB and IRF3 through TLR3 and also through an internal pathway involving RIG-I and homologous proteins.
Specific Aim 3 A: we will identify cellular proteins that function as dominant negative regulators of the response of TLR3-responsive promoters to dsRNA.
Specific Aim 3 B: we will isolate dominant mutants in which an IRF3-responsive promoter has been activated constitutively, to identify positively acting proteins.
Specific Aim 3 C: we will investigate how newly identified novel components or negative regulators of pathways that respond to dsRNA function in signaling. Our combination of robust selective systems and powerful new genetic methods can add much to the current understanding of pathways that drive and regulate important mediators of innate immunity. Uncontrolled inflammation is a major health problem that impacts many different diseases, including cancer. We use novel genetic methods to study the normal positive and negative controls that keep inflammatory pathways in check when they are not needed and activate them when they are. Our findings have the potential to lead to new therapeutic approaches in several disease settings.
|Stark, George R; Darnell Jr, James E (2012) The JAK-STAT pathway at twenty. Immunity 36:503-14|
|Sears, Nathaniel; Sen, Ganes C; Stark, George R et al. (2011) Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation. J Biol Chem 286:33037-44|
|Wan, Youzhong; Kim, Tae Whan; Yu, Minjia et al. (2011) The dual functions of IL-1 receptor-associated kinase 2 in TLR9-mediated IFN and proinflammatory cytokine production. J Immunol 186:3006-14|
|Guo, Canhui; Stark, George R (2011) FER tyrosine kinase (FER) overexpression mediates resistance to quinacrine through EGF-dependent activation of NF-kappaB. Proc Natl Acad Sci U S A 108:7968-73|
|Lu, Tao; Jackson, Mark W; Wang, Benlian et al. (2010) Regulation of NF-kappaB by NSD1/FBXL11-dependent reversible lysine methylation of p65. Proc Natl Acad Sci U S A 107:46-51|
|Lu, Tao; Jackson, Mark W; Singhi, Aatur D et al. (2009) Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB. Proc Natl Acad Sci U S A 106:16339-44|
|Dasgupta, Maupali; Agarwal, Mukesh K; Varley, Patrick et al. (2008) Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB. Cell Cycle 7:2249-56|
|Kandel, Eugene S; Lu, Tao; Wan, Youzhong et al. (2005) Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB. Proc Natl Acad Sci U S A 102:6425-30|
|Lu, Tao; Stark, George R (2004) Cytokine overexpression and constitutive NFkappaB in cancer. Cell Cycle 3:1114-7|