In response to activating signals, NFKB and STATS are reversibly methylated on specific lysine residues by chromatin remodeling enzymes, in ways that profoundly affect their functions. We propose that reversible methylation occurs, in concert with histone methylation, when the factors are bound to specific promoters, altering the strength and duration of inducible gene expression, giving plasticity to the dependent biological responses, and helping to explain specific responses to different pro-inflammatory signals.
Aim 1. We will conduct global gene expression analyses in cells expressing STATS or the p65 subunit of NFKB in which the lysine methylation sites have been mutated and will also evaluate whether different activators of NFKB (IL-6 versus TNF versus TLRs) impose different patterns of phosphorylation on p65 as """"""""bar codes"""""""" that in turn affect lysine methylation patterns at different cohorts of target genes.
Aim 2. We will extend initial observations that methylafions take place on promoter-bound STATS and NFKB as part of an ordered sequence of events leading to transcriptional activation. Extensive use will be made of chromatin immunoprecipitation (CHIP) assays to determine the fime courses of events at specific promoters that are regulated differentially by lysine methylation of STATS or p65. We also will use innovative mass spectrometric approaches to identify unknown proteins that assemble on specific promoters as a function of time following activation of STATS or NFKB.
Aim 3. For lysine residues that are not in direct contact with DNA, methylation may facilitate the binding of accessory proteins that alter function. These proteins will be identified by using a GST protein domain microarray and by immunopreciptation assays. K218 and K221 of p65 do contact DNA, so we will explore these interactions by studying the binding of the pure methylated protein to DNA directly.
Aim 4. IRFS, TIS7 and IRAK4 are important modulators of NFKB activafion and funcfion. We will determine how each affects the phosphorylation and methylation of p65 and the dependent patterns of NpKB-dependent gene expression. Examples of genes whose activafion is modulated will then be studied in detail as above, to define promoter-specific mechanisms. In Projects 1, 3 and 4, the ways in which the activation of these genes by NFKB is modulated will also be studied in primary mouse cells and in mouse tumor models. In this way, the mechanistic analyses of Project 2 will connect directly to the biological analyses of the other three projects.

Public Health Relevance

Recent evidence has shown us that promoter-bound transcription factors are modified by the same enzymes that modify histones, with important functional consequences. The modifications are gene-specific, and a full understanding of how they occur, and of how they affect the responses to major pro-inflammatory cytokines (IL-1, IL-6, TNF, TLRs) through their activation of NFKB and STATS, is important for understanding fully how the interactions of cells of the immune system with epithelial cells lead to cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA062220-19
Application #
8607130
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
19
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Herjan, Tomasz; Hong, Lingzi; Bubenik, Jodi et al. (2018) IL-17-receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling. Nat Immunol 19:354-365
Veleeparambil, Manoj; Poddar, Darshana; Abdulkhalek, Samar et al. (2018) Constitutively Bound EGFR-Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal. J Immunol 200:2809-2818
Nan, Jing; Wang, Yuxin; Yang, Jinbo et al. (2018) IRF9 and unphosphorylated STAT2 cooperate with NF-?B to drive IL6 expression. Proc Natl Acad Sci U S A 115:3906-3911
Sarvestani, Samaneh K; Signs, Steven A; Lefebvre, Veronique et al. (2018) Cancer-predicting transcriptomic and epigenetic signatures revealed for ulcerative colitis in patient-derived epithelial organoids. Oncotarget 9:28717-28730
Cai, Gang; Zhu, Liang; Chen, Xing et al. (2018) TRAF4 binds to the juxtamembrane region of EGFR directly and promotes kinase activation. Proc Natl Acad Sci U S A 115:11531-11536
Liu, Weiwei; Liu, Xia'nan; Li, Yu et al. (2017) LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection. J Exp Med 214:3051-3066
Zepp, Jarod A; Zhao, Junjie; Liu, Caini et al. (2017) IL-17A-Induced PLET1 Expression Contributes to Tissue Repair and Colon Tumorigenesis. J Immunol 199:3849-3857
Zhou, Hao; Bulek, Katarzyna; Li, Xiao et al. (2017) IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs. Elife 6:
Wang, Chenhui; Zhang, Cun-Jin; Martin, Bradley N et al. (2017) IL-17 induced NOTCH1 activation in oligodendrocyte progenitor cells enhances proliferation and inflammatory gene expression. Nat Commun 8:15508
Wang, Yuxin; Nan, Jing; Willard, Belinda et al. (2017) Negative regulation of type I IFN signaling by phosphorylation of STAT2 on T387. EMBO J 36:202-212

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