Ataxia-Telangiectasia, Mutated (ATM)/Nuclear Factor κ light chain enhancer of activated B cells (NFκB) signaling controls basal and DNA damage-induced transglutaminase 2 expression.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that cross-links proteins and its overexpression, linked to a drug resistant phenotype, is commonly observed in cancer cells. Further, up-regulation of TG2 expression occurs during response to various forms of cell stress; however, the molecular mechanisms that drive inducible expression of the TG2 gene (TGM2) require elucidation. Here we show that genotoxic stress induces TG2 expression through the Ataxia-Telangiectasia, Mutated (ATM)/Nuclear Factor κ light chain enhancer of activated B cells (NFκB) signaling pathway. We further document that NFκB is both necessary and sufficient to drive constitutive TG2 expression in cultured cell lines. Additionally, shRNA-mediated knockdown or pharmacological inhibition of the ATM kinase results in reduced constitutive TG2 expression and NFκB transcriptional activity. We document that the NFκB subunit p65 (RelA) interacts with two independent consensus NFκB binding sites within the TGM2 promoter, that mutation of either site or pharmacological inhibition of NFκB reduces TGM2 promoter activity, and genotoxic stress drives heightened association of p65 with the TGM2 promoter. Finally, we observed that knockdown of either p65 or ATM in MDA-MB-468 breast cancer cells expressing recombinant TG2 partially reduces resistance to doxorubicin, indicating that the drug resistance linked to overexpression of TG2 functions, in part, through p65 and ATM. This work establishes a novel ATM-dependent signaling loop where TG2 and NFκB activate each other resulting in sustained activation of NFκB and acquisition of a drug-resistant phenotype.

Exonuclease 1 (Exo1) is required for activating response to S(N)1 DNA methylating agents.

S(N)1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O(6) position of guanine (O(6)meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O(6)meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S(N)1 methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S(N)1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.