SUMMARY The TET2 DNA dioxygenase regulates gene expression by catalyzing demethylation of 5-methylcytosine, thus epigenetically modulating the genome. TET2 does not contain a sequence-specific DNA-binding domain, and how it is recruited to specific genomic sites is not fully understood. Here we carried out a mammalian two-hybrid screen and identified multiple transcriptional regulators potentially interacting with TET2. The SMAD nuclear interacting protein 1 (SNIP1) physically interacts with TET2 and bridges TET2 to bind several transcription factors, including c-MYC. SNIP1 recruits TET2 to the promoters of c-MYC target genes, including those involved in DNA damage response and cell viability. TET2 protects cells from DNA damage-induced apoptosis dependending on SNIP1. Our observations uncover a mechanism for targeting TET2 to specific promoters through a ternary interaction with a co-activator and many sequence-specific DNA-binding factors. This study also reveals a TET2-SNIP1-c-MYC pathway in mediating DNA damage response, thereby connecting epigenetic control to maintenance of genome stability.

SUMMARY MyD88, the intracellular adaptor of most TLRs, mediates either pro-inflammatory or immunosuppressive signaling that contributes to chronic inflammation-associated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of MyD88 interactome, we found that in endotoxin-tolerant macrophages PP2Ac enhances its association with MyD88, and is constitutively activated. Knockdown of PP2Ac prevents suppression of pro-inflammatory genes and resistance to apoptosis. Through sitespecific dephosphorylation constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex, and broadly suppresses the activities of multiple pro-inflammatory/proapoptotic pathways as well, shifting pro-inflammatory MyD88 signaling to a pro-survival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence pro-inflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions.

Inflammation is tightly regulated by nuclear factor-kappa B (NF-kappaB), and if left unchecked excessive NF-kappaB activation for cytokine overproduction can lead to various pathogenic consequences including carcinogenesis. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), can be used to explore possible mechanisms whereby unknown functional pathways modulate the NF-kappaB activity for regulating TNF-alpha-induced inflammation. Given the multifunctional nature of 14-3-3 family proteins and the recent finding of their presence in the TNF-alpha/NF-kappaB pathway network, we used a dual-tagging quantitative proteomic method to first profile the TNF-alpha-inducible interacting partners of 14-3-3 epsilon, the least characterized 14-3-3 isomer in the family. For the first time, we found that TNF-alpha stimulation enhances the interactions between 14-3-3 epsilon and some key components in the mitogen-activated protein kinase (MAPK) signal module which is located at the immediate upstream of NF-kappaB, including transforming growth factor-beta activated kinase-1 (TAK1) and its interacting protein, protein phosphatase 2C beta (PPM1B). By using confocal laser scanning, we observed the TNF-alpha-induced colocalizations among 14-3-3 epsilon, TAK1, and protein phosphatase 2C beta (PPM1B), and these interactions were also TNF-alpha-inducible in different cell types. Further, we found that during the full course of the cellular response to TNF-alpha, the interactions between 14-3-3 epsilon and these two proteins were dynamic and were closely correlated with the time course-dependent changes in NF-kappaB activity, suggesting that these 14-3-3 epsilon interactions are the critical points of convergence for TNF-alpha signaling for modulating NF-kappaB activity. We then postulated a mechanistic view describing how 14-3-3 epsilon coordinates its dynamic interactions with TAK1 and PPM1B for differentially modulating TNF-alpha-induced changes in NF-kappaB activity. By using bioinformatics tools, we constructed the network involving most of the 14-3-3 epsilon interacting proteins identified in our proteomic study. We revealed that 14-3-3 epsilon coordinates the cross talks between the MAPK signal module and other molecular pathways/biological processes primarily including protein metabolism and synthesis, DNA repair, and cell cycle regulation where pharmacological targets for therapeutic intervention could be systematically located.

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