MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.icroRNAs (miRNAs) are small, noncoding RNAs, 19-24 nt in length, with gene-expression regulatory functions (1, 2) and are expressed aberrantly in most types of cancer (3, 4). MiRNAs also have been detected in the blood of cancer patients (5, 6) and can serve as circulating biomarkers (7). It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells (8) by canonical binding to their target messenger RNAs (8, 9). More recently, it has been demonstrated that, in addition to their role as gene-expression regulators, miRNAs also directly interact with proteins (10).Members of the Toll-like receptor (TLR) family (namely, murine TLR7 and human TLR8) can recognize and bind viral single-stranded RNA (ssRNA) sequences on dendritic cells and B lymphocytes, leading to cell activation and cytokine production (11,12). TLRs are a family of receptors through which the mammalian innate immune system recognizes the presence of invading pathogens (13,14). Both murine TLR7 and human TLR8 bind to and are activated by 20-nt-long ssRNAs, which represent physiological ligands for these two receptors (12), located in intracellular endosomes. Circulating mature miRNAs are 19-24 nt in length and could represent tumor-released ligands of TLR7 and TLR8 involved in intercellular communication in the tumor microenvironment. Results and Discussion Identification of Specific miRNAs Released in Cancer Cell-DerivedExosomes. To identify which miRNAs are present in tumor-secreted exosomes, we isolated exosomes from the supernatant of A-549 and SK-MES lung cancer cell lines. First, we assessed the purified supernatant exosome fraction for enrichment in CD9 and CD63, two known exosome markers (SI Appendix, Fig. S1A) (8,15). By performing NanoString analysis, we observed that nine miRNAs (miR-16, -21, -27b, -29a, -133a, -193a-3p, -544, -563, and -1283) were present in exosomes derived from ...

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of Kto-Q mutations in the REL homology domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/ 221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/ 221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.protein methylation | histone modifying enzyme M embers of the nuclear factor κB (NF-κB) family are central coordinators of innate and adaptive immune responses. Of the five family members in mammals [RelA (p65), RelB, c-Rel, NF-κB 1 (p50), and NF-κB2 (p52)], the p65/p50 heterodimer functions most often, in the "classic" signaling pathway (1). In unstimulated cells, NF-κB dimers are retained in an inactive state in the cytoplasm through binding to a member of the Inhibitor of κB (IκB) family. Activation of NF-κB is catalyzed by IκB kinase (IKK), followed by the phosphorylation and degradation of IκB, the phosphorylation of NF-κB itself, and the liberation of phosphorylated NF-κB dimers, which translocate to the nucleus and activate the transcription of target genes (2). The p65 subunit of NF-κB can be phosphorylated on more than a dozen different serine and threonine residues in response to activating stimuli, leading to differential gene regulation and dependent biological effects. Mutation of these phosphorylation sites modulates activity, affecting the ability of p65 to transactivate some promoters, but leaving others essentially unaffected (3). It seems likely that additional posttranslational modifications of p65 also occur in a signal-dependent manner, and that differential posttranslational modifications of p65 are likely to be responsible for the differences in p65 function that have been observed in response to different activating stimuli (4). The activity of NF-κB is regulated by many different stimuli in all cell types, with many different functional consequences (4). Regardless of the stimulus, all pathways leading to NF-κB activation involve posttranslational modifications of IKK, IκB, and NF-κB itself, including phosphorylation, ubiquitination, acetylation, sumoylation, and nitrosylation, and the specific modifications that occur depend on the nature of the inducing stimulus (5). Recent dis...

We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis.

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