NAT10 promotes cancer metastasis by modulating p300/CBP activity through chromatin-associated tRNA [RNA-ChIP]

Acetylation of proteins and RNA is crucial for development and cancer progression. NAT10 is the sole RNA acetylase responsible for N4-acetylcytidine (ac4C) modification of various RNAs. Our study reveals that NAT10 loss significantly reduces lung metastasis in breast cancer mouse models. NAT10 interacts with a mechanosensitive, metastasis-susceptibility protein complex at the nuclear pore. Mechanistically, NAT10-mediated acetylation of chromatin-associated tRNAs enhances p300/CBP activity. Without NAT10, acetylation of these tRNAs decreases, leading to p300/CBP inactivation and mislocalization. As a result, NAT10 depletion disrupts enhancer organization, altering gene transcription critical for metastasis, including reduced chemokines that recruit myeloid cells and create a less metastasis-prone tumor microenvironment. Our findings highlight the distinct role of NAT10 in acetylated tRNA-dependent regulation of enhancer function in metastatic tumor cells and its impact on tumor-immune interactions influencing metastasis. Overall design: We performed p300 RNA immunoprecipitation followed by sequencing of chromatin-associated RNAs (RNA-ChIP) of WT and Nat10 knockout (KO-c43) 4T1 mouse mammary tumor cells. We followed the protocol described in RNA Chromatin Immunoprecipitation (RNA-ChIP) kit (Active Motif). Briefly, 4T1 cells were cultured in 15 cm dishes at a seeding density of 4 × 106 cells per dish. After 48 hours, the cells were fixed with 1% formaldehyde for 15 minutes at room temperature. The fixation was quenched by adding 1x Glycine Stop-Fix solution. Cells were then harvested by scraping, followed by centrifugation at 3,500 rpm for 10 minutes at 4°C. For nuclei isolation, the cell pellets were lysed in 1 ml of ice-cold Complete Lysis Buffer for 30 minutes and then homogenized using a Dounce homogenizer with 50 strokes. The release of nuclei was confirmed under a brightfield microscope. The lysates were centrifuged at 5,000 rpm for 10 minutes at 4°C, and the nuclear pellet was resuspended in Complete Shearing Buffer containing RNase inhibitors. Chromatin was sheared using a Bioruptor Plus sonicator (Diagenode) for 3 cycles (30 seconds on, 30 seconds off) at 4°C. The sheared chromatin samples were then centrifuged at 18,000 x g for 10 minutes at 4°C, and the supernatant containing the chromatin fraction was transferred to fresh tubes. The concentration of chromatin was determined by isolating RNA from an aliquot of the chromatin preparation. RNA-ChIP reactions were performed using three biological replicates for both WT and KO samples, with each reaction containing the equivalent of 6 µg of RNA from chromatin. Immunoprecipitation was conducted in a 150 µl reaction volume, consisting of 25 µl of pre-washed Protein G Magnetic Beads, RNA-IP buffer, RNase inhibitors, Protease Inhibitor Cocktail, and 2 µg of rabbit p300 antibody (Abcam). The reaction mixture was incubated overnight on a rotator at 4°C. The beads were then washed four times with Complete RNA-ChIP Wash Buffer 1, followed by two washes with Complete RNA-ChIP Wash Buffer 2. RNA was eluted from the beads with Complete RNA-ChIP Elution Buffer by rotating on an end-to-end rotator for 15 minutes at room temperature. The supernatant containing the eluted RNA was transferred to a fresh tube after magnetic separation of the beads. Both the immunoprecipitated RNA and input RNA samples were treated with proteinase K and reverse cross-linked at 65°C for 1.5 hours. The RNA was then purified using TriPure Isolation Reagent (Millipore-Sigma). To remove residual DNA, the purified RNA was treated with DNase I, followed by further purification using the RNA Clean & Concentrator-5 Kit (Zymo).