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. 2022 Jun 27;54(6):834–847. doi: 10.1038/s12276-022-00791-3

Fig. 5. TAS1 regulates H3K27ac enrichment in the TMPO promoter by recruiting FUS and p300 to form condensates.

Fig. 5

a, b Enrichment of the TMPO promoter by ChIP using an anti-H3K27ac antibody in KYSE150 and TE-11 cells with or without TAS1 KD were evaluated. The TMPO promoter level in the 10% input sample is set to 1. Primer locations in the TMPO promoter are shown at the bottom of Supplementary Fig. 5d. The primer set P3 was used to obtain the results shown (n = 3). c FUS in cell lysates was pulled down by biotin-labeled TAS1 but not its antisense RNA. d TAS1 binding proteins were detected using MS2-TRAP and WB analysis. TAS1-bound FUS was captured on anti-Flag antibody-conjugated affinity agarose beads; IP complexes were separated and identified using specific antibodies. e RIP assays indicated that TAS1 in ESCC cell lysates was enriched by FUS-specific antibodies. f, g ChIRP-purified DNA and proteins were analyzed using qPCR and western blotting, respectively. Odd, Even and Scr. denote the odd- and even-ranked corresponding probes for TAS1 and the negative control probes provided by RiboBio. The TMPO promoter region represented by P3 was enriched by the TAS1 probes. FUS and p300 proteins were also precipitated by the TAS1 probes in ESCC cells. The locations of the primers in the TMPO promoter are shown at the bottom of Supplementary Fig. 5d. h IF and FISH assays showed that TAS1, FUS and p300 were colocalized mostly in the nucleus and existed as puncta. Scale bar: 5 μm. i IF and FISH assays showed a reduction in the number of colocalized puncta formed by TAS1, FUS and p300 after TAS1 silencing in TE-11 cells. Scale bar: 5 μm. The data are presented as the mean±S.D. values. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.