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. 2018 Sep 18;293(44):17154–17165. doi: 10.1074/jbc.RA118.003055

Figure 5.

Figure 5.

lncRNA GAS8-AS1 activates GAS8 expression via recruiting the MLL1/WDR5 complex to the GAS8 promoter and modulating promoter H3K4me3. A, knockdown of GAS8-AS1 with its siRNAs in HepG2 or SMMC7721 cells leads to significantly decreased mRNA and protein expression of GAS8. B, lncRNA GAS8-AS1 predominantly locates in the nuclear fraction in HCC cells. C, DNA fragments a, b, c, d, and e around the promoter region of GAS8 are detected using ChIP-qPCR. D, ChIP-qPCR results showed reduced H3K4me3 and RNA Pol II occupancy at the GAS8 promoter after silenced expression of endogenous lncRNA GAS8-AS1. E, RNA pulldown was performed by incubating in vitro–transcribed biotinylated transcripts of GAS8-AS1 or antisense GAS8-AS1 (negative control) with nucleus HepG2 cell extracts followed by detection of the presence of the GAS8-AS1 RNP complex by Western blotting. Both the GAS8-AS1 RNP complex components, MLL1 and WDR5, were detected in GAS8-AS1 pulldown, but not in GAS8-AS1 antisense pulldown. F, knockdown of GAS8-AS1 significantly inhibits MLL1/WDR5-mediated H3K4me3 of the GAS8 promoter in HepG2 cells. G, RNA-IP assays suggested that GAS8-AS1 may physically interact with the MLL1/WDR5 complex. Results of the mean of triplicate assays with S.D. (error bars) are presented. *, p < 0.05; **, p < 0.01; ***, p < 0.001.