Research Paper Volume 12, Issue 14 pp 14754—14774

Figure 6. DSCAM-AS1 regulated DCTPP1 transcription by modulating histone acetylation. (A) The epigenetic regulation of DCTPP1 expression was analyzed using the UCSC Genome Browser Gateway. The DCTPP1 promoter was influenced by histone 3 acetylation at lysine 27. (B) As indicated by catRAPID, DSCAM-AS1 was predicted to bind to two key histone acetylases: KAT5 and EP300. The interaction matrix showed that bases 0 to 200 and 800 to 900 of DSCAM-AS1 probably have strong associations with both the KAT5 and EP300 proteins. (C) In the RIP assay, anti-KAT5, anti-EP300 antibodies, and IgG were used to separate KAT5-RNA, EP300-RNA, and IgG-RNA complexes from the cell lysate. DSCAM-AS1 was successfully detected in the KAT5-RNA and EP300-RNA complexes but not in the IgG-RNA complex. (D) In the RNA pull-down assay, bio-DSCAM and bio-DSCAM-AS1 were used to detect proteins that could bind to DSCAM mRNA and lncRNA DSCAM-AS1. Western blotting indicated that DSCAM-AS1, but not DSCAM mRNA, bound to KAT5 and EP300 proteins. (E) Results from ChIP assay showed that acetylated histone 3 bound to the DCTPP1 promoter. However, DSCAM-AS1 knockdown decreased acetylated histone 3 enrichment in the DCTPP1 promoter. (F) In FISH and immunofluorescence assays, both DSCAM-AS1 and DCTPP1 were observed in the cytoplasm and cell nucleus (F). **P<0.01 and ***P<0.001.