Fig. 4

LAMP5-AS1 regulates LAMP5 expression through the binding of DOT1L to the LAMP5 locus. (a) Western blotting show that DOT1L was enriched by LAMP5-AS1 pull-down assays. Antisense of LAMP5-AS1 sequence indicates the negative control. FT: flow through. (b) Western blotting for the protein levels of H3K79me2, H3K79me3, and LAMP5 in MLL leukemia cells transduced by LAMP5-AS1 siRNA and control. H3 and beta-actin as the internal control. The H3K79me2 or H3K79me3/ H3, LAMP5/Actin densitometric ratio were recorded by ImageJ, respectively. (c) ChIP-seq profiles (shown in GSE150483) of H3K79me2 and H3K79me3 at the LAMP5 genomic loci in LAMP5-AS1 knockdown (red) compared with control (blue) MOLM-13 cells. The y-axis scales represent read density per million sequenced reads. (d, e) ChIP Quantitative RT-PCR assays show H3K79me2 (d) and H3K79me3 (e) on the LAMP5 gene locus clearly declined upon LAMP5-AS1 knockdown in MLL leukemia cells. Error bars reflect ± SEM (**, p < 0.01; ***, p < 0.001) from three independent experiments. (f) Diagram depicts the procedure of ChIRP. (g) Quantitative RT-PCR for the LAMP5-AS1(upper, left) and LAMP5 locus (upper, right) enrichment in ChIRP experiment captured by LAMP5-AS1 probes. GAPDH and NC-probe were used to be the negative control. Western blotting shows the DOT1L and H3K79me2 protein levels that captured by LAMP5-AS1 probes in ChIRP experiment. GAPDH protein as the negative control. (h) RNA and DNA FISH and IF experiments showed that LAMP5-AS1 and DOT1L co-localized at LAMP5 locus in the cell nucleus. (i) Proposed model for LAMP5-AS1 regulate LAMP5 in MLL leukemia. LAMP5-AS1 regulates LAMP5 by directly interacting with DOT1L