Figure 3.

SNHG1 promotes ZCCHC10 promoter methylation by recruiting DNMT1 and DNMT3B in acute myeloid leukemia. (A) Schematic representation of the ZCCHC10 gene promoter. The CpG island was predicted by MethPrimer software and encompasses nucleotides -128 to +461 relative to the transcription start site (+1). The binding sites of SNHG1 in the sense or antisense strands of ZCCHC10 DNA are shown above and below the promoter representation, respectively. (B-E) 293 cells were transfected with empty vector, wild-type SNHG1 or mutant SNHG1. A total of 24 h post-transfection, the methylation status of ZCCHC10 was determined by (B) nested methylation-specific PCR, the mRNA levels of SNHG1 and ZCCHC10 were determined by (C and D) RT-qPCR, and the protein level of ZCCHC10 was determined by western blotting (E). (F and G) Chromatin immunoprecipitation assays were performed in (F) U937 and (G) ML2 cells expressing shNC or shSNHG1-2 using IgG (negative control), an anti-DNMT1 antibody, or an anti-DNMT3B antibody. Input and precipitated DNA were analyzed by semi-quantitative RT-qPCR. (H) RNA immunoprecipitation assays were performed in ML2 cells using IgG (negative control), an anti-DNMT1 antibody, or an anti-DNMT3B antibody. Input and precipitated RNA were reverse-transcribed into cDNA and analyzed by RT-qPCR. ^**P<0.01, ^***P<0.001 and ^****P<0.0001. DNMT, DNA methyltransferase; RT-qPCR, reverse transcription-quantitative PCR; sh-, short hairpin; NC, negative control.