Fig. 4: ASXL1 mutation leads to aberrant genome methylation patterns.

A Methylation DNA immunoprecipitation demonstrates widespread increased cytosine methylation in the ASXL1 mutant cells compared to the homozygous corrected cells. Analysis of this data shown as percent control demonstrates significantly more (p-value = 0.0003, t test) cytosine methylation in the ASXL1 mutant cells compared to the corrected cells. B Evaluation of the methylation DNA immunoprecipitation data alongside the RNA sequencing data demonstrates a clear difference in expression between genes with methylation peaks in the gene bodies versus those with peaks in the gene promoter. Specifically, increased methylation of the gene body was associated with increased gene expression, while increased methylation of the promoter region of genes was associated with decreased gene expression. C To validate the results in the primary samples, DNA methylation in a cohort of MDS ASXL1 mutant and wild-type peripheral blood mononuclear samples was assessed. The HELP assay revealed increased methylation in ASXL1 mutant samples particularly in the gene bodies, consistent with the results seen in the isogenic cell lines. D Genome-wide analysis of ASXL1 mutant and wild-type patient samples for gene body methylation demonstrate lower average methylation angles among the ASXL1 mutant samples (n = 7) compared to the wild-type samples (n = 9), indicating increased methylation in the gene body of the ASXL1 mutant patient samples, consistent with the results seen in the isogenic cell lines. E IGV plots for the BCL2 and BCLXL promoter regions of the ASXL1 mutant and wild-type patient samples demonstrate hypomethylation in the ASXL1 mutant samples (MDS 1–7, n = 7) compared to the wild type samples (MDS 8–16, n = 9).