ASXL1 deficiency alters H3K27me3 methylation on gene promoters. (A) ASXL1^flox/flox and ASXL1^cKO BMMs from 12-week-old male mice were cultured with M-CSF and 50 ng/mL RANKL for 2 days. Representative immunoblot of bulk histone methylation for H3K27me3 on acid extracted histones. Total H3 was used as a loading control (n = 3 independent experiments). (B) ASXL1^flox/flox (WT) and ASXL1^cKO (KO) osteoclast were chromatin immunoprecipitated using H3K27me3 or H3K4me3 antibody followed by DNA sequencing. Model based analysis of ChIP-seq data (MACS2) for H3K27me3 methylation. Left: Venn diagram made with http://jolars.co/eulerr/. Right: Heat map for H3K27me3 on chr1 in ASXL1^flox/flox and ASXL1^cKO osteoclast (+3 kb of transcription start site [TSS]). (C) Characterization of H3K27me3 binding sites in various genomic regions in ASXL1^flox/flox and ASXL1^cKO osteoclasts. (D) Gene sets uniquely enriched in KO mice with H3K27me3 loss using Kyoto Encyclopedia of Genes and Genomes pathway analysis. (E) MA plots for H3K27me3 (up) and H3K4me3 (down) to visualize genes (data points) that are being identified as differentially bound (red). Osteoclast relevant genes identified in blue. (F) H3K27me3 peaks at individual loci (highlighted blue bar), NFATc1, itgb3, and Mmp14; WT (ASXL1^flox/flox) and KO (ASXL1^cKO). (G) Motif enrichment analysis of novel H3K4me3 binding sites in ASXL1^cKO osteoclasts.