<i>Setd2</i> deficiency altered genome-wide H3K36me3 occupancy and downstream gene expression.

(A) Oil Red O staining of BMSCs, which were isolated from Setd2fl/fl mice and infected with lentivirus expressing GFP and Cre, induced by adipogenesis medium for 6 days. Scale bar = 1 mm (upper); scale bar = 100 μm (lower). (B) Quantitative analysis of Oil Red O staining area. Results are presented as the mean ± SD, n = 6. (C) Western blot analysis of SETD2 and H3K36me1/2/3 level in BMSCs isolated from Setd2fl/fl mice and infected with lentivirus expressing GFP and Cre. (D) Heat map of RNA-seq data to compare the gene expression of BMSCs from control (Setd2fl/fl BMSCs expressing GFP) and Setd2-knockout cells (Setd2fl/fl BMSCs expressing Cre). The genes are ordered by clustering tightness. Each row represents a single RNA-seq data. (E) Analysis of the occupancy of H3K36me3 ChIP-seq peaks in gene bodies and intergenic regions. The lack of Setd2 caused the decrease of peaks located in gene bodies and the increase in intergenic regions. (F) Normalized read density of H3K36me3 ChIP-seq signals of Setd2fl/fl BMSCs expressing GFP or Cre from 5 kb upstream of the TSS to 5 kb downstream of the TES. (G) Venn diagram illustration of H3K36me3 peaks of Setd2fl/fl BMSCs expressing GFP or Cre and their overlap with differential expression genes determined by RNA-seq. Data used in the generation of this figure can be found in S1 Data. BMSC, bone marrow mesenchymal stem cell; ChIP-seq, chromatin immunoprecipitation sequencing; GFP, green fluorescent protein; H3K36me3, H3 lysine 36 trimethylation; RNA-seq, RNA sequencing; SETD2, SET-domain-containing 2; TES, transcription end site; TSS, transcription start site.