Fig 7. SETD2 regulates the transcription level of Lbp via altering the trimethylation level of H3K36.
(A) ChIP-seq profiles of H3K36me3 occupancy to the locus of Lbp gene in BMSCs from Setd2fl/fl BMSCs expressing GFP or Cre recombinase. Scale bar: 5 kb. All panels have the same signal scale of 0–5 RPM on the y-axis. (B) ChIP-qPCR analysis of H3K36me3 occupancy to the locus of Lbp gene, using IgG as the control. The location of primer #1 and #2 are indicated in the diagram of A. (C) qPCR analysis of the expression of Lbp heterogeneous nuclear RNA introns. The location of primers is indicated in the upper diagram. (D) ChIP-qPCR analysis of RNA polymerase II binding to the locus of Lbp genes. The location of primers for ChIP-qPCR (black bars) is indicated in the upper diagram. (E) Western blot of H3K36me3 in WT and Setd2 KO BMSCs transfected with tSetd2 WT and tSetd2 R1599H mutation lentivirus. (F) qPCR analysis of Lbp in WT and Setd2 KO BMSCs infected with WT tSetd2 and tSetd2 R1599H mutation lentivirus. Results are presented as the mean ± SD, n = 4. (G) Morphological images of adipocyte differentiation at day 6 from BMSCs of WT and Prx1-Cre, Setd2fl/fl mice infected with GFP, WT tSetd2 and tSetd2 R1599H mutation lentivirus. Differentiated adipocytes were stained with Oil Red O. Scale bar = 1 mm. (H) Quantitative analysis of Oil Red O staining area. Results are presented as the mean ± SD, n = 3. (I) Analysis of Cebpα, Perilipin via qPCR at day 6 of adipocyte differentiation after indicated treatment. (J) Graphic model. H3K36me3 mediated by SETD2 regulates BMSC lineage commitment by promoting Lbp expression. Data used in the generation of this figure can be found in S1 Data. BMSC, bone marrow mesenchymal stem cell; Cebpα, CCAAT enhancer binding protein α; ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; H3K36me3, H3 lysine 36 trimethylation; IgG, immunoglobulin G; KO, knockout; Lbp, lipopolysaccharide-binding protein; qPCR, quantitative PCR; SETD2, SET-domain-containing 2; WT, wild-type.