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. 2022 Jun 9;13:3190. doi: 10.1038/s41467-022-30940-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Schema showing the calculation of the H3K36me3 index. b Dot plot showing the first screening results. Genes, KO of which changed H3K36me3 indexes less than 15% (gray), decreased or increased the H3K36me3 indexes in blue and red, respectively. c Dot plot showing the second screening results. color scheme as in (b). d Protein levels in WT and Smyd5 KO mESCs. Setd2 KO #1 mESCs were used as a positive control for H3K36me3. Cell extracts were analyzed by Western blotting using the specified antibodies. Two independent experiments were performed. Source data are provided as a Source Data file. e The normalized read distribution profiles of H3K36me3 CUT&Tag spanning 5 Kb of gene bodies in WT and Smyd5 KO mESCs. TSS, transcription start site. TES, transcription end site. f Heatmaps of H3K36me3 levels detected by CUT&Tag around gene body regions in WT and Smyd5 KO mESCs. 5 Kb windows spanning the TSS to TES of all genes determined by NCBI RefSeq were plotted. g IGV tracks presenting the enrichment of H3K36me3 by H3K36me3 CUT&Tag in WT and Smyd5 KO mESCs. Red boxes: promoter regions. Blue boxes: gene body regions. h HMT assays of full length SMYD5, SET domain of SUV420H1, and SET domain of SETD2 proteins against nucleosome or octamer substrates. Upper panel: Western blotting. Lower panel: the input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Star indicated the non-specific proteins. i End-point HMT assays of SMYD5 against an equal amount of WT, H3K36M, H4K20M, and H3K36M, H4K20M mutant octamers. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. j HMT assays of full length SMYD5 against different amounts of WT and H3K36M octamer substrates. Upper panel: H3K36me3 western blot. Lower panel, input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Source data are provided as a Source Data file. k End-point HMT assays of SMYD5 against the different amounts of H4K20M and H3K36M/H4K20M octamers. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. Source data are provided as a Source Data file. l ESI-TOF mass spectrometry analysis of Histone H3.