Fig 2: SETD6-mediated methylation of BRD4 at K99 regulates the expression of genes involved in mRNA translation.(A and B) MDA-MB-231 (A) CRISPR control and SETD6 KO/KD or (B) stably expressing Flag BRD4 wild-type or Flag BRD4 K99R cells were subjected to Western blot indicating SETD6 (A) or Flag BRD4 protein levels (B) in the cells (top). Bottom: Heatmap of differentially expressed up- and down-regulated genes from RNA-seq in all the indicated cells. Orange and blue colors represent higher and lower expression, respectively. (C) Common up-regulated (27) CRISPR SETD6 KO/KD–dependent and BRD4 K99R–dependent genes were analyzed using the DAVID database for biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (on the right). Pathways shown are based on the Benjamini-corrected P value of <0.05. (D) Validation of RNA-seq experiments. mRNA was extracted from MDA-MB-231 cells, and transcript levels were determined by quantitative polymerase chain reaction (qPCR). mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and then to empty cells. Error bars are SEM. Statistical analysis was performed for three experimental repeats using one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: Synthetic vp22-RelA peptide interacts specifically with SETD6 in vitro(A) Sequence of vp22-RelA and vp22 peptides. (B) 1 µM of the vp22-RelA and vp22 peptides (analytes) were allowed to flow over the GLM chip bound His-SETD6. Binding was analyzed using SPR spectroscopy with the ProteOn XPR36 system. (C) The dissociation constant (KD) was determined from the sensorgram of the equilibrium binding phase calculated by allowing 5 different concentrations of the vp22-RelA analyte to flow over the chip bound His-SETD6. (D) Upper panel - Illustration of the ELISA sandwich experiment performed in the lower panel. ELISA-based analysis of the interaction between recombinant GST-SETD6 and the biotinylated peptides. The 96-well plate was coated with 100 µg streptavidin to which 20 µM biotinylated peptides were bound. Then peptides were covered with 0.5 µg GST-SETD6 or GST. Signal detection was obtained using anti-GST antibody followed by secondary HRP-conjugated antibody (error bars, s.e.m.).

Fig 4: SETD6 methylates TWIST1 at K33. (A) Illustration of TWIST1 sequence and domains with the identified methylated lysine residues (NLS-nuclear localization sequence, bHLH-basic-helix-loop-helix). (B) In vitro methylation assay was performed with recombinant His-TWIST1 and GST-SETD6 with or without SAM followed by mass spectrometry analysis. MS spectra of TWIST1 QQPPSGKR peptide is shown. The MS spectra was visualized with PEAKS software. Major y- and b-ions are displayed as red, and blue, respectively. The m/z ratio of labelled ions are shown above each ion label. Mono-methylated lysine is indicated as a lowercase ‘k’. For the parent ion, mass-to-charge ratio (m/z), overall charge, and PEAKS score (-10lgP) are shown above the spectrum. (C) MS results were validated by in vitro methylation assay with the indicated recombinant proteins in the presence of 3H-labeled SAM. (D) semi-in vitro methylation assay. HEK293T SETD6 KO cells were transfected with FLAG-TWIST1 WT or K33R mutant. Chromatin fractions were immunoprecipitated with FLAG-M2 beads and were subjected to radioactive in vitro methylation assay with recombinant SETD6. Autoradiogram used to detect 3H-labeled proteins and Coomassie staining to detect all proteins. (E) TWIST1 peptides (Unmodified and K33me1) were subjected to SDS-PAGE followed by western blot analysis with K33me1 antibody and coomassie staining. (F) Western blot analysis of U251 stably expressing empty vector, FLAG-TWIST1 WT or K33R cells with the indicated antibodies. (G) HEK293T or U251 (H) control and SETD6 KO cells were transfected with FLAG-TWIST1 and immunoprecipitated with FLAG-M2 beads followed by Western blot analysis with the indicated antibodies.

Fig 5: Methylated TWIST1 binds LINC-PINT gene region. (A) Venn diagram showing lncRNAs regulated by both SETD6 and TWIST1, which identified in the previous RNA-seq experiments. (B) Graph of top 8 shared lncRNAs (FC > 1.5) with their FC in SETD6 KO cells (x-axis) and cells stably express FLAG-TWIST1 (y-axis). (C) LINC-PINT genomic region. Layered H3K4me3, H3K27Ac and TF clusters were extracted from the UCSC genome browser. The two TWIST1 binding sites (BS1 and BS2) used for ChIP assays are shown. E-boxes inside each binding site are indicated. (D) ChIP assays of U251 SETD6 KO cells immunoprecipitated with TWIST1 antibody or beads as negative control. (E) ChIP assay of U251 stably expressing empty vector, FLAG-TWIST1 WT or K33R cells immunoprecipitated with FLAG-M2 beads. (D, E) Graphs show % input of the quantified DNA. Error bars are SEM. Statistical significance was calculated using two-way ANOVA for three experimental repeats (ns, non-significant, * P < 0.05, **P < 0.01, ***P < 0.001).