PPARγ binds SETD6 promoter and activates its expression.

(A) Top: Sequence logo of the PPARγ response element (PPRE) from JASPAR. Bottom: Schematic representation of PPARγ binding to a predicted PPRE site within the SETD6 promoter (B) Capture of a genome browser showing the enrichment of PPARγ at the SETD6 promoter in HepG2 cells with open chromatin state represented by H3K4me3, H3K27ac, and ATAC-seq tracks. (C) ChIP assay with Flag-PPARγ antibody or beads as negative control in HepG2 cells followed by qPCR with primers flanking the predicted binding site at the SETD6 promoter. Graphs show % input of the quantified DNA. (D) RNA was extracted from HepG2 cells transfected with control or Flag-PPARγ WT. Transcript levels of SETD6 were determined by qPCR. Error bars are SEM. Statistical analysis was performed for three experimental repeats using one-way ANOVA (*p < 0.05, ****p < 0.0001). (E)– dual-luciferase assay in HepG2 cell transfect with an increasing amount of Flag-PPARγ. About 24 h post-transfection, the whole cell lysates were subjected to dual-luciferase assay (Promega). Relative luminescence was calculated after normalization of the firefly luciferase signal over Renilla luciferase control. Error bars are SD. Statistical analysis was performed for three experimental repeats. *p ≤ 0.03

Physical interaction between PPARγ and SETD6 in-vitro and in cells

(A) ELISA-based analysis of the interaction between recombinant GST-SETD6 and the indicated recombinant proteins. ****p < 0.0001. (B) HEK293T cells were transfected with the indicated plasmids followed by immunoprecipitation with Flag antibody of the chromatin fraction. Samples were then subjected to WB analysis using the indicated antibodies. (C) Endogenous SETD6 was immunoprecipitated from chromatin isolated from HepG2 cells followed by western blot with the indicated antibodies. (D) Left-Representative images of PLA detecting GFP-SETD6 and Flag-PPARγ proximity in HepG2 cells. Red dots represent PLA signal. Scale bar = 10 micron. Right-PLA signal quantification for each sample. Statistical analysis was performed using student’s t-test (**** p<0.0001).

SETD6 methylates PPARγ at K170 in-vitro and in cells.

(A) In vitro methylation assay in the presence of 3H-labeled SAM and the indicated purified proteins. Coomassie stain of the recombinant proteins used in the reactions is shown at the bottom. Schematic representation of PPARg domain structure. The methylated residue (K170) identified by mass spectrometry is shown in red. DBD-DNA binding domain; LBD-ligand binding domain. (B) Endogenous PPARγ was immunoprecipitated from HepG2 cells followed by WB with the indicated antibodies (C) Flag-PPARγ was over-expressed followed by immunoprecipitation using pan-methyl antibody in control (CT) and SETD6 KO cells followed by western blot with indicated antibodies. (D) 0.25ug of PPARγ peptides (un-modified and K170me1) were spotted on a nitrocellulose membrane followed by incubation with anti-PPARγ K170me1 antibody or Streptavidin-HRP. (E) Endogenous PPARγ was immunoprecipitated from control and KO SETD6 HepG2 cells followed by western blot with the indicated antibodies.

PPARγ binds and activates SETD6 expression in a K170 methylation dependent manner

(A) – dual-luciferase assay in Control and SETD6 KO HepG2 cells. 24 h post-transfection, the whole cell lysates were subjected to dual-luciferase assay (Promega). Relative luminescence was calculated after normalization of the firefly luciferase signal over Renilla luciferase control. Error bars are SD. Statistical analysis was performed for three experimental repeats. ***p < 0.001. (B) Left-WB analysis with the indicated antibodies for stable cells expressing Flag-WT or Flag-K170R PPARγ. Right-Transcript levels of the SETD6 were determined by qPCR of stably expressing HepG2 cells-Empty, Flag-PPARγ WT, and Flag-PPARγ K170R mutant. mRNA levels were normalized to GAPDH and then to Empty. (C) Chromatin immunoprecipitation (ChIP) assay. The chromatin fraction of HepG2 SETD6 CRISPR CT, KO1, and KO2 cells were immunoprecipitated with magnetic beads conjugated with anti-PPARγ antibody. The bound DNA was purified and amplified by qPCR. (D) Same as C for cells stably expression of Empty, Flag-PPARγ WT, and Flag-PPARγ K170R mutant. Graphs for C and D show the percent input of the quantified DNA. Two-way ANOVA analysis was performed; error bars are S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

SETD6 positively regulates lipid droplets formation:

(A) WB analysis with the indicated antibodies for HepG2 control (2 clones) and SETD6 KO cells (3 clones) (B) Heatmap showing up– and down-regulated genes from RNA-sequencing analysis of 2 SETD6 control and 3 KO HepG2 cells independent clones. Yellow and blue colors represent high and low expression levels, respectively. (C) Selected pathways from gene ontology (GO) analysis of the differentially expressed genes were analyzed. Circle size represents the count of differentially expressed genes related to each pathway. (D) Illustration of the system to monitor lipid droplets accumulation over time: HepG2 cells are treated with oleic acid (OA) and stained with Hoechst (nucleus) and BODIPY (neutral lipids), followed by live cell imaging. (E) Top-Representative images of HepG2 CRISPR CT (control), KO1, and KO2 challenged with 300 mM of OA (20 H time point) and stained with Hoechst (nucleus) and BODIPY (neutral lipids). Bottom-Mean green fluorescence was calculated as fluorescence signal divided by cell count. Data is analyzed from three beacons per well in three wells. Statistical analysis was performed using one-way ANOVA. ns: non-significant; *p < 0.05; **p < 0.01. (F) WB analysis for CT and SETD6 KO with or without over-expression of HA-SETD6. (G) Top-Representative images of HepG2 CRISPR CT (control) and SETD6 KO without or with over-expression of HA-SETD6, challenged with 300 mM of OA (20 H time point) and stained with Hoechst (nucleus) and BODIPY (neutral lipids). Bottom-Mean green fluorescence was calculated as fluorescence signal divided by cell count. Data is analyzed from three beacons per well in three wells. Statistical analysis was performed using one-way ANOVA. ns: non-significant; ***p < 0.001; ****p < 0.0001.

PPARγ methylation at K170 positively regulates gene expression and lipid droplets formation.

(A) Heatmap showing up– and down-regulated genes from RNA-sequencing analysis of HepG2 cells stably expression PPARg-WT and PPARg K170R mutant. independent clones. Yellow and blue colors represent high and low expression levels, respectively. (B) Selected pathways from gene ontology (GO) analysis of the differentially expressed genes were analyzed. Circle size represents the count of differentially expressed genes related to each pathway. (C) ChIP analysis for HepG2 control and SETD6 KO cells (two clones) that were immunoprecipitated with PPARg antibody. The bound DNA was purified and amplified by qPCR using specific primers to MOGAT1 and PLIN2 gene promoter regions. Graphs show the percent input of the quantified DNA. Two-way ANOVA analysis was performed; error bars are S.E.M. ****p < 0.0001. (D) Same as for C with HepG2 cells stably exprssing Empty, Flag-PPARγ2 WT, and Flag-PPARγ2 K170R mutant that were immunoprecipitated with FLAG conjugated magnetic beads. error bars are S.E.M. ns: non-significant; *p < 0.05; **p < 0.01; ****p < 0.0001. (E) Top-Representative images of HepG2 cells stably expressing Empty, PPARγ WT or PPARγ K170R mutant, challenged with 300 mM of OA (20 H time point) and stained with Hoechst (nucleus) and BODIPY (neutral lipids). Bottom-Mean green fluorescence was calculated as fluorescence signal divided by cell count. Data is analyzed from three beacons per well in three wells. Statistical analysis was performed using one-way ANOVA. ****p < 0.0001. (F) A Schematic representation of our proposed working model.

Primers for cloning and mutagenesis

Primers for qPCR

Primers for ChIP – qPCR

MS/MS spectra showing monomethylation of recombinant PPARγ at lysine-170 (LKme1LIYDR, m/zobserved =467.789 (z=+2)) after in vitro methylation by SETD6.

The MS spectra was visualized with PDV software (v.2.0.0 (40). The y– and b-ions are annotated and displayed as red, and blue, respectively.

Top: OA accumulation curve of HepG2 cells over 20 hours challenged with 300 µM, 600 µM, and 900 µM of OA.

900 µM DMSO served as a control treatment. Mean green fluorescence was calculated as fluorescence signal divided by cell count. Data is analyzed from five beacons per well, with three wells per OA or DMSO treatment. Bottom-Representative images of last time point (20 H) with three concentrations of OA. For each image, a magnified area of interest is shown (black boxes).

Transcript levels of the indicated genes were determined by qPCR of stably expressing HepG2 cells-Empty, Flag-PPARγ WT, and Flag-PPARγ K170R mutant.

mRNA levels were normalized to GAPDH and then to Empty. error bars are S.E.M. **p < 0.01; ***p < 0.0001.